Learning from the master: targets and functions of the CtrA response regulator in Brucella abortus and other alpha-proteobacteria

Learning from the master: targets and functions of the CtrA response regulator in Brucella... Abstract The α-proteobacteria are a fascinating group of free-living, symbiotic and pathogenic organisms, including the Brucella genus, which is responsible for a worldwide zoonosis. One common feature of α-proteobacteria is the presence of a conserved response regulator called CtrA, first described in the model bacterium Caulobacter crescentus, where it controls gene expression at different stages of the cell cycle. Here, we focus on Brucella abortus and other intracellular α-proteobacteria in order to better assess the potential role of CtrA in the infectious context. Comparative genomic analyses of the CtrA control pathway revealed the conservation of specific modules, as well as the acquisition of new factors during evolution. The comparison of CtrA regulons also suggests that specific clades of α-proteobacteria acquired distinct functions under its control, depending on the essentiality of the transcription factor. Other CtrA-controlled functions, for instance motility and DNA repair, are proposed to be more ancestral. Altogether, these analyses provide an interesting example of the plasticity of a regulation network, subject to the constraints of inherent imperatives such as cell division and the adaptations to diversified environmental niches. CtrA, Brucella, cell cycle, alpha-proteobacteria, infection, regulation network evolution INTRODUCTION Brucella species are responsible for brucellosis, a major and worldwide zoonosis. In animals, it occurs as a chronic infection that is characterized by epididymitis in males or placentitis and abortion in pregnant females (Carvalho Neta et al.2010). Humans are accidental hosts of Brucella melitensis, Brucella abortus and Brucella suis, in which they are responsible for a debilitating disease known as undulant fever or Malta fever (Moreno and Moriyon 2006). Usually, human infections happen through the ingestion of contaminated dairy products or by exposure to infected animals. Another major way of infection is through the aerosol route, which is why Brucella strains are subjected to strict regulations in laboratories (Yagupsky and Baron 2005). There are currently no vaccines available for humans and the only treatment is the use of a combination of antibiotics (Moreno and Moriyon 2006). This review aims at summarizing what is known about Brucella abortus infectious process in host cells, with a particular emphasis on its cell-cycle regulation. Indeed, B. abortus has been reported to stall its cell cycle in the G1 phase, which corresponds to a non-replicating stage, for up to 8 h at the onset of infection of HeLa cells or RAW 264.7 macrophages (Deghelt et al.2014). This review therefore focuses on the master regulator CtrA, a transcription factor particularly well conserved in α-proteobacteria and known to regulate the Caulobacter crescentus cell cycle (Laub, Chen and McAdams 2002; Brilli et al.2010). Up to now, the only comparative studies about the CtrA regulons of different α-proteobacteria were mainly based on bioinformatics predictions (Hallez et al.2004; Brilli et al.2010). As literature on CtrA has been dramatically increasing over the last years, it is now possible to compile experimental data. We thus give an overview of the conservation of specific modules in the CtrA regulon, as well as the acquisition of new factors that occurred during evolution, while focusing more particularly on intracellular bacteria. Brucella inside host cells Brucella intracellular trafficking A whole genome-based phylogeny study revealed that brucellosis probably appeared in wildlife populations in the past 86 000 to 296 000 years (Foster et al.2009). It thus happened before livestock domestication, even though this crucial step in history probably played a role in allowing the worldwide spreading of these pathogens (Foster et al.2009). Even though they can be cultivated on artificial media, it is established that Brucella need to enter inside their host cells in order to complete a successful infection process (Moreno and Moriyon 2006). This is why they are now considered as facultatively extracellular intracellular parasites (Moreno and Moriyon 2002). The mechanism by which Brucella manage to invade their host organism is not very clear but they seem to cross the mucosal barrier, which could imply an interaction with epithelial cells (Roop et al.2009). The role of these cells has not been deciphered yet but epithelial HeLa cells have been effectively used as models for Brucella infection in non-professional phagocytes (Pizarro-Cerda et al.1998; Castaneda-Roldan et al.2004; Starr et al.2008). Once inside its host, Brucella could also get internalized by professional phagocytes such as macrophages or dendritic cells. There, the bacterium can survive and multiply before disseminating in the organism (Archambaud et al.2010). Surprisingly, B. melitensis has also been reported to be able to invade murine erythrocytes during infection, which suggests that other cellular and in vivo models of infection should be developed to fully understand Brucella pathogenesis (Vitry et al.2014). The entry of Brucella into epithelial or phagocytic cells occurs within minutes after cell-to-cell contact (Pizarro-Cerda et al.1998). Once internalized, the bacterium stays in a membrane-bound Brucella-containing vacuole (BCV) that interacts with the endocytic pathway (therefore termed eBCV) (Fig. 1). Early endosomal markers, such as Rab5, are rapidly followed by the acquisition of late endosomal markers, typically lysosomal membrane-associated protein-1 (LAMP1) (Pizarro-Cerda et al.1998). Transient interactions with lysosomes have also been reported (Starr et al.2008). This eventually leads to eBCV acidification, which is deleterious to many bacteria, but nonetheless necessary for Brucella to reach their replicative niche and survive in the long-term (Porte, Liautard and Kohler 1999; Boschiroli et al.2002a; Celli et al.2003; Starr et al.2008). Indeed, the acidic pH of the eBCV has been linked to the capacity of the pathogen to induce the expression of the virB operon (Boschiroli et al.2002a). These genes code for a type IV secretion system (T4SS) that is essential for the bacteria to reach their proliferation niche (Boschiroli et al.2002b). Figure 1. View largeDownload slide Schematic representation of B. abortus trafficking inside host cells. Once inside its host cell, B. abortus extensively interacts with the endocytic pathway. The compartment in which it resides at that stage can be referred to as the endocytic Brucella-containing vacuole (eBCV). In HeLa cells and RAW 264.7, during this first step of the infection, the bacterium is blocked in G1 and its growth is arrested. After a transient interaction with the lysosomes and thanks to its type IV secretion system VirB, the bacterium reaches its replicative niche (rBCV), which is part of the endoplasmic reticulum (ER) in most cell types. Later on, bacteria are found in autophagy-dependent vacuoles (aBCV) and are proposed to reinfect neighbor cells. Figure 1. View largeDownload slide Schematic representation of B. abortus trafficking inside host cells. Once inside its host cell, B. abortus extensively interacts with the endocytic pathway. The compartment in which it resides at that stage can be referred to as the endocytic Brucella-containing vacuole (eBCV). In HeLa cells and RAW 264.7, during this first step of the infection, the bacterium is blocked in G1 and its growth is arrested. After a transient interaction with the lysosomes and thanks to its type IV secretion system VirB, the bacterium reaches its replicative niche (rBCV), which is part of the endoplasmic reticulum (ER) in most cell types. Later on, bacteria are found in autophagy-dependent vacuoles (aBCV) and are proposed to reinfect neighbor cells. The Brucella replicative niche (rBCV) has been known for years to derive from the endoplasmic reticulum (ER), in both HeLa cells and macrophages (Pizarro-Cerda et al.1998; Celli et al.2003). It is only recently that the rBCV was shown to actually be part of the endoplasmic reticulum (Sedzicki et al.2018) (Fig. 1). The transition from eBCV to rBCV is not clearly understood yet, but it has been suggested that its maturation could occur at the ER exit sites (Celli, Salcedo and Gorvel 2005; Celli 2015). Several ER-associated functions have been linked to Brucella infection, such as the unfolded protein response IRE1α signaling pathway (Qin et al.2008; Smith et al.2013; Taguchi et al.2015), some autophagy-associated factors such as ATG9 and WIPI (Taguchi et al.2015) and the early secretory trafficking depending on the Sar1/coat protein complex II (Celli, Salcedo and Gorvel 2005; Taguchi et al.2015). Since the T4SS is essential for Brucella to reach the rBCV, it is expected that the maturation of the BCV would be mediated by the delivery of bacterial effectors inside the host cell. One such effector is BspB, shown to target the Golgi apparatus by interacting with the oligomeric Golgi tethering complex (Miller et al.2017). This leads to the redirecting of Golgi-derived vesicles to the BCV by remodeling the ER-Golgi secretory trafficking (Miller et al.2017). It is important to note that there exist alternatives to the ER-derived replicative niche since opsonized B. abortus proliferate in a non-acidic LAMP1-positive compartment in the human monocytic cell line THP-1 (Bellaire, Roop and Cardelli 2005) and in endosomal inclusions in extravillous trophoblasts (Salcedo et al.2013). Once the number of bacteria within a cell reaches a critical level, destruction of the host cell can be observed (Moreno and Moriyon 2006). Another means for Brucella to spread from one cell to its neighbors has been shown by Starr et al (2012). The formation of a compartment with autophagic features (aBCV) could be the key to this important step of the infection (Fig. 1). Indeed, autophagy-deficient Brucella are not able to perform cell-to-cell spreading when cellular infections are prolonged for long periods, typically 72 h (Starr et al.2012). Interestingly, only the initiation complex of autophagy seems to be needed by Brucella to promote reinfection (Starr et al.2012). Indeed, markers of the elongation phase of autophagy such as ATG5 and LC3 were not found to be associated to the aBCV (Starr et al.2012). It should be noted that autophagy is particularly important at birth. At that time, the transplacental nutrient supply is no longer available, which suggests that autophagy is strongly activated in the neonate in order to adapt to the early neonatal starvation period (Kuma et al.2004). The use of this process by the bacteria could therefore be relevant for their spreading inside newborn calves. Growth and replication of Brucella B. abortus possesses two distinct chromosomes (Chain et al.2005). Surprisingly, bacteria with multipartite genomes are not uncommon, at about 10% of the sequenced species (Val et al.2014). Contrarily to plasmids that are known to initiate replication several times during the bacterial cell cycle, chromids (also known as megaplasmids) code for essential genes and initiate their replication only once per cell cycle, like chromosomes (Pinto, Pappas and Winans 2012; Val et al.2014). In B. abortus, the large and circular chromosome (I) is 2.1 Mb long and possesses a ParAB segregation system with three centromere-like parS sites, while the small chromosome (II) of 1.2 Mb is a chromid, with its replication being controlled by a RepABC system (see Pinto, Pappas and Winans 2012 for a review on this segregation system). The repABC operon also contains two centromere-like sequences called repS (Livny, Yamaichi and Waldor 2007; Deghelt et al.2014). The chromosomal replication status of B. abortus, and thus the stage of its cell cycle, can be followed with fluorescent reporters of the segregation markers ParB and RepB, as well as with fluorescent reporters allowing the localization of the replication origins (ori) and terminators (ter). Both chromosomes are oriented along the cell length, with oriI and terI associated with the poles, whereas oriII and terII are usually found closer to the midcell (Deghelt et al.2014). This is in agreement with what has been found in Sinorhizobium meliloti, another α-proteobacterium. Indeed, this bacterium possesses a tripartite genome with one primary chromosome (3.65 Mb) and two chromids (1.35 Mb for pSymA and 1.68 Mb for pSymB) (Galibert et al.2001). Both chromids are segregated by a RepABC system and their ori are not anchored to the poles (Kahng and Shapiro 2003; Frage et al.2016). S. meliloti is capable of colonizing the soil rhizosphere as a free-living bacterium, but also of invading the roots of leguminous plants as an intracellular symbiotic nitrogen-fixing bacterium, involving complex interactions between the bacterium and its host (Gibson et al.2006). Similarly to C. crescentus and B. abortus, free-living S. meliloti regulate their cell cycle so that replication of their genome occurs once-and-only-once per cell division (Mergaert et al.2006). In both B. abortus and S. meliloti, a temporal coordination of replication and segregation was found, as the initiation of replication of their chromids is always delayed compared to the main chromosome (Deghelt et al.2014; Frage et al.2016). In Brucella, the replication of oriI starts before oriII and both chromosomes would finish their replication at approximately the same time (Deghelt et al.2014). Note that the size of the chromids does not seem to be the determining factor for the temporal regulation of their replication initiation. Indeed, in S. meliloti, it has been proposed that the smaller pSymA initiates its replication when the ori of the main chromosome has reached the new pole and that it is followed by the bigger pSymB, which behaves in a similar manner after the pSymA ori has been replicated (Frage et al.2016). Two main phases can be observed during HeLa cells infection by B. abortus. Indeed, when the bacterium is transiting within the eBCV, it is unable to proliferate, which reflects the fact that the number of colony forming unit (CFU) is stable during this non-proliferative stage (Comerci et al.2001; Starr et al.2008; Deghelt et al.2014). The second phase occurs when Brucella reaches its ER-derived proliferative niche, with the number of CFU increasing drastically (Pizarro-Cerda et al.1998; Celli et al.2003; Starr et al.2008). Thanks to fluorescent reporter systems that can track ori's, it has been possible to follow the B. abortus cell cycle inside host cells. One interesting observation was that during the non-proliferative stage of the trafficking in HeLa cells and RAW 264.7 macrophages, the bacteria are blocked in G1 (only one focus of oriI), similarly to what happens in the carbon-starved swarmer cells of C. crescentus, a free living α-proteobacterium (Lesley and Shapiro 2008; Deghelt et al.2014). As Brucella exhibits asymmetric growth like other Rhizobiales (Brown et al.2012), it is also possible to use Texas Red Succinimidyl Ester—a fluorescent compound that covalently binds amine groups on the bacterial surface—as a mean to follow the bacterium unipolar growth (Brown et al.2012) inside host cells (Deghelt et al.2014). These techniques brought to light the fact that the bacteria found within the eBCV at early times after infection are predominantly non-growing newborn cell types. This term refers to bacteria that recently divided but did not yet initiate chromosome replication (Deghelt et al.2014). They stay in this state for up to 8 h before resuming their growth and chromosome replication when they still reside within an eBCV (Deghelt et al.2014). Roles and regulation of CtrA B. abortus CtrA regulation is similar to that of C. crescentus Since the invasive B. abortus are mainly in the G1 phase of their cell cycle (Deghelt et al.2014), it is possible that transcription factors involved in cell-cycle regulation could be also important for Brucella virulence. One such factor is CtrA. This transcription factor is very well conserved amongst α-proteobacteria (Brilli et al.2010) and has been best studied in the model organism C. crescentus. This remarkable bacterium possesses two distinct life forms. One is a sessile stalked form, which allows the bacterium to adhere to surfaces when it is in a nutrient-rich environment. The other form is a motile swarmer cell that is used for scouting and colonizing new favourable environments but that is not competent for replication (Ausmees and Jacobs-Wagner 2003; Quardokus and Brun 2003). Importantly, C. crescentus divides asymmetrically into its two phenotypically different daughter cells after each cell division. This is why this bacterium is considered as an excellent model for bacterial cell cycle studies. In this context, the transcription factor CtrA has been found to be of utmost importance as it is a master regulator of C. crescentus cell cycle (Quon, Marczynski and Shapiro 1996) (Fig. 2). Indeed, one of CtrA many targets is the ori, thus preventing the DnaA protein from initiating the replication of C. crescentus chromosome as long as CtrA is present (Quon et al.1998; Siam and Marczynski 2000). As CtrA needs to be phosphorylated to be active, a tight regulatory network based on two-component regulators is in charge of its synthesis, phosphorylation and degradation (Quon, Marczynski and Shapiro 1996; Domian, Quon and Shapiro 1997; Wu, Ohta and Newton 1998; Biondi et al.2006; Tsokos, Perchuk and Laub 2011). Figure 2. View largeDownload slide Models for CtrA regulation in two α-proteobacteria. The schemes represented here are mainly based on C. crescentus CtrA regulation (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), therefore it is important to take into consideration the fact that the phosphorylation cascade events might not happen exactly as depicted. In the case of B. abortus, data were obtained from Willett et al. (2015) and Francis et al. (2017). Green arrows correspond to confirmed CtrA targets that are positively regulated by the transcription factor. Blue rounded arrows correspond to targets that are bound by CtrA on their promoter, but for which the effect of this binding still remains unknown. Figure 2. View largeDownload slide Models for CtrA regulation in two α-proteobacteria. The schemes represented here are mainly based on C. crescentus CtrA regulation (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), therefore it is important to take into consideration the fact that the phosphorylation cascade events might not happen exactly as depicted. In the case of B. abortus, data were obtained from Willett et al. (2015) and Francis et al. (2017). Green arrows correspond to confirmed CtrA targets that are positively regulated by the transcription factor. Blue rounded arrows correspond to targets that are bound by CtrA on their promoter, but for which the effect of this binding still remains unknown. In C. crescentus, the dual CckA enzyme that possesses both kinase and phosphatase activities regulates the phosphorylation level of CtrA and CpdR, a response regulator stimulating CtrA proteolysis when dephosphorylated (Jenal and Fuchs 1998; Jacobs et al.2003; Biondi et al.2006). CckA does so by interacting with the phosphotransferase ChpT (Biondi et al.2006). The kinase activity of CckA is inhibited by the phosphorylated form of the response regulator DivK, which is stabilized by the atypical histidine kinase DivL (Tsokos, Perchuk and Laub 2011; Childers et al.2014). DivK phosphorylation is itself regulated by the histidine kinase DivJ and by the phosphatase PleC (Wu, Ohta and Newton 1998; Wheeler and Shapiro 1999). PleC is also able to phosphorylate the diguanylate cyclase PleD, which in turn will synthesize cyclic di-GMP (Paul et al.2008). The binding of this secondary messenger to CckA will force CckA to switch from its kinase to its phosphatase mode, thus preventing the phosphorylation of CtrA (Lori et al.2015). Cyclic di-GMP also binds to PopA, which interacts with RcdA, another protein involved in CtrA proteolysis at the stalked cell pole (Ozaki et al.2014; Smith et al.2014). Interestingly, several genes coding for proteins regulating CtrA are also part of its regulon, including divK (Laub et al.2000) and divJ (Fumeaux et al.2014) in C. crescentus. At the transcription level, ctrA is regulated by two proteins in C. crescentus. One of them is CtrA itself (Domian, Reisenauer and Shapiro 1999). The other one is GcrA, an unconventional transcription factor that binds to the housekeeping σ70 factor (Haakonsen, Yuan and Laub 2015). Since gcrA transcription is repressed by CtrA and ctrA transcription is activated by GcrA, the two transcription factors are present temporally and spatially out-of-phase during the cell cycle of C. crescentus (Holtzendorff et al.2004). Of note, DnaA also participates to gcrA transcription (Collier 2012). The spatio-temporal regulation of CtrA is particularly well adapted to C. crescentus aquatic free-living lifestyle, but it appears to be surprisingly conserved in other α-proteobacteria with very different ways of life (Brilli et al.2010; Pini et al.2015; Schallies et al.2015; Willett et al.2015). In B. abortus, the core actors involved in the CtrA regulatory network, defined here as the PleC/DivJ/DivK and CckA/ChpT/CtrA two-component systems, are conserved (Hallez et al.2004; Brilli et al.2010) (Fig. 2). Another gene, called pdhS for PleC/DivJ homologue sensor, has also been found to be part of this network (Hallez et al.2004). Both pleC and divK from B. abortus are able to heterocomplement the corresponding deletion mutants in C. crescentus, which suggests that their function is conserved between both organisms (Hallez et al.2007). In addition, in B. abortus DivK has been found by yeast two-hybrid experiment to bind to DivJ, PleC, DivL and PdhS (Hallez et al.2007). Nevertheless, the localization of PleC is different between B. abortus and C. crescentus and DivJ was not found to be crucially involved in DivK phosphorylation (Hallez et al.2007). Indeed, in a ΔdivJ background, DivK did not lose its phosphorylation-dependent polar localization (Hallez et al.2007), while a loss-of-function of pdhS generates delocalization of DivK-YFP (Van der Henst et al.2012). As pdhS is an essential gene and depletion strains were not available at that time, its involvement in DivK phosphorylation could only be suggested through indirect experiments. PdhS was also shown to accumulate at the old pole of the large cells, which is the same localization as the phosphorylated form of DivK (Hallez et al.2007). Of note, this localization is similar to the one of DivJ in C. crescentus, which suggests a common function between B. abortus PdhS and C. crescentus DivJ (Hallez et al.2007). As PdhS is cytoplasmic in B. abortus, it is possible that its function is shared with DivJ depending on the time and/or space of DivK phosphorylation (Hallez et al.2007). The polar localization of PdhS is conserved at 48 h post-infection in bovine macrophages (Hallez et al.2007), but nothing is known about the role of DivJ in this context and at later times of the infection. As for the phosphorelay going from CckA to CtrA and CpdR via ChpT, it has been confirmed to be functional in B. abortus (Willett et al.2015). As was the case for C. crescentus (Laub et al.2000; Fumeaux et al.2014), several genes predicted to be involved in CtrA regulation have been found by ChIP-seq to be potentially part of CtrA regulon in B. abortus, including ctrA itself, divK, divJ, divL, chpT, cpdR and rcdA (Francis et al.2017). If all of these genes are indeed regulated by B. abortus CtrA, it would mean that the control of this transcription factor is more complex in this bacterium than in C. crescentus (Fig. 2). One tempting hypothesis is that the regulation of B. abortus CtrA could reflect a need for the bacterium to precisely regulate its cell cycle depending on its intracellular environment. B. abortus CtrA controls the expression of genes involved in envelope biogenesis The impact of B. abortus CtrA levels inside host cells could potentially be very important, as this transcription factor seems to be involved in both cell-cycle regulation and bacterial envelope composition (Francis et al.2017). Actually, one striking feature of B. abortus CtrA regulon is the high number of genes involved in envelope biogenesis (Francis et al.2017). Indeed, in addition to revealing the direct interaction between CtrA and the promoters of genes involved in the regulation of lipopolysaccharide and peptidoglycan synthesis, a ChIP-seq experiment also showed that promoters of genes coding for abundant outer membrane proteins (OMP) are also bound by CtrA (Francis et al.2017). CtrA-dependent regulation of these genes was supported by western blots against Omp2b and Omp25, two major OMPs of B. abortus that were found at lower levels in a CtrA depletion strain (Francis et al.2017). In addition, Omp25 homogenous localization patterns are perturbed when CtrA is absent (Francis et al.2017). This modification of the envelope composition could potentially have an impact on the bacterial fitness inside its host cell (Cha et al.2012). Indeed, the levels of Omp2b have been shown to decrease after 20–40 h during the course of macrophage infection by B. abortus (Lamontagne et al.2009). As for omp25, its disruption in B. abortus led to an increased sensitivity to Polymyxin B in vitro (Manterola et al.2007). In HeLa cells and murine macrophages, this strain was not shown to be involved in virulence, as its intracellular replication was similar to that of the wild type (Manterola et al.2007). In BALB/C mice, however, results were different in two independent experiments. In the first, a B. abortus omp25 mutant was injected intravenously at 5 × 104 CFU and led to an attenuation of virulence at 18 weeks post-infection, when compared to the wild type strain (Edmonds, Cloeckaert and Elzer 2002). In the second experiment, mice were infected via the intraperitoneal route with 105 CFU and behaved like the wild-type strain even after 24 weeks (Manterola et al.2007). This suggests that the route of infection could be an important factor to take into account when studying B. abortus infection. In this respect, it would be more appropriate to use a more physiological mode of infection in future research, such as the intranasal one (Hanot Mambres et al.2016). Note that the omp25 mutant seems to have different phenotypes depending on the Brucella species in which it is studied. Indeed, in B. suis, the disruption of this gene had much more dramatic consequences, as it was already attenuated from 1 to 8 weeks post-infection, before to be completely cleared from mice spleen (Edmonds, Cloeckaert and Elzer 2002). The authors suggested that this might be due to the fact that the B. suis strain that they used is a naturally occurring rough strain, thus the loss of the structural Omp25 could be more damaging in this case (Edmonds, Cloeckaert and Elzer 2002). Another difference between the two Brucella strains is that in B. suis, Omp25 is involved in the inhibition of the production of the pro-inflammatory TNF-α cytokine in human macrophages (Jubier-Maurin et al.2001; Luo et al.2017), whereas in B. abortus, it could be involved in the activation of the synthesis of TNF-α in human trophoblastic cells (Zhang et al.2017). The regulation of genes involved in the structure of the bacterial envelope by CtrA is probably not exclusive to B. abortus as many other α-proteobacteria do seem to regulate such genes via CtrA (Laub et al.2000; Brilli et al.2010). Nevertheless, it could have a more important impact on intracellular bacteria, as their envelope will be presented at the interface with their host cell and could therefore impact the host immune response and the survival of the bacteria inside them. For instance, the obligate intracellular Ehrlichia chaffeensis is thought to regulate the expression of its pal gene, coding for a major outer membrane stabilizing protein, through CtrA (Cheng et al.2011). This α-proteobacterium is the causative agent of the life-threatening human monocytic ehrlichiosis and has a developmental cycle comprising two forms in mammalian cells (Zhang et al.2007). The small dense-cored cells (with dense nucleoids) attach to and enter into the host cells, then differentiate into larger reticulate cells (defined by uniformly dispersed nucleoids) that can multiply for 48 h before transforming back into dense-cored cells at 72 h post-infection in order to prepare for reinfection (Zhang et al.2007). Interestingly, both pal and ctrA gene expressions were found to be highest when bacteria were in their dense-cored infectious form (Cheng et al.2011). Conversely, CtrA was found to be rapidly degraded, following a proline and glutamine uptake, after bacterial entry in host cells (Cheng, Lin and Rikihisa 2014). Pal is known to be immunogenic in dogs infected with E. chaffeensis and bacteria treated with anti-Pal antibodies were shown to be less infectious in vitro (Cheng et al.2011). Note that E. chaffeensis has been reported to lack lipopolysaccharide (Lin and Rikihisa 2003). S. meliloti also interacts tightly with host cells during nodulation. Very interestingly, it appears that B. abortus and S. meliloti do share some similarities in their potential CtrA targets. Indeed, CtrA binds to the promoter of several genes coding for L,D-transpeptidases homologs in B. abortus (BAB1_0047, BAB1_0138, BAB1_0589, BAB1_0978, BAB1_1159 and BAB1_1867) and S. meliloti (SMc00150, SMc01200, SMc01575 and SMc01769). L,D-transpeptidases are required to cross-link peptides in the peptidoglycan, and appear to be localized at the specific growth sites in the Rhizobiale Agrobacterium tumefaciens (Cameron et al.2014), another Rhizobiale displaying a unipolar growth (Brown et al.2012). Still, B. abortus seems to have evolved to possess more complex regulation of envelope biogenesis than S. meliloti: all but one (BAB1_0785) L,D-transpeptidase gene promoters are bound by CtrA in B. abortus (Francis et al.2017), as opposed to four out of seven in the case of S. meliloti (the others being SMc02636, SMc02582 and SMc00039) (Pini et al.2015). These L,D-transpeptidases should be further investigated in order to understand how they are truly regulated, as well as their specific roles in peptidoglycan growth and homeostasis. To determine if the envelope biogenesis regulation can directly be linked to the lifestyle of the bacteria would of course require more experimental data from other intracellular bacteria. The expression of ctrA is not crucial for B. abortus trafficking to the rBCV The regulation of genes according to the stage of B. abortus cell cycle can be observed through the use of a fluorescent-based reporter system. Such genes are ccrM and the repABC operon, and both are probably regulated by CtrA as the activities of their promoters were abolished when their respective CtrA-binding boxes were mutated (Francis et al.2017). As the activity of the promoter of the repABC operon seems to be inverted compared to the one of ctrA, it is expected that CtrA acts as a negative regulator of chromosome II replication (Francis et al.2017). Knowing that B. abortus first has to go through a non-replicative phase inside the eBCV, one can expect that CtrA would be important during this specific stage of the infection. However, CtrA was not found to be essential for the ability of B. abortus to infect cells in the models of infection tested thus far. Indeed, a study performed with a thermo-sensitive allele of B. abortus ctrA concluded that the transcription factor is not required for the entry of the bacterium inside THP-1 macrophages (Willett et al.2015). Inside HeLa cells, the CtrA depletion phenotypes of B. abortus were also visible around or after 10 h post-infection (Francis et al.2017). Furthermore, a B. abortus CtrA depletion strain was able to reach its rBCV replicative niche in the same proportion as the wild type strain (Francis et al.2017). This supports the view that CtrA function is dispensable for B. abortus trafficking inside these host cells. Nevertheless, at 48 h post-infection, CtrA depletion strains underwent a clear drop of CFU in both cellular infection models (Willett et al.2015; Francis et al.2017). The defects leading to bacterial cell death are unclear but could be explored by the analysis of suppressor mutants. The fact that CtrA might only be necessary at late time points during Brucella infection does make sense, though, as its level needs to be regulated more particularly during late phases of other intracellular α-proteobacteria life cycle. For example, in the obligate intracellular pathogen E. chaffeensis, CtrA is important during the late stage of intracellular growth (Cheng et al.2011). It has been observed that the ctrA gene is up-regulated at 72 h post-infection, which corresponds to the time when the bacteria differentiate back from large reticulate cells to small infectious dense-cored cells in order to prepare to spread from the present to the next host cells (Zhang et al.2007; Cheng et al.2011). Similarly, in S. meliloti, CtrA levels are high before infection, then a decrease of ctrA transcription coincides with bacteroid differentiation within the nodule (Roux et al.2014) and the CtrA protein is absent in mature bacteroids (Pini et al.2013). It is possible that CtrA is only required when Brucella find themselves in a particular situation. For instance, C. crescentus is known to regulate CtrA levels in response to stresses affecting its envelope, and this in a CckA-dependent manner, but independently of DivK and cyclic-di-GMP (Heinrich, Sobetzko and Jonas 2016). In E. chaffeensis, surE has been proposed to code for a protein involved in bacterial growth under stress and it is thought to be part of CtrA regulon (Cheng et al.2011). It is therefore possible that the in vitro models of infection tested thus far for B. abortus CtrA function do not reflect the environment and the stresses that they would have to face inside a living animal. In this respect, an in vivo model of infection might prove to be much more relevant for deciphering the potential impact of CtrA depletion in B. abortus. Hypotheses emerging from analyses of CtrA regulons Regulation of the cell cycle in different α-proteobacteria As several groups are now focusing on CtrA functions in other α-proteobacteria, more data about this transcription factor have become available over the last years. Despite their very diverse ways of life, α-proteobacteria do share some common traits, including the presence of a gene coding for CtrA (Brilli et al.2010). The comparison of the CtrA regulons in different bacteria, in light of their respective evolutionary lineage and lifestyle, might lead to interesting hypotheses regarding CtrA functions in B. abortus and other intracellular bacteria. Indeed, comparison of the direct CtrA regulons, when they are available, clearly indicate that CtrA binds to promoters of orthologous genes, suggesting that the control of these promoters by CtrA is a conserved feature. With that in mind, we compiled the experimental data that were available for this transcription factor in different α-proteobacteria. Data were collected in a hierarchical manner: (i) direct binding of CtrA to its target promoter, typically by ChIP-seq data, was considered first, (ii) when no such data were available, mRNA-level studies (for example microarrays) were used, (iii) bioinformatics predictions were taken into account only if no experimental data were found. For the determination of the predicted targets, we used RSAT (van Helden 2003). All data were compiled in Fig. 3. It is important to keep in mind that the approaches based on protein binding, mRNA levels or bioinformatics are not a direct proof of gene regulation and that they should be considered with reserve. This section is thus more prospective since meta-analyses usually generate working hypotheses that, if they lead to correlation, could indicate that similar processes are at play. Importantly, the absence of correlation does not necessarily mean the opposite. Hypotheses proposed here might therefore be challenged in the future. Figure 3. View largeDownload slide Comparison between CtrA targets in different α-proteobacteria. Data were collected in a hierarchical manner. Information about the direct binding of CtrA were found for C. crescentus (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), B. abortus (Francis et al.2017), S. meliloti (Pini et al.2015; Ichida and Long 2016), E. chaffeensis (Cheng et al.2011) and R. prowazekii (Brassinga et al.2002). Data concerning the mRNA-level of potential CtrA targets were then collected for S. melonis (Francez-Charlot, Kaczmarczyk and Vorholt 2015), D. shibae (Wang et al.2014), R. capsulatus (Mercer et al.2010; Leung, Brimacombe and Beatty 2013) and M. magneticum (Greene et al.2012). Finally, when no experimental data were available, we considered bioinformatics predictions. In the case of Rickettsiales and A. tumefaciens, some were already available (Hallez et al.2004; Ioannidis et al.2007; Pinto, Pappas and Winans 2012). For S. meliloti, prediction on ftsZ1 promoter was based on Ichida and Long (2016), with one substitution allowed. To complete these data, we also predicted CtrA targets for A. tumefaciens, E. chaffeensis, Wolbachia wMel and R. prowazekii. To do so, we used RSAT (van Helden 2003) and considered genes based on the presence in their promoter of either a 9-mer (TTAAN7TTAAC) or a 8-mer (TTAACCAT) CtrA-binding box (Marczynski and Shapiro 1992; Laub, Chen and McAdams 2002) with one substitution allowed. Note that we considered that ftsZ was potentially regulated by CtrA when it was the case for the ddl gene, as ftsZ is probably in operon with this gene in S. meliloti, B. abortus and A. tumefaciens. In C. crescentus, CtrA was found to bind upstream the ruvCAB operon, itself located upstream the tolQRAB operon. We considered that the spacing between these two operons did not allow the prediction of a CtrA control on the tolQRAB operon. The ORF of each gene analyzed here are available in supporting information. See the text for a detailed discussion about this figure. ‘OM constr.’ stands for outer membrane constriction. Figure 3. View largeDownload slide Comparison between CtrA targets in different α-proteobacteria. Data were collected in a hierarchical manner. Information about the direct binding of CtrA were found for C. crescentus (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), B. abortus (Francis et al.2017), S. meliloti (Pini et al.2015; Ichida and Long 2016), E. chaffeensis (Cheng et al.2011) and R. prowazekii (Brassinga et al.2002). Data concerning the mRNA-level of potential CtrA targets were then collected for S. melonis (Francez-Charlot, Kaczmarczyk and Vorholt 2015), D. shibae (Wang et al.2014), R. capsulatus (Mercer et al.2010; Leung, Brimacombe and Beatty 2013) and M. magneticum (Greene et al.2012). Finally, when no experimental data were available, we considered bioinformatics predictions. In the case of Rickettsiales and A. tumefaciens, some were already available (Hallez et al.2004; Ioannidis et al.2007; Pinto, Pappas and Winans 2012). For S. meliloti, prediction on ftsZ1 promoter was based on Ichida and Long (2016), with one substitution allowed. To complete these data, we also predicted CtrA targets for A. tumefaciens, E. chaffeensis, Wolbachia wMel and R. prowazekii. To do so, we used RSAT (van Helden 2003) and considered genes based on the presence in their promoter of either a 9-mer (TTAAN7TTAAC) or a 8-mer (TTAACCAT) CtrA-binding box (Marczynski and Shapiro 1992; Laub, Chen and McAdams 2002) with one substitution allowed. Note that we considered that ftsZ was potentially regulated by CtrA when it was the case for the ddl gene, as ftsZ is probably in operon with this gene in S. meliloti, B. abortus and A. tumefaciens. In C. crescentus, CtrA was found to bind upstream the ruvCAB operon, itself located upstream the tolQRAB operon. We considered that the spacing between these two operons did not allow the prediction of a CtrA control on the tolQRAB operon. The ORF of each gene analyzed here are available in supporting information. See the text for a detailed discussion about this figure. ‘OM constr.’ stands for outer membrane constriction. In Fig. 3, it appears at first sight that the bacteria reported in this table belong to three distinct categories. The first one comprises A. tumefaciens, S. meliloti, B. abortus and C. crescentus, i.e. Rhizobiales and Caulobacterales. These bacteria have the common characteristic that the gene coding for CtrA is essential (Barnett et al.2001; Christen et al.2011; Figueroa-Cuilan et al.2016; Francis et al.2017). Moreover, at the exception of A. tumefaciens, their cell cycle has been shown to be regulated by CtrA (Quon et al.1998; Reisenauer, Quon and Shapiro 1999; Laub, Chen and McAdams 2002; Pinto, Pappas and Winans 2012; Pini et al.2015; Francis et al.2017). Therefore, it is not surprising to see that CtrA binds promoters for many genes involved in chromosome replication and segregation, as well as cell division. It is interesting to note that these processes are achieved through different genes in different bacteria and that it reflects their evolutionary lineage. Indeed, both A. tumefaciens and S. meliloti, which are closely related, seem to directly regulate their main chromosomal origin partitioning genes (parAB) through CtrA, while B. abortus is proposed to regulate its chromosome I replication through dnaA. As for C. crescentus, CtrA is directly binding to the ori as discussed earlier. It also seems that when a bacterium in this clade possesses a secondary chromosome, CtrA regulates its partition via the repABC operon (Pinto, Pappas and Winans 2012; Pini et al.2015; Francis et al.2017). One feature that is shared between Rhizobiales and C. crescentus is the predicted regulation of ftsK and ftsQ by CtrA, FtsK being involved in the segregation of the ter (Stouf, Meile and Cornet 2013) and FtsQ in the Z ring formation and constriction (Carson, Barondess and Beckwith 1991). The Z ring is composed of the tubulin-like FtsZ proteins, triggering the invagination of the cytoplasmic membrane, thus leading to septation and cytokinesis of the bacterium (Lutkenhaus and Addinall 1997). Interestingly, the ftsZ gene seems to be a target of CtrA in the first group of bacteria in Fig. 3. Note that in the case of A. tumefaciens, two copies of ftsZ exist and it is only the promoter region of the second one (Atu2086) that is predicted to possess an 8-mer CtrA binding site. There are also two ftsZ genes encoded in S. meliloti genome, and in this case, only the promoter of ftsZ1 is predicted to have a S. meliloti–specific CtrA-binding box with a AACCAT motif (Ichida and Long 2016), with one substitution (AGCCAT). However, neither of the two ftsZ promoters was found to be bound by ChIP-seq, so it was concluded that S. meliloti regulates its cell division through the control of minC and minD expression (Pini et al.2015). Indeed, MinC and MinD participate to the proper localization of the Z ring in Escherichia coli (de Boer, Crossley and Rothfield 1989) and many other bacteria. Interestingly, CtrA seems to systematically bind the promoter the minCDE operon in Rhizobiales. Note that this operon is absent in C. crescentus, where it is replaced by mipZ (Thanbichler and Shapiro 2006). Altogether, these analyses indicate that a number of crucial targets involved in different stages of the cell cycle were placed under the control of CtrA in the ancestors of Rhizobiales. In C. crescentus, some genes that are regulated by CtrA need to be expressed at very specific times. The most recent model to explain how CtrA-dependent genes are regulated in a timely manner is based on two other transcription factors, SciP and MucR (Fumeaux et al.2014). Genes that need to be expressed only in G2 phase cells, such as the DNA methyltransferase ccrM, are repressed by SciP in G1 phase cells even when CtrA is present (Gora et al.2010). On the other hand, genes that need to be expressed only in G1 phase cells are repressed by MucR in predivisional bacteria (Fumeaux et al.2014). From Fig. 3 it is clear that mucR is never predicted to be regulated by CtrA, in contrast to sciP, which is potentially regulated in S. meliloti, B. abortus and C. crescentus. The absence of a conserved CtrA-binding box in A. tumefaciens sciP promoter is surprising but does not mean that the gene is not regulated by CtrA in this bacterium. Another important player in C. crescentus gene expression regulation is the methyltransferase CcrM. As discussed above, ccrM temporal regulation by CtrA is important since it must only be present in late predivisional cells, where it methylates the newly synthesized DNA strands on GANTC sites precisely before cell division occurs (Stephens et al.1996; Reisenauer, Quon and Shapiro 1999). This means that, according to their chromosomal position away from the origin of replication, genes stay hemi-methylated for a different amount of time during the cell cycle (Marczynski 1999). This could have an important impact on gene expression at the whole genome level (Kozdon et al.2013). In fact, the transcription of several genes of C. crescentus has been observed to change in response to the methylation state of their promoters (Collier and Shapiro 2007; Gonzalez and Collier 2013). Remarkably, the promoter of ctrA is one of those (Reisenauer and Shapiro 2002). This is also supported by the fact that ccrM overexpression leads to abnormal chromosome content (Zweiger, Marczynski and Shapiro 1994). In the first group, ccrM appears to be systematically regulated by CtrA. This suggests that DNA methylation could be important for cell-cycle regulation in these bacteria. Of note, the function of GcrA has been demonstrated to be sensitive to CcrM DNA methylation in C. crescentus (Fioravanti et al.2012). As mentioned earlier, GcrA controls ctrA expression in this bacterium and vice versa (Holtzendorff et al.2004; Haakonsen, Yuan and Laub 2015). The regulation of gcrA by CtrA in C. crescentus was predicted by Brilli et al. (2010) to be an exception in α-proteobacteria. Experimental data obtained since then seem to be in agreement with this prediction, as gcrA has not yet been found to be part of the direct CtrA regulon in B. abortus (Francis et al.2017), S. meliloti (Pini et al.2015) or A. tumefaciens (Fig. 3). The second group of bacteria in Fig. 3 ranges from Sphingomonas melonis to Magnetospirillum magneticum. In this group, CtrA is not essential and the cell cycle progression does not seem to be coupled to this transcription factor (Greene et al.2012). Nevertheless, it is notable that some bacteria in this category could be at an evolutionary crossroads between the first and third group, as S. melonis (Francez-Charlot, Kaczmarczyk and Vorholt 2015) and Rhodospirillum centenum are both predicted to regulate the cell cycle transcription factor SciP. Of note, CtrA levels are regulated by quorum sensing in Dinoroseobacter shibae (Wang et al.2014), Rhodobacter capsulatus (Mercer et al.2010) and Ruegaria (Zan et al.2013). Despite not being involved in the cell-cycle regulation of these bacteria, the role of CtrA as a regulator of bacterial development, like in C. crescentus, seems to be conserved in this class. Indeed, cells of ctrA deletion strains are elongated (Wang et al.2014; Francez-Charlot, Kaczmarczyk and Vorholt 2015), which is reminiscent to the observation made with α-proteobacteria depletion strains when ctrA is essential (Reisenauer and Shapiro 2002; Figueroa-Cuilan et al.2016; Francis et al.2017). One can wonder how the cell cycle is regulated in α-proteobacteria that do not rely on CtrA for this function. Interestingly, bacteria from other clades developed similar strategies than C. crescentus to avoid replication over-initiation (Wolanski, Jakimowicz and Zakrzewska-Czerwinska 2014). For example, the gram-positive species Streptomyces coelicolor relies on AdpA to directly bind to its ori and inhibit binding of the replication machinery (Wolanski, Jakimowicz and Zakrzewska-Czerwinska 2012), whereas the γ-proteobacterium E. coli uses SeqA for a similar purpose (Nievera et al.2006). It is thus possible that α-proteobacteria inherited a similar mechanism from a more distantly related ancestor. When the regulation of DNA replication was put under the control of CtrA (in bacteria of the first group in Fig. 3 for example), this ancestral system could have progressively become obsolete, whereas it kept its role in the other α-proteobacteria. Importantly, essential vital functions may be regulated by redundant mechanisms to ensure their precise control. In the case of chromosome replication, redundant mechanisms are indeed often at play, such as proteolytic degradation of DnaA, its titration, the modulation of its activity by other proteins or a tight control of the expression of the dnaA gene (Wolanski, Jakimowicz and Zakrzewska-Czerwinska 2014). Only future studies will be able to reveal the real actors of cell-cycle regulation in the second group of α-proteobacteria but GcrA could be a good candidate for this function. Indeed, in C. crescentus, the transcription factor GcrA is known to participate in cell-cycle regulation by regulating CtrA expression but also, amongst others, by inhibiting the expression of dnaA when it is not necessary (Holtzendorff et al.2004). As a gene coding for GcrA is predicted to be present in the α-proteobacteria of the second group, it is possible that GcrA still retains this particular function in these bacteria. Note that M. magneticum does not seem to possess a gcrA gene, but this particular bacterium is apparently an exception, as all other Rhodospirillales studied thus far do possess one (Brilli et al.2010). A gene predicted to code for CcrM was also found in the genome of all the bacteria of the second group (Brilli et al.2010). If the ability of GcrA to differentiate genes during the cell cycle according to their CcrM-dependent methylation status is conserved in these bacteria, the two proteins might be sufficient to adequately control expression of cell cycle-related genes. The third group of bacteria in Fig. 3 is composed of the Rickettsiales E. chaffeensis, Wolbachia wMel (the endosymbiont of Drosophila melanogaster) and Rickettsia prowazekii. Not much is known about CtrA regulon and essentiality in these bacteria. However, the prediction that genes involved in cell division and chromosome replication are regulated by CtrA in these organisms is in agreement with the fact that the ori of R. prowazekii is bound by CtrA (Brassinga et al.2002) and that the promoter of the E. chaffeensis pal gene has been found as a target of the transcription factor (Cheng et al.2011). Moreover, eight strains of Wolbachia, in addition to other Rickettsiales, were found to possess DnaA binding sites and up to five CtrA consensus binding sites per ori (Ioannidis et al.2007). It is thus possible that ctrA regulates the cell cycle and is essential in this group of bacteria (Fig. 4). If this is confirmed in the future, it could mean that CtrA gained the function of regulating the cell cycle twice during evolution: once before the emergence of Rickettsiales and once before the appearance of Caulobacterales and Rhizobiales (Fig. 4). An alternative hypothesis would be that the regulation of the cell cycle was an ancestral function of CtrA that was lost early, but that seems less likely as it would imply the re-acquisition of this function in C. crescentus and Rhizobiales. Note that the third group in Fig. 3 is quite different from the first group, as they do not possess genes coding for the other cell cycle transcription factors such as SciP, MucR or GcrA. Nonetheless, they were found to possess a CckA/CtrA two-component system (Christensen and Serbus 2015). Furthermore, all Wolbachia CckA proteins were found to share a common conserved PAS domain, which could suggest that this protein has a ‘sensor’ capacity with a possibly conserved signal (Christensen and Serbus 2015). This potentially intrinsic ability of CckA to detect environmental signals has also been suggested to explain how C. crescentus is able to regulate CckA independently of the PleC/DivJ/DivK two-component system under stress (Heinrich, Sobetzko and Jonas 2016). Figure 4. View largeDownload slide Phylogenetic tree of α-proteobacteria based on CtrA. The tree was constructed with CtrA sequences from the genome of A. tumefaciens C58, S. meliloti 1021, B. abortus 2308, C. crescentus CB15, S. melonis TY, R. capsulatus SB 1003, D. shibae DFL 12, M. magneticum AMB-1, R. centenum SW ATCC 51521, R. prowazekii Rp22, Wolbachia wMel and E. chaffeensis Arkansas (https://biocyc.org/). Sequences were aligned with Clustal Omega (version 1.2.4; http://www.clustal.org/omega/) and curated manually with Jalview (http://www.jalview.org/download). The tree was created with PhyML (Guindon and Gascuel 2003) with the following settings: Starting tree = BioNJ; Tree Topology = SPR Move; Boostrap = 100 replicates; Substitution model = WAG. The final figure was formatted on iTol (https://itol.embl.de/), then on Inkscape. This tree is in agreement with previously published results (Williams, Sobral and Dickerman 2007; Greene et al.2012). Numbers correspond to bootstrap values. The unit of branch length are nucleotide substitutions per site (the number of changes divided by the length of the sequence). This figure suggests a possible timing, along evolution, of major events concerning genes that were taken under the control of CtrA. Note that cell cycle regulation seems to always coincide with a regulation of ftsZ and mismatch repair genes. Figure 4. View largeDownload slide Phylogenetic tree of α-proteobacteria based on CtrA. The tree was constructed with CtrA sequences from the genome of A. tumefaciens C58, S. meliloti 1021, B. abortus 2308, C. crescentus CB15, S. melonis TY, R. capsulatus SB 1003, D. shibae DFL 12, M. magneticum AMB-1, R. centenum SW ATCC 51521, R. prowazekii Rp22, Wolbachia wMel and E. chaffeensis Arkansas (https://biocyc.org/). Sequences were aligned with Clustal Omega (version 1.2.4; http://www.clustal.org/omega/) and curated manually with Jalview (http://www.jalview.org/download). The tree was created with PhyML (Guindon and Gascuel 2003) with the following settings: Starting tree = BioNJ; Tree Topology = SPR Move; Boostrap = 100 replicates; Substitution model = WAG. The final figure was formatted on iTol (https://itol.embl.de/), then on Inkscape. This tree is in agreement with previously published results (Williams, Sobral and Dickerman 2007; Greene et al.2012). Numbers correspond to bootstrap values. The unit of branch length are nucleotide substitutions per site (the number of changes divided by the length of the sequence). This figure suggests a possible timing, along evolution, of major events concerning genes that were taken under the control of CtrA. Note that cell cycle regulation seems to always coincide with a regulation of ftsZ and mismatch repair genes. In light of what can be observed in these three groups of bacteria, a hypothesis can be proposed about why B. abortus CtrA was not found to be important for infecting cells (Willett et al.2015; Francis et al.2017). It is possible that B. abortus does not rely solely on CtrA to regulate its cell cycle. Maybe another transcription factor, such as GcrA, directs this important function during the first phase of the infection, when B. abortus are in their non-replicative stage. Since the function of B. abortus CtrA during later times post-infection was not investigated, it would be interesting to test whether this transcription factor is required for reinfection, as in E. chaffeensis (Cheng et al.2011). It is also possible that CtrA is only necessary during infection when CckA responds to specific environmental stressors, similar to its role in C. crescentus (Heinrich, Sobetzko and Jonas 2016). These hypotheses are of course speculative and will need to be tested in the future. Could DNA repair regulation be an ancestral function of CtrA? Elucidation of the targets of M. magneticum CtrA suggests that motility is an ancestral trait of α-proteobacteria (Greene et al.2012). The authors of that study also proposed that the transition to the intracellular lifestyle of E. chaffeensis and R. prowazekii led to the loss of flagellar and chemotaxis genes and thus the loss of this regulation function (Greene et al.2012). Alternatively, the regulation of motility could have occurred on an evolutionary branch that is further away from the Rickettsiales (Fig. 4). Brucella was also considered as a non-motile and non-flagellated intracellular bacterium but it retains flagellar genes that appear to be important during infection (Halling 1998). Indeed, flagellin was shown to modulate the host response and bacterial proliferation in a mouse model of infection (Terwagne et al.2013). This is an interesting example of alteration of a given protein during evolution, from its initial function as a flagellin into a host protective factor (Shames and Finlay 2010; Terwagne et al.2013). The flagella of Bartonella bacilliformis, a close phylogenetic relative of Brucella, were also found to be required for entry into host erythrocytes (Scherer, DeBuron-Connors and Minnick 1993), illustrating that the function of these organelles diverged quickly during the course of evolution. In a transcriptomic study focusing on the M. magneticum CtrA regulon, the promoters of genes coding for proteins involved in motility were not predicted to be enriched amongst CtrA targets, even though the regulation of such genes by CtrA is proposed to be ancestral in α-proteobacteria (Greene et al.2012). Another functional category that could have been underestimated is DNA repair. Indeed, all α-proteobacteria studied in the context of CtrA regulation have at least one DNA repair gene predicted or effectively shown to be part of CtrA regulon. In the first and third groups of Fig. 3, one interesting observation is that promoters of genes coding for the Mismatch Repair (MR) system appear to be systematically targeted by CtrA. The MR system, composed of MutH, MutL and MutS proteins, is dependent on the Dam-related methylation status of E. coli DNA. Indeed, in this γ-proteobacterium, MutH is able to discern the parental DNA strand (that serves as template) from the newly synthesized one by recognizing the non-methylated state of the new DNA (Yamaguchi, Dao and Modrich 1998; Kunkel and Erie 2005). Thus, after MutS has detected the distortion in the helix caused by a base mismatch, MutL is recruited to allow the interaction between MutS and MutH. After MutH has excised the base, an exonuclease degrades a portion of DNA on the mutated and non-methylated strand, which is later repaired by the DNA polymerase III and a ligase (Kunkel and Erie 2005). In B. abortus and other α-proteobacteria, a homologous gene coding for MutH is missing (Martins-Pinheiro, Marques and Menck 2007; Guarne 2012). However, it has been proposed that in such a case, MutL is able to also perform the MutH function (Kadyrov et al.2006; Pillon et al.2010). In C. crescentus, mutS expression has been found to be cell cycle regulated (Laub et al.2000). Moreover, mutS was over-expressed in a CtrA depletion background, suggesting that it is under the control of CtrA (Laub et al.2000). As for mutL, it is not cell cycle regulated but its promoter has been found to be potentially linked to CtrA, based on a DNA microarray experiment (Laub, Chen and McAdams 2002). It should be noted that in C. crescentus, another gene is oriented opposite of mutL, so it is possible that this gene is the one being regulated by CtrA. However, the promoter of either mutL or mutS is consistently predicted to be bound by CtrA in other α-proteobacteria where CtrA probably regulates their cell cycle (Fig. 3, groups 1 and 3). One hypothesis would therefore be that the regulation of the MR system along the cell cycle would insure the correct timing for the utilization of this DNA repair mechanism. Indeed, the use of MR system is not always favourable, as it is known to result in enhanced mutagenesis in bacteria treated with e.g. alkylating agents (Nakano et al.2017). Note that in C. crescentus, CtrA directly activates the expression of the gene coding for S-adenosylmethionine (SAM) synthase, the enzyme responsible for the production of SAM (Laub, Chen and McAdams 2002). SAM is a methyl-donor for CcrM but it is also known to be a weak aspecific endogenous alkylating agent (Rydberg and Lindahl 1982). As for B. abortus, the promoter of tagA, which codes for a protein specifically involved in repairing alkylated DNA (Mielecki and Grzesiuk 2014), is directly bound by CtrA (Francis et al.2017). Incidentally, it would be interesting to know if TagA is required during B. abortus cellular infection, as it would suggest that alkylating stress is met by the bacterium inside its host. Knowing that several other genes involved in DNA repair are cell cycle regulated in C. crescentus, including the gene coding for the SOS repressor LexA (Laub et al.2000), it would also be interesting to investigate whether bacteria are more prone to DNA damage during certain stages of their cell cycle or not. In C. crescentus, both mutL and mutS, in addition to ctrA, are considered to be part of the GcrA regulon, as their promoters are all bound by GcrA in a ChIP-seq experiment and their expression changes in cells depleted in GcrA compared to wild-type cells (Haakonsen, Yuan and Laub 2015). As GcrA is able to sense CcrM-dependent methylation on DNA (Fioravanti et al.2013) and since this specific type of methylation is cell cycle regulated in C. crescentus (Stephens et al.1996) and probably also in B. abortus (Francis et al.2017), an interesting hypothesis would be that the regulation of the MR system by CtrA is a way to prevent it from functioning after full methylation has occurred and thus to avoid the cleavage of the wrong strand. However, CcrM-dependent methylation has been shown to be dispensable for C. crescentus to perform proper MR, as suggested by the frequency of rifampicin resistant mutants (Gonzalez et al.2014). This either means that the MR system is independent of CcrM methylation in C. crescentus (Gonzalez et al.2014), or alternatively that there are redundant DNA repair systems that prevent mismatches from occurring in C. crescentus, such as a robust base excision repair system (Martins-Pinheiro, Marques and Menck 2007). Two other genes that are supposedly involved in DNA repair could often be regulated by CtrA: dprA, also known as smf, and radC (Fig. 3). Both dprA and radC code for proteins with enigmatic functions. DprA interacts with RecA and is known to be essential for natural competence (Kidane et al.2012; Yadav et al.2013; Le et al.2017). However, the gene coding for this protein is conserved in many bacteria that are not naturally competent. In the α-proteobacterium R. capsulatus, DprA is involved in the regulation of Gene Transfer Agent (GTA) production in a CtrA-dependent manner (Brimacombe, Ding and Beatty 2014). A GTA system is analogous to phage transduction but it is unable to fully self-propagate and contains random segments of the host DNA that spread from one cell to another (Lang, Zhaxybayeva and Beatty 2012). As for RadC, and despite numerous efforts, its real function has remained elusive (Ogura et al.2002; Peterson et al.2004; Attaiech et al.2008). It is also known to be specifically expressed in naturally competent bacteria (Ogura et al.2002; Peterson et al.2004; Redfield et al.2005; Vickerman et al.2007). In the Rickettsiale pathogen Wolbachia, there exist three homologues of radC and all of them are associated with a cluster of genes that are distantly related to phage repressors (Wu et al.2004). Therefore, one hypothesis would be that RadC and DprA are not involved in competence in these bacteria but rather in coping with phage-derived DNA integration. Also, note that each α-proteobacterium seems to possess its own specific CtrA-dependent DNA repair targets. For example, D. shibae CtrA could regulate recA expression (Wang et al.2014), while it seems to be uvrB in the case of R. capsulatus (Mercer et al.2010). As for E. chaffeensis, it has a perfect 8-mer CtrA binding box in the promoter of its mfd gene, which codes for the Transcription Repair Coupling Factor that affects nucleotide excision repair (Selby and Sancar 1993). Thus, each bacterium probably optimized the different cellular functions regulated by CtrA through evolution according to its specific lifestyle. The clade of α-proteobacteria is composed of organisms with very different phenotypes and lifestyles. As proposed by others (Greene et al.2012), it seems that motility is an ancestral trait of CtrA regulon in these organisms. In addition to this observation, our review has proposed that DNA repair could also be a common target of this transcription factor. More precisely, the CtrA regulon seems to have evolved to couple the MR system to cell-cycle regulation in some bacteria and to modulate the levels of DprA and RadC in others. It also appears that each bacterium has selected the regulation of some specific DNA repair genes under the control of CtrA, which could reflect the kind of stresses that they meet in their respective environments. Yet, in B. abortus and most other α-proteobacteria, there is a gap in the literature on DNA repair. In the case of B. abortus, addressing this question would undoubtedly help to better understand what type of stresses are met by the bacterium inside its host cells and thus, to better understand the infectious process itself. In this regard, it would be very interesting to know if the blockage in G1 in HeLa cells and RAW 264.7 macrophages (Deghelt et al.2014) is linked to more efficient resistance to DNA damage. Concluding remarks One surprising conclusion about CtrA is that it is neither involved in the ability of B. abortus to enter inside its host cells, nor in its capacity to reach its replicative niche (Willett et al.2015; Francis et al.2017). As the survival of a B. abortus CtrA depletion strain decreases after 48 h post-infection (Willett et al.2015; Francis et al.2017), investigation of the impact of the protein at later times could lead to interesting discoveries and might provide answers to some open questions. For example, is the ability of CtrA to modify the bacterial envelope a way to modulate its pathogenicity? Is CtrA necessary for the cell-to-cell spreading of B. abortus, like in E. chaffeensis (Cheng et al.2011)? The fact remains that B. abortus does need to tightly regulate its cell cycle at early times post-infection, so that it is blocked in the G1 phase for up to 8 h after the entry inside HeLa cells and RAW 264.7 macrophages (Deghelt et al.2014). As this process is apparently independent of CtrA (Francis et al.2017), the question remains as to what is the molecular mechanism ensuring this G1 blockage. As B. abortus was suggested to face starvation while it is inside the eBCV, it is possible that the ppGpp-dependent starvation response is involved at that stage (Dozot et al.2006). In favour of this hypothesis, ppGpp has been found to regulate DnaA stability and initiation of DNA replication in carbon-starved C. crescentus (Lesley and Shapiro 2008), as well as to modulate the cell cycle when C. crescentus is unable to synthesize fatty acids (Stott et al.2015) or when it senses a decrease in intracellular glutamine concentration (Ronneau et al.2016). Another transcription factor that is supposed to be involved in cell-cycle regulation, such as GcrA, could also perform this function. Another important question to answer is to know whether this blockage occurs in all eukaryotic cell types, or if it is specific to infection of HeLa cells and RAW 264.7 macrophages. Conversely, one could wonder why B. abortus has evolved to favour a delay in its cell cycle progression during infection. One hypothesis would be that avoiding DNA replication as long as the bacterium is in the endosomal pathway is a way to prevent the fixation of mutations. Indeed, the passage through the eBCV is thought to be very stressful for the bacterium, which could cause DNA damage (Roop et al.2009). In addition, at that stage, most proteins—including those for DNA repair—are very weakly produced (Lamontagne et al.2009). It would therefore be more advantageous for B. abortus to wait for the storm to pass and repair its DNA when it is safe to do so. Otherwise, there is a risk that replication forks would stall and eventually collapse, which would lead to cell death (Cox et al.2000). As for the absence of growth itself during the first hours of the infection, it could be a way for B. abortus to limit its pathogen associated molecular pattern production and thus limit its recognition by host cells. Not growing could also be a way for the bacterium to avoid using too many resources while it still resides in the eBCV, which is usually considered a nutrient-poor environment (Roop et al.2009). The question of whether the G1 block is a common strategy to other intracellular pathogens also merits attention. Indeed, Legionella, Salmonella, Chlamydia and Francisella have also been reported to display a biphasic infection, with a relatively long non-proliferative period followed by a phase of massive proliferation (Salcedo and Holden 2005). There is a crucial need to address these fundamental issues in the future, as only a good knowledge of bacterial biology and infectious processes will allow us to combat pathogens that are becoming more and more resistant to antibiotic treatments. SUPPLEMENTARY DATA Supplementary data are available at FEMSRE online. Acknowledgements We thank J. Van Helden for uploading Rhodospirillum centenum genome on RSAT on request. We thank the members of the URBM for stimulating discussions. FUNDING We thank UNamur (https://www.unamur.be/) for financial and logistic support. 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Zweiger G , Marczynski G , Shapiro L . A Caulobacter DNA methyltransferase that functions only in the predivisional cell . J Mol Biol 1994 ; 235 : 472 – 85 . © FEMS 2018. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Reviews Oxford University Press

Learning from the master: targets and functions of the CtrA response regulator in Brucella abortus and other alpha-proteobacteria

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Abstract

Abstract The α-proteobacteria are a fascinating group of free-living, symbiotic and pathogenic organisms, including the Brucella genus, which is responsible for a worldwide zoonosis. One common feature of α-proteobacteria is the presence of a conserved response regulator called CtrA, first described in the model bacterium Caulobacter crescentus, where it controls gene expression at different stages of the cell cycle. Here, we focus on Brucella abortus and other intracellular α-proteobacteria in order to better assess the potential role of CtrA in the infectious context. Comparative genomic analyses of the CtrA control pathway revealed the conservation of specific modules, as well as the acquisition of new factors during evolution. The comparison of CtrA regulons also suggests that specific clades of α-proteobacteria acquired distinct functions under its control, depending on the essentiality of the transcription factor. Other CtrA-controlled functions, for instance motility and DNA repair, are proposed to be more ancestral. Altogether, these analyses provide an interesting example of the plasticity of a regulation network, subject to the constraints of inherent imperatives such as cell division and the adaptations to diversified environmental niches. CtrA, Brucella, cell cycle, alpha-proteobacteria, infection, regulation network evolution INTRODUCTION Brucella species are responsible for brucellosis, a major and worldwide zoonosis. In animals, it occurs as a chronic infection that is characterized by epididymitis in males or placentitis and abortion in pregnant females (Carvalho Neta et al.2010). Humans are accidental hosts of Brucella melitensis, Brucella abortus and Brucella suis, in which they are responsible for a debilitating disease known as undulant fever or Malta fever (Moreno and Moriyon 2006). Usually, human infections happen through the ingestion of contaminated dairy products or by exposure to infected animals. Another major way of infection is through the aerosol route, which is why Brucella strains are subjected to strict regulations in laboratories (Yagupsky and Baron 2005). There are currently no vaccines available for humans and the only treatment is the use of a combination of antibiotics (Moreno and Moriyon 2006). This review aims at summarizing what is known about Brucella abortus infectious process in host cells, with a particular emphasis on its cell-cycle regulation. Indeed, B. abortus has been reported to stall its cell cycle in the G1 phase, which corresponds to a non-replicating stage, for up to 8 h at the onset of infection of HeLa cells or RAW 264.7 macrophages (Deghelt et al.2014). This review therefore focuses on the master regulator CtrA, a transcription factor particularly well conserved in α-proteobacteria and known to regulate the Caulobacter crescentus cell cycle (Laub, Chen and McAdams 2002; Brilli et al.2010). Up to now, the only comparative studies about the CtrA regulons of different α-proteobacteria were mainly based on bioinformatics predictions (Hallez et al.2004; Brilli et al.2010). As literature on CtrA has been dramatically increasing over the last years, it is now possible to compile experimental data. We thus give an overview of the conservation of specific modules in the CtrA regulon, as well as the acquisition of new factors that occurred during evolution, while focusing more particularly on intracellular bacteria. Brucella inside host cells Brucella intracellular trafficking A whole genome-based phylogeny study revealed that brucellosis probably appeared in wildlife populations in the past 86 000 to 296 000 years (Foster et al.2009). It thus happened before livestock domestication, even though this crucial step in history probably played a role in allowing the worldwide spreading of these pathogens (Foster et al.2009). Even though they can be cultivated on artificial media, it is established that Brucella need to enter inside their host cells in order to complete a successful infection process (Moreno and Moriyon 2006). This is why they are now considered as facultatively extracellular intracellular parasites (Moreno and Moriyon 2002). The mechanism by which Brucella manage to invade their host organism is not very clear but they seem to cross the mucosal barrier, which could imply an interaction with epithelial cells (Roop et al.2009). The role of these cells has not been deciphered yet but epithelial HeLa cells have been effectively used as models for Brucella infection in non-professional phagocytes (Pizarro-Cerda et al.1998; Castaneda-Roldan et al.2004; Starr et al.2008). Once inside its host, Brucella could also get internalized by professional phagocytes such as macrophages or dendritic cells. There, the bacterium can survive and multiply before disseminating in the organism (Archambaud et al.2010). Surprisingly, B. melitensis has also been reported to be able to invade murine erythrocytes during infection, which suggests that other cellular and in vivo models of infection should be developed to fully understand Brucella pathogenesis (Vitry et al.2014). The entry of Brucella into epithelial or phagocytic cells occurs within minutes after cell-to-cell contact (Pizarro-Cerda et al.1998). Once internalized, the bacterium stays in a membrane-bound Brucella-containing vacuole (BCV) that interacts with the endocytic pathway (therefore termed eBCV) (Fig. 1). Early endosomal markers, such as Rab5, are rapidly followed by the acquisition of late endosomal markers, typically lysosomal membrane-associated protein-1 (LAMP1) (Pizarro-Cerda et al.1998). Transient interactions with lysosomes have also been reported (Starr et al.2008). This eventually leads to eBCV acidification, which is deleterious to many bacteria, but nonetheless necessary for Brucella to reach their replicative niche and survive in the long-term (Porte, Liautard and Kohler 1999; Boschiroli et al.2002a; Celli et al.2003; Starr et al.2008). Indeed, the acidic pH of the eBCV has been linked to the capacity of the pathogen to induce the expression of the virB operon (Boschiroli et al.2002a). These genes code for a type IV secretion system (T4SS) that is essential for the bacteria to reach their proliferation niche (Boschiroli et al.2002b). Figure 1. View largeDownload slide Schematic representation of B. abortus trafficking inside host cells. Once inside its host cell, B. abortus extensively interacts with the endocytic pathway. The compartment in which it resides at that stage can be referred to as the endocytic Brucella-containing vacuole (eBCV). In HeLa cells and RAW 264.7, during this first step of the infection, the bacterium is blocked in G1 and its growth is arrested. After a transient interaction with the lysosomes and thanks to its type IV secretion system VirB, the bacterium reaches its replicative niche (rBCV), which is part of the endoplasmic reticulum (ER) in most cell types. Later on, bacteria are found in autophagy-dependent vacuoles (aBCV) and are proposed to reinfect neighbor cells. Figure 1. View largeDownload slide Schematic representation of B. abortus trafficking inside host cells. Once inside its host cell, B. abortus extensively interacts with the endocytic pathway. The compartment in which it resides at that stage can be referred to as the endocytic Brucella-containing vacuole (eBCV). In HeLa cells and RAW 264.7, during this first step of the infection, the bacterium is blocked in G1 and its growth is arrested. After a transient interaction with the lysosomes and thanks to its type IV secretion system VirB, the bacterium reaches its replicative niche (rBCV), which is part of the endoplasmic reticulum (ER) in most cell types. Later on, bacteria are found in autophagy-dependent vacuoles (aBCV) and are proposed to reinfect neighbor cells. The Brucella replicative niche (rBCV) has been known for years to derive from the endoplasmic reticulum (ER), in both HeLa cells and macrophages (Pizarro-Cerda et al.1998; Celli et al.2003). It is only recently that the rBCV was shown to actually be part of the endoplasmic reticulum (Sedzicki et al.2018) (Fig. 1). The transition from eBCV to rBCV is not clearly understood yet, but it has been suggested that its maturation could occur at the ER exit sites (Celli, Salcedo and Gorvel 2005; Celli 2015). Several ER-associated functions have been linked to Brucella infection, such as the unfolded protein response IRE1α signaling pathway (Qin et al.2008; Smith et al.2013; Taguchi et al.2015), some autophagy-associated factors such as ATG9 and WIPI (Taguchi et al.2015) and the early secretory trafficking depending on the Sar1/coat protein complex II (Celli, Salcedo and Gorvel 2005; Taguchi et al.2015). Since the T4SS is essential for Brucella to reach the rBCV, it is expected that the maturation of the BCV would be mediated by the delivery of bacterial effectors inside the host cell. One such effector is BspB, shown to target the Golgi apparatus by interacting with the oligomeric Golgi tethering complex (Miller et al.2017). This leads to the redirecting of Golgi-derived vesicles to the BCV by remodeling the ER-Golgi secretory trafficking (Miller et al.2017). It is important to note that there exist alternatives to the ER-derived replicative niche since opsonized B. abortus proliferate in a non-acidic LAMP1-positive compartment in the human monocytic cell line THP-1 (Bellaire, Roop and Cardelli 2005) and in endosomal inclusions in extravillous trophoblasts (Salcedo et al.2013). Once the number of bacteria within a cell reaches a critical level, destruction of the host cell can be observed (Moreno and Moriyon 2006). Another means for Brucella to spread from one cell to its neighbors has been shown by Starr et al (2012). The formation of a compartment with autophagic features (aBCV) could be the key to this important step of the infection (Fig. 1). Indeed, autophagy-deficient Brucella are not able to perform cell-to-cell spreading when cellular infections are prolonged for long periods, typically 72 h (Starr et al.2012). Interestingly, only the initiation complex of autophagy seems to be needed by Brucella to promote reinfection (Starr et al.2012). Indeed, markers of the elongation phase of autophagy such as ATG5 and LC3 were not found to be associated to the aBCV (Starr et al.2012). It should be noted that autophagy is particularly important at birth. At that time, the transplacental nutrient supply is no longer available, which suggests that autophagy is strongly activated in the neonate in order to adapt to the early neonatal starvation period (Kuma et al.2004). The use of this process by the bacteria could therefore be relevant for their spreading inside newborn calves. Growth and replication of Brucella B. abortus possesses two distinct chromosomes (Chain et al.2005). Surprisingly, bacteria with multipartite genomes are not uncommon, at about 10% of the sequenced species (Val et al.2014). Contrarily to plasmids that are known to initiate replication several times during the bacterial cell cycle, chromids (also known as megaplasmids) code for essential genes and initiate their replication only once per cell cycle, like chromosomes (Pinto, Pappas and Winans 2012; Val et al.2014). In B. abortus, the large and circular chromosome (I) is 2.1 Mb long and possesses a ParAB segregation system with three centromere-like parS sites, while the small chromosome (II) of 1.2 Mb is a chromid, with its replication being controlled by a RepABC system (see Pinto, Pappas and Winans 2012 for a review on this segregation system). The repABC operon also contains two centromere-like sequences called repS (Livny, Yamaichi and Waldor 2007; Deghelt et al.2014). The chromosomal replication status of B. abortus, and thus the stage of its cell cycle, can be followed with fluorescent reporters of the segregation markers ParB and RepB, as well as with fluorescent reporters allowing the localization of the replication origins (ori) and terminators (ter). Both chromosomes are oriented along the cell length, with oriI and terI associated with the poles, whereas oriII and terII are usually found closer to the midcell (Deghelt et al.2014). This is in agreement with what has been found in Sinorhizobium meliloti, another α-proteobacterium. Indeed, this bacterium possesses a tripartite genome with one primary chromosome (3.65 Mb) and two chromids (1.35 Mb for pSymA and 1.68 Mb for pSymB) (Galibert et al.2001). Both chromids are segregated by a RepABC system and their ori are not anchored to the poles (Kahng and Shapiro 2003; Frage et al.2016). S. meliloti is capable of colonizing the soil rhizosphere as a free-living bacterium, but also of invading the roots of leguminous plants as an intracellular symbiotic nitrogen-fixing bacterium, involving complex interactions between the bacterium and its host (Gibson et al.2006). Similarly to C. crescentus and B. abortus, free-living S. meliloti regulate their cell cycle so that replication of their genome occurs once-and-only-once per cell division (Mergaert et al.2006). In both B. abortus and S. meliloti, a temporal coordination of replication and segregation was found, as the initiation of replication of their chromids is always delayed compared to the main chromosome (Deghelt et al.2014; Frage et al.2016). In Brucella, the replication of oriI starts before oriII and both chromosomes would finish their replication at approximately the same time (Deghelt et al.2014). Note that the size of the chromids does not seem to be the determining factor for the temporal regulation of their replication initiation. Indeed, in S. meliloti, it has been proposed that the smaller pSymA initiates its replication when the ori of the main chromosome has reached the new pole and that it is followed by the bigger pSymB, which behaves in a similar manner after the pSymA ori has been replicated (Frage et al.2016). Two main phases can be observed during HeLa cells infection by B. abortus. Indeed, when the bacterium is transiting within the eBCV, it is unable to proliferate, which reflects the fact that the number of colony forming unit (CFU) is stable during this non-proliferative stage (Comerci et al.2001; Starr et al.2008; Deghelt et al.2014). The second phase occurs when Brucella reaches its ER-derived proliferative niche, with the number of CFU increasing drastically (Pizarro-Cerda et al.1998; Celli et al.2003; Starr et al.2008). Thanks to fluorescent reporter systems that can track ori's, it has been possible to follow the B. abortus cell cycle inside host cells. One interesting observation was that during the non-proliferative stage of the trafficking in HeLa cells and RAW 264.7 macrophages, the bacteria are blocked in G1 (only one focus of oriI), similarly to what happens in the carbon-starved swarmer cells of C. crescentus, a free living α-proteobacterium (Lesley and Shapiro 2008; Deghelt et al.2014). As Brucella exhibits asymmetric growth like other Rhizobiales (Brown et al.2012), it is also possible to use Texas Red Succinimidyl Ester—a fluorescent compound that covalently binds amine groups on the bacterial surface—as a mean to follow the bacterium unipolar growth (Brown et al.2012) inside host cells (Deghelt et al.2014). These techniques brought to light the fact that the bacteria found within the eBCV at early times after infection are predominantly non-growing newborn cell types. This term refers to bacteria that recently divided but did not yet initiate chromosome replication (Deghelt et al.2014). They stay in this state for up to 8 h before resuming their growth and chromosome replication when they still reside within an eBCV (Deghelt et al.2014). Roles and regulation of CtrA B. abortus CtrA regulation is similar to that of C. crescentus Since the invasive B. abortus are mainly in the G1 phase of their cell cycle (Deghelt et al.2014), it is possible that transcription factors involved in cell-cycle regulation could be also important for Brucella virulence. One such factor is CtrA. This transcription factor is very well conserved amongst α-proteobacteria (Brilli et al.2010) and has been best studied in the model organism C. crescentus. This remarkable bacterium possesses two distinct life forms. One is a sessile stalked form, which allows the bacterium to adhere to surfaces when it is in a nutrient-rich environment. The other form is a motile swarmer cell that is used for scouting and colonizing new favourable environments but that is not competent for replication (Ausmees and Jacobs-Wagner 2003; Quardokus and Brun 2003). Importantly, C. crescentus divides asymmetrically into its two phenotypically different daughter cells after each cell division. This is why this bacterium is considered as an excellent model for bacterial cell cycle studies. In this context, the transcription factor CtrA has been found to be of utmost importance as it is a master regulator of C. crescentus cell cycle (Quon, Marczynski and Shapiro 1996) (Fig. 2). Indeed, one of CtrA many targets is the ori, thus preventing the DnaA protein from initiating the replication of C. crescentus chromosome as long as CtrA is present (Quon et al.1998; Siam and Marczynski 2000). As CtrA needs to be phosphorylated to be active, a tight regulatory network based on two-component regulators is in charge of its synthesis, phosphorylation and degradation (Quon, Marczynski and Shapiro 1996; Domian, Quon and Shapiro 1997; Wu, Ohta and Newton 1998; Biondi et al.2006; Tsokos, Perchuk and Laub 2011). Figure 2. View largeDownload slide Models for CtrA regulation in two α-proteobacteria. The schemes represented here are mainly based on C. crescentus CtrA regulation (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), therefore it is important to take into consideration the fact that the phosphorylation cascade events might not happen exactly as depicted. In the case of B. abortus, data were obtained from Willett et al. (2015) and Francis et al. (2017). Green arrows correspond to confirmed CtrA targets that are positively regulated by the transcription factor. Blue rounded arrows correspond to targets that are bound by CtrA on their promoter, but for which the effect of this binding still remains unknown. Figure 2. View largeDownload slide Models for CtrA regulation in two α-proteobacteria. The schemes represented here are mainly based on C. crescentus CtrA regulation (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), therefore it is important to take into consideration the fact that the phosphorylation cascade events might not happen exactly as depicted. In the case of B. abortus, data were obtained from Willett et al. (2015) and Francis et al. (2017). Green arrows correspond to confirmed CtrA targets that are positively regulated by the transcription factor. Blue rounded arrows correspond to targets that are bound by CtrA on their promoter, but for which the effect of this binding still remains unknown. In C. crescentus, the dual CckA enzyme that possesses both kinase and phosphatase activities regulates the phosphorylation level of CtrA and CpdR, a response regulator stimulating CtrA proteolysis when dephosphorylated (Jenal and Fuchs 1998; Jacobs et al.2003; Biondi et al.2006). CckA does so by interacting with the phosphotransferase ChpT (Biondi et al.2006). The kinase activity of CckA is inhibited by the phosphorylated form of the response regulator DivK, which is stabilized by the atypical histidine kinase DivL (Tsokos, Perchuk and Laub 2011; Childers et al.2014). DivK phosphorylation is itself regulated by the histidine kinase DivJ and by the phosphatase PleC (Wu, Ohta and Newton 1998; Wheeler and Shapiro 1999). PleC is also able to phosphorylate the diguanylate cyclase PleD, which in turn will synthesize cyclic di-GMP (Paul et al.2008). The binding of this secondary messenger to CckA will force CckA to switch from its kinase to its phosphatase mode, thus preventing the phosphorylation of CtrA (Lori et al.2015). Cyclic di-GMP also binds to PopA, which interacts with RcdA, another protein involved in CtrA proteolysis at the stalked cell pole (Ozaki et al.2014; Smith et al.2014). Interestingly, several genes coding for proteins regulating CtrA are also part of its regulon, including divK (Laub et al.2000) and divJ (Fumeaux et al.2014) in C. crescentus. At the transcription level, ctrA is regulated by two proteins in C. crescentus. One of them is CtrA itself (Domian, Reisenauer and Shapiro 1999). The other one is GcrA, an unconventional transcription factor that binds to the housekeeping σ70 factor (Haakonsen, Yuan and Laub 2015). Since gcrA transcription is repressed by CtrA and ctrA transcription is activated by GcrA, the two transcription factors are present temporally and spatially out-of-phase during the cell cycle of C. crescentus (Holtzendorff et al.2004). Of note, DnaA also participates to gcrA transcription (Collier 2012). The spatio-temporal regulation of CtrA is particularly well adapted to C. crescentus aquatic free-living lifestyle, but it appears to be surprisingly conserved in other α-proteobacteria with very different ways of life (Brilli et al.2010; Pini et al.2015; Schallies et al.2015; Willett et al.2015). In B. abortus, the core actors involved in the CtrA regulatory network, defined here as the PleC/DivJ/DivK and CckA/ChpT/CtrA two-component systems, are conserved (Hallez et al.2004; Brilli et al.2010) (Fig. 2). Another gene, called pdhS for PleC/DivJ homologue sensor, has also been found to be part of this network (Hallez et al.2004). Both pleC and divK from B. abortus are able to heterocomplement the corresponding deletion mutants in C. crescentus, which suggests that their function is conserved between both organisms (Hallez et al.2007). In addition, in B. abortus DivK has been found by yeast two-hybrid experiment to bind to DivJ, PleC, DivL and PdhS (Hallez et al.2007). Nevertheless, the localization of PleC is different between B. abortus and C. crescentus and DivJ was not found to be crucially involved in DivK phosphorylation (Hallez et al.2007). Indeed, in a ΔdivJ background, DivK did not lose its phosphorylation-dependent polar localization (Hallez et al.2007), while a loss-of-function of pdhS generates delocalization of DivK-YFP (Van der Henst et al.2012). As pdhS is an essential gene and depletion strains were not available at that time, its involvement in DivK phosphorylation could only be suggested through indirect experiments. PdhS was also shown to accumulate at the old pole of the large cells, which is the same localization as the phosphorylated form of DivK (Hallez et al.2007). Of note, this localization is similar to the one of DivJ in C. crescentus, which suggests a common function between B. abortus PdhS and C. crescentus DivJ (Hallez et al.2007). As PdhS is cytoplasmic in B. abortus, it is possible that its function is shared with DivJ depending on the time and/or space of DivK phosphorylation (Hallez et al.2007). The polar localization of PdhS is conserved at 48 h post-infection in bovine macrophages (Hallez et al.2007), but nothing is known about the role of DivJ in this context and at later times of the infection. As for the phosphorelay going from CckA to CtrA and CpdR via ChpT, it has been confirmed to be functional in B. abortus (Willett et al.2015). As was the case for C. crescentus (Laub et al.2000; Fumeaux et al.2014), several genes predicted to be involved in CtrA regulation have been found by ChIP-seq to be potentially part of CtrA regulon in B. abortus, including ctrA itself, divK, divJ, divL, chpT, cpdR and rcdA (Francis et al.2017). If all of these genes are indeed regulated by B. abortus CtrA, it would mean that the control of this transcription factor is more complex in this bacterium than in C. crescentus (Fig. 2). One tempting hypothesis is that the regulation of B. abortus CtrA could reflect a need for the bacterium to precisely regulate its cell cycle depending on its intracellular environment. B. abortus CtrA controls the expression of genes involved in envelope biogenesis The impact of B. abortus CtrA levels inside host cells could potentially be very important, as this transcription factor seems to be involved in both cell-cycle regulation and bacterial envelope composition (Francis et al.2017). Actually, one striking feature of B. abortus CtrA regulon is the high number of genes involved in envelope biogenesis (Francis et al.2017). Indeed, in addition to revealing the direct interaction between CtrA and the promoters of genes involved in the regulation of lipopolysaccharide and peptidoglycan synthesis, a ChIP-seq experiment also showed that promoters of genes coding for abundant outer membrane proteins (OMP) are also bound by CtrA (Francis et al.2017). CtrA-dependent regulation of these genes was supported by western blots against Omp2b and Omp25, two major OMPs of B. abortus that were found at lower levels in a CtrA depletion strain (Francis et al.2017). In addition, Omp25 homogenous localization patterns are perturbed when CtrA is absent (Francis et al.2017). This modification of the envelope composition could potentially have an impact on the bacterial fitness inside its host cell (Cha et al.2012). Indeed, the levels of Omp2b have been shown to decrease after 20–40 h during the course of macrophage infection by B. abortus (Lamontagne et al.2009). As for omp25, its disruption in B. abortus led to an increased sensitivity to Polymyxin B in vitro (Manterola et al.2007). In HeLa cells and murine macrophages, this strain was not shown to be involved in virulence, as its intracellular replication was similar to that of the wild type (Manterola et al.2007). In BALB/C mice, however, results were different in two independent experiments. In the first, a B. abortus omp25 mutant was injected intravenously at 5 × 104 CFU and led to an attenuation of virulence at 18 weeks post-infection, when compared to the wild type strain (Edmonds, Cloeckaert and Elzer 2002). In the second experiment, mice were infected via the intraperitoneal route with 105 CFU and behaved like the wild-type strain even after 24 weeks (Manterola et al.2007). This suggests that the route of infection could be an important factor to take into account when studying B. abortus infection. In this respect, it would be more appropriate to use a more physiological mode of infection in future research, such as the intranasal one (Hanot Mambres et al.2016). Note that the omp25 mutant seems to have different phenotypes depending on the Brucella species in which it is studied. Indeed, in B. suis, the disruption of this gene had much more dramatic consequences, as it was already attenuated from 1 to 8 weeks post-infection, before to be completely cleared from mice spleen (Edmonds, Cloeckaert and Elzer 2002). The authors suggested that this might be due to the fact that the B. suis strain that they used is a naturally occurring rough strain, thus the loss of the structural Omp25 could be more damaging in this case (Edmonds, Cloeckaert and Elzer 2002). Another difference between the two Brucella strains is that in B. suis, Omp25 is involved in the inhibition of the production of the pro-inflammatory TNF-α cytokine in human macrophages (Jubier-Maurin et al.2001; Luo et al.2017), whereas in B. abortus, it could be involved in the activation of the synthesis of TNF-α in human trophoblastic cells (Zhang et al.2017). The regulation of genes involved in the structure of the bacterial envelope by CtrA is probably not exclusive to B. abortus as many other α-proteobacteria do seem to regulate such genes via CtrA (Laub et al.2000; Brilli et al.2010). Nevertheless, it could have a more important impact on intracellular bacteria, as their envelope will be presented at the interface with their host cell and could therefore impact the host immune response and the survival of the bacteria inside them. For instance, the obligate intracellular Ehrlichia chaffeensis is thought to regulate the expression of its pal gene, coding for a major outer membrane stabilizing protein, through CtrA (Cheng et al.2011). This α-proteobacterium is the causative agent of the life-threatening human monocytic ehrlichiosis and has a developmental cycle comprising two forms in mammalian cells (Zhang et al.2007). The small dense-cored cells (with dense nucleoids) attach to and enter into the host cells, then differentiate into larger reticulate cells (defined by uniformly dispersed nucleoids) that can multiply for 48 h before transforming back into dense-cored cells at 72 h post-infection in order to prepare for reinfection (Zhang et al.2007). Interestingly, both pal and ctrA gene expressions were found to be highest when bacteria were in their dense-cored infectious form (Cheng et al.2011). Conversely, CtrA was found to be rapidly degraded, following a proline and glutamine uptake, after bacterial entry in host cells (Cheng, Lin and Rikihisa 2014). Pal is known to be immunogenic in dogs infected with E. chaffeensis and bacteria treated with anti-Pal antibodies were shown to be less infectious in vitro (Cheng et al.2011). Note that E. chaffeensis has been reported to lack lipopolysaccharide (Lin and Rikihisa 2003). S. meliloti also interacts tightly with host cells during nodulation. Very interestingly, it appears that B. abortus and S. meliloti do share some similarities in their potential CtrA targets. Indeed, CtrA binds to the promoter of several genes coding for L,D-transpeptidases homologs in B. abortus (BAB1_0047, BAB1_0138, BAB1_0589, BAB1_0978, BAB1_1159 and BAB1_1867) and S. meliloti (SMc00150, SMc01200, SMc01575 and SMc01769). L,D-transpeptidases are required to cross-link peptides in the peptidoglycan, and appear to be localized at the specific growth sites in the Rhizobiale Agrobacterium tumefaciens (Cameron et al.2014), another Rhizobiale displaying a unipolar growth (Brown et al.2012). Still, B. abortus seems to have evolved to possess more complex regulation of envelope biogenesis than S. meliloti: all but one (BAB1_0785) L,D-transpeptidase gene promoters are bound by CtrA in B. abortus (Francis et al.2017), as opposed to four out of seven in the case of S. meliloti (the others being SMc02636, SMc02582 and SMc00039) (Pini et al.2015). These L,D-transpeptidases should be further investigated in order to understand how they are truly regulated, as well as their specific roles in peptidoglycan growth and homeostasis. To determine if the envelope biogenesis regulation can directly be linked to the lifestyle of the bacteria would of course require more experimental data from other intracellular bacteria. The expression of ctrA is not crucial for B. abortus trafficking to the rBCV The regulation of genes according to the stage of B. abortus cell cycle can be observed through the use of a fluorescent-based reporter system. Such genes are ccrM and the repABC operon, and both are probably regulated by CtrA as the activities of their promoters were abolished when their respective CtrA-binding boxes were mutated (Francis et al.2017). As the activity of the promoter of the repABC operon seems to be inverted compared to the one of ctrA, it is expected that CtrA acts as a negative regulator of chromosome II replication (Francis et al.2017). Knowing that B. abortus first has to go through a non-replicative phase inside the eBCV, one can expect that CtrA would be important during this specific stage of the infection. However, CtrA was not found to be essential for the ability of B. abortus to infect cells in the models of infection tested thus far. Indeed, a study performed with a thermo-sensitive allele of B. abortus ctrA concluded that the transcription factor is not required for the entry of the bacterium inside THP-1 macrophages (Willett et al.2015). Inside HeLa cells, the CtrA depletion phenotypes of B. abortus were also visible around or after 10 h post-infection (Francis et al.2017). Furthermore, a B. abortus CtrA depletion strain was able to reach its rBCV replicative niche in the same proportion as the wild type strain (Francis et al.2017). This supports the view that CtrA function is dispensable for B. abortus trafficking inside these host cells. Nevertheless, at 48 h post-infection, CtrA depletion strains underwent a clear drop of CFU in both cellular infection models (Willett et al.2015; Francis et al.2017). The defects leading to bacterial cell death are unclear but could be explored by the analysis of suppressor mutants. The fact that CtrA might only be necessary at late time points during Brucella infection does make sense, though, as its level needs to be regulated more particularly during late phases of other intracellular α-proteobacteria life cycle. For example, in the obligate intracellular pathogen E. chaffeensis, CtrA is important during the late stage of intracellular growth (Cheng et al.2011). It has been observed that the ctrA gene is up-regulated at 72 h post-infection, which corresponds to the time when the bacteria differentiate back from large reticulate cells to small infectious dense-cored cells in order to prepare to spread from the present to the next host cells (Zhang et al.2007; Cheng et al.2011). Similarly, in S. meliloti, CtrA levels are high before infection, then a decrease of ctrA transcription coincides with bacteroid differentiation within the nodule (Roux et al.2014) and the CtrA protein is absent in mature bacteroids (Pini et al.2013). It is possible that CtrA is only required when Brucella find themselves in a particular situation. For instance, C. crescentus is known to regulate CtrA levels in response to stresses affecting its envelope, and this in a CckA-dependent manner, but independently of DivK and cyclic-di-GMP (Heinrich, Sobetzko and Jonas 2016). In E. chaffeensis, surE has been proposed to code for a protein involved in bacterial growth under stress and it is thought to be part of CtrA regulon (Cheng et al.2011). It is therefore possible that the in vitro models of infection tested thus far for B. abortus CtrA function do not reflect the environment and the stresses that they would have to face inside a living animal. In this respect, an in vivo model of infection might prove to be much more relevant for deciphering the potential impact of CtrA depletion in B. abortus. Hypotheses emerging from analyses of CtrA regulons Regulation of the cell cycle in different α-proteobacteria As several groups are now focusing on CtrA functions in other α-proteobacteria, more data about this transcription factor have become available over the last years. Despite their very diverse ways of life, α-proteobacteria do share some common traits, including the presence of a gene coding for CtrA (Brilli et al.2010). The comparison of the CtrA regulons in different bacteria, in light of their respective evolutionary lineage and lifestyle, might lead to interesting hypotheses regarding CtrA functions in B. abortus and other intracellular bacteria. Indeed, comparison of the direct CtrA regulons, when they are available, clearly indicate that CtrA binds to promoters of orthologous genes, suggesting that the control of these promoters by CtrA is a conserved feature. With that in mind, we compiled the experimental data that were available for this transcription factor in different α-proteobacteria. Data were collected in a hierarchical manner: (i) direct binding of CtrA to its target promoter, typically by ChIP-seq data, was considered first, (ii) when no such data were available, mRNA-level studies (for example microarrays) were used, (iii) bioinformatics predictions were taken into account only if no experimental data were found. For the determination of the predicted targets, we used RSAT (van Helden 2003). All data were compiled in Fig. 3. It is important to keep in mind that the approaches based on protein binding, mRNA levels or bioinformatics are not a direct proof of gene regulation and that they should be considered with reserve. This section is thus more prospective since meta-analyses usually generate working hypotheses that, if they lead to correlation, could indicate that similar processes are at play. Importantly, the absence of correlation does not necessarily mean the opposite. Hypotheses proposed here might therefore be challenged in the future. Figure 3. View largeDownload slide Comparison between CtrA targets in different α-proteobacteria. Data were collected in a hierarchical manner. Information about the direct binding of CtrA were found for C. crescentus (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), B. abortus (Francis et al.2017), S. meliloti (Pini et al.2015; Ichida and Long 2016), E. chaffeensis (Cheng et al.2011) and R. prowazekii (Brassinga et al.2002). Data concerning the mRNA-level of potential CtrA targets were then collected for S. melonis (Francez-Charlot, Kaczmarczyk and Vorholt 2015), D. shibae (Wang et al.2014), R. capsulatus (Mercer et al.2010; Leung, Brimacombe and Beatty 2013) and M. magneticum (Greene et al.2012). Finally, when no experimental data were available, we considered bioinformatics predictions. In the case of Rickettsiales and A. tumefaciens, some were already available (Hallez et al.2004; Ioannidis et al.2007; Pinto, Pappas and Winans 2012). For S. meliloti, prediction on ftsZ1 promoter was based on Ichida and Long (2016), with one substitution allowed. To complete these data, we also predicted CtrA targets for A. tumefaciens, E. chaffeensis, Wolbachia wMel and R. prowazekii. To do so, we used RSAT (van Helden 2003) and considered genes based on the presence in their promoter of either a 9-mer (TTAAN7TTAAC) or a 8-mer (TTAACCAT) CtrA-binding box (Marczynski and Shapiro 1992; Laub, Chen and McAdams 2002) with one substitution allowed. Note that we considered that ftsZ was potentially regulated by CtrA when it was the case for the ddl gene, as ftsZ is probably in operon with this gene in S. meliloti, B. abortus and A. tumefaciens. In C. crescentus, CtrA was found to bind upstream the ruvCAB operon, itself located upstream the tolQRAB operon. We considered that the spacing between these two operons did not allow the prediction of a CtrA control on the tolQRAB operon. The ORF of each gene analyzed here are available in supporting information. See the text for a detailed discussion about this figure. ‘OM constr.’ stands for outer membrane constriction. Figure 3. View largeDownload slide Comparison between CtrA targets in different α-proteobacteria. Data were collected in a hierarchical manner. Information about the direct binding of CtrA were found for C. crescentus (Laub et al.2000; Laub, Chen and McAdams 2002; Fumeaux et al.2014), B. abortus (Francis et al.2017), S. meliloti (Pini et al.2015; Ichida and Long 2016), E. chaffeensis (Cheng et al.2011) and R. prowazekii (Brassinga et al.2002). Data concerning the mRNA-level of potential CtrA targets were then collected for S. melonis (Francez-Charlot, Kaczmarczyk and Vorholt 2015), D. shibae (Wang et al.2014), R. capsulatus (Mercer et al.2010; Leung, Brimacombe and Beatty 2013) and M. magneticum (Greene et al.2012). Finally, when no experimental data were available, we considered bioinformatics predictions. In the case of Rickettsiales and A. tumefaciens, some were already available (Hallez et al.2004; Ioannidis et al.2007; Pinto, Pappas and Winans 2012). For S. meliloti, prediction on ftsZ1 promoter was based on Ichida and Long (2016), with one substitution allowed. To complete these data, we also predicted CtrA targets for A. tumefaciens, E. chaffeensis, Wolbachia wMel and R. prowazekii. To do so, we used RSAT (van Helden 2003) and considered genes based on the presence in their promoter of either a 9-mer (TTAAN7TTAAC) or a 8-mer (TTAACCAT) CtrA-binding box (Marczynski and Shapiro 1992; Laub, Chen and McAdams 2002) with one substitution allowed. Note that we considered that ftsZ was potentially regulated by CtrA when it was the case for the ddl gene, as ftsZ is probably in operon with this gene in S. meliloti, B. abortus and A. tumefaciens. In C. crescentus, CtrA was found to bind upstream the ruvCAB operon, itself located upstream the tolQRAB operon. We considered that the spacing between these two operons did not allow the prediction of a CtrA control on the tolQRAB operon. The ORF of each gene analyzed here are available in supporting information. See the text for a detailed discussion about this figure. ‘OM constr.’ stands for outer membrane constriction. In Fig. 3, it appears at first sight that the bacteria reported in this table belong to three distinct categories. The first one comprises A. tumefaciens, S. meliloti, B. abortus and C. crescentus, i.e. Rhizobiales and Caulobacterales. These bacteria have the common characteristic that the gene coding for CtrA is essential (Barnett et al.2001; Christen et al.2011; Figueroa-Cuilan et al.2016; Francis et al.2017). Moreover, at the exception of A. tumefaciens, their cell cycle has been shown to be regulated by CtrA (Quon et al.1998; Reisenauer, Quon and Shapiro 1999; Laub, Chen and McAdams 2002; Pinto, Pappas and Winans 2012; Pini et al.2015; Francis et al.2017). Therefore, it is not surprising to see that CtrA binds promoters for many genes involved in chromosome replication and segregation, as well as cell division. It is interesting to note that these processes are achieved through different genes in different bacteria and that it reflects their evolutionary lineage. Indeed, both A. tumefaciens and S. meliloti, which are closely related, seem to directly regulate their main chromosomal origin partitioning genes (parAB) through CtrA, while B. abortus is proposed to regulate its chromosome I replication through dnaA. As for C. crescentus, CtrA is directly binding to the ori as discussed earlier. It also seems that when a bacterium in this clade possesses a secondary chromosome, CtrA regulates its partition via the repABC operon (Pinto, Pappas and Winans 2012; Pini et al.2015; Francis et al.2017). One feature that is shared between Rhizobiales and C. crescentus is the predicted regulation of ftsK and ftsQ by CtrA, FtsK being involved in the segregation of the ter (Stouf, Meile and Cornet 2013) and FtsQ in the Z ring formation and constriction (Carson, Barondess and Beckwith 1991). The Z ring is composed of the tubulin-like FtsZ proteins, triggering the invagination of the cytoplasmic membrane, thus leading to septation and cytokinesis of the bacterium (Lutkenhaus and Addinall 1997). Interestingly, the ftsZ gene seems to be a target of CtrA in the first group of bacteria in Fig. 3. Note that in the case of A. tumefaciens, two copies of ftsZ exist and it is only the promoter region of the second one (Atu2086) that is predicted to possess an 8-mer CtrA binding site. There are also two ftsZ genes encoded in S. meliloti genome, and in this case, only the promoter of ftsZ1 is predicted to have a S. meliloti–specific CtrA-binding box with a AACCAT motif (Ichida and Long 2016), with one substitution (AGCCAT). However, neither of the two ftsZ promoters was found to be bound by ChIP-seq, so it was concluded that S. meliloti regulates its cell division through the control of minC and minD expression (Pini et al.2015). Indeed, MinC and MinD participate to the proper localization of the Z ring in Escherichia coli (de Boer, Crossley and Rothfield 1989) and many other bacteria. Interestingly, CtrA seems to systematically bind the promoter the minCDE operon in Rhizobiales. Note that this operon is absent in C. crescentus, where it is replaced by mipZ (Thanbichler and Shapiro 2006). Altogether, these analyses indicate that a number of crucial targets involved in different stages of the cell cycle were placed under the control of CtrA in the ancestors of Rhizobiales. In C. crescentus, some genes that are regulated by CtrA need to be expressed at very specific times. The most recent model to explain how CtrA-dependent genes are regulated in a timely manner is based on two other transcription factors, SciP and MucR (Fumeaux et al.2014). Genes that need to be expressed only in G2 phase cells, such as the DNA methyltransferase ccrM, are repressed by SciP in G1 phase cells even when CtrA is present (Gora et al.2010). On the other hand, genes that need to be expressed only in G1 phase cells are repressed by MucR in predivisional bacteria (Fumeaux et al.2014). From Fig. 3 it is clear that mucR is never predicted to be regulated by CtrA, in contrast to sciP, which is potentially regulated in S. meliloti, B. abortus and C. crescentus. The absence of a conserved CtrA-binding box in A. tumefaciens sciP promoter is surprising but does not mean that the gene is not regulated by CtrA in this bacterium. Another important player in C. crescentus gene expression regulation is the methyltransferase CcrM. As discussed above, ccrM temporal regulation by CtrA is important since it must only be present in late predivisional cells, where it methylates the newly synthesized DNA strands on GANTC sites precisely before cell division occurs (Stephens et al.1996; Reisenauer, Quon and Shapiro 1999). This means that, according to their chromosomal position away from the origin of replication, genes stay hemi-methylated for a different amount of time during the cell cycle (Marczynski 1999). This could have an important impact on gene expression at the whole genome level (Kozdon et al.2013). In fact, the transcription of several genes of C. crescentus has been observed to change in response to the methylation state of their promoters (Collier and Shapiro 2007; Gonzalez and Collier 2013). Remarkably, the promoter of ctrA is one of those (Reisenauer and Shapiro 2002). This is also supported by the fact that ccrM overexpression leads to abnormal chromosome content (Zweiger, Marczynski and Shapiro 1994). In the first group, ccrM appears to be systematically regulated by CtrA. This suggests that DNA methylation could be important for cell-cycle regulation in these bacteria. Of note, the function of GcrA has been demonstrated to be sensitive to CcrM DNA methylation in C. crescentus (Fioravanti et al.2012). As mentioned earlier, GcrA controls ctrA expression in this bacterium and vice versa (Holtzendorff et al.2004; Haakonsen, Yuan and Laub 2015). The regulation of gcrA by CtrA in C. crescentus was predicted by Brilli et al. (2010) to be an exception in α-proteobacteria. Experimental data obtained since then seem to be in agreement with this prediction, as gcrA has not yet been found to be part of the direct CtrA regulon in B. abortus (Francis et al.2017), S. meliloti (Pini et al.2015) or A. tumefaciens (Fig. 3). The second group of bacteria in Fig. 3 ranges from Sphingomonas melonis to Magnetospirillum magneticum. In this group, CtrA is not essential and the cell cycle progression does not seem to be coupled to this transcription factor (Greene et al.2012). Nevertheless, it is notable that some bacteria in this category could be at an evolutionary crossroads between the first and third group, as S. melonis (Francez-Charlot, Kaczmarczyk and Vorholt 2015) and Rhodospirillum centenum are both predicted to regulate the cell cycle transcription factor SciP. Of note, CtrA levels are regulated by quorum sensing in Dinoroseobacter shibae (Wang et al.2014), Rhodobacter capsulatus (Mercer et al.2010) and Ruegaria (Zan et al.2013). Despite not being involved in the cell-cycle regulation of these bacteria, the role of CtrA as a regulator of bacterial development, like in C. crescentus, seems to be conserved in this class. Indeed, cells of ctrA deletion strains are elongated (Wang et al.2014; Francez-Charlot, Kaczmarczyk and Vorholt 2015), which is reminiscent to the observation made with α-proteobacteria depletion strains when ctrA is essential (Reisenauer and Shapiro 2002; Figueroa-Cuilan et al.2016; Francis et al.2017). One can wonder how the cell cycle is regulated in α-proteobacteria that do not rely on CtrA for this function. Interestingly, bacteria from other clades developed similar strategies than C. crescentus to avoid replication over-initiation (Wolanski, Jakimowicz and Zakrzewska-Czerwinska 2014). For example, the gram-positive species Streptomyces coelicolor relies on AdpA to directly bind to its ori and inhibit binding of the replication machinery (Wolanski, Jakimowicz and Zakrzewska-Czerwinska 2012), whereas the γ-proteobacterium E. coli uses SeqA for a similar purpose (Nievera et al.2006). It is thus possible that α-proteobacteria inherited a similar mechanism from a more distantly related ancestor. When the regulation of DNA replication was put under the control of CtrA (in bacteria of the first group in Fig. 3 for example), this ancestral system could have progressively become obsolete, whereas it kept its role in the other α-proteobacteria. Importantly, essential vital functions may be regulated by redundant mechanisms to ensure their precise control. In the case of chromosome replication, redundant mechanisms are indeed often at play, such as proteolytic degradation of DnaA, its titration, the modulation of its activity by other proteins or a tight control of the expression of the dnaA gene (Wolanski, Jakimowicz and Zakrzewska-Czerwinska 2014). Only future studies will be able to reveal the real actors of cell-cycle regulation in the second group of α-proteobacteria but GcrA could be a good candidate for this function. Indeed, in C. crescentus, the transcription factor GcrA is known to participate in cell-cycle regulation by regulating CtrA expression but also, amongst others, by inhibiting the expression of dnaA when it is not necessary (Holtzendorff et al.2004). As a gene coding for GcrA is predicted to be present in the α-proteobacteria of the second group, it is possible that GcrA still retains this particular function in these bacteria. Note that M. magneticum does not seem to possess a gcrA gene, but this particular bacterium is apparently an exception, as all other Rhodospirillales studied thus far do possess one (Brilli et al.2010). A gene predicted to code for CcrM was also found in the genome of all the bacteria of the second group (Brilli et al.2010). If the ability of GcrA to differentiate genes during the cell cycle according to their CcrM-dependent methylation status is conserved in these bacteria, the two proteins might be sufficient to adequately control expression of cell cycle-related genes. The third group of bacteria in Fig. 3 is composed of the Rickettsiales E. chaffeensis, Wolbachia wMel (the endosymbiont of Drosophila melanogaster) and Rickettsia prowazekii. Not much is known about CtrA regulon and essentiality in these bacteria. However, the prediction that genes involved in cell division and chromosome replication are regulated by CtrA in these organisms is in agreement with the fact that the ori of R. prowazekii is bound by CtrA (Brassinga et al.2002) and that the promoter of the E. chaffeensis pal gene has been found as a target of the transcription factor (Cheng et al.2011). Moreover, eight strains of Wolbachia, in addition to other Rickettsiales, were found to possess DnaA binding sites and up to five CtrA consensus binding sites per ori (Ioannidis et al.2007). It is thus possible that ctrA regulates the cell cycle and is essential in this group of bacteria (Fig. 4). If this is confirmed in the future, it could mean that CtrA gained the function of regulating the cell cycle twice during evolution: once before the emergence of Rickettsiales and once before the appearance of Caulobacterales and Rhizobiales (Fig. 4). An alternative hypothesis would be that the regulation of the cell cycle was an ancestral function of CtrA that was lost early, but that seems less likely as it would imply the re-acquisition of this function in C. crescentus and Rhizobiales. Note that the third group in Fig. 3 is quite different from the first group, as they do not possess genes coding for the other cell cycle transcription factors such as SciP, MucR or GcrA. Nonetheless, they were found to possess a CckA/CtrA two-component system (Christensen and Serbus 2015). Furthermore, all Wolbachia CckA proteins were found to share a common conserved PAS domain, which could suggest that this protein has a ‘sensor’ capacity with a possibly conserved signal (Christensen and Serbus 2015). This potentially intrinsic ability of CckA to detect environmental signals has also been suggested to explain how C. crescentus is able to regulate CckA independently of the PleC/DivJ/DivK two-component system under stress (Heinrich, Sobetzko and Jonas 2016). Figure 4. View largeDownload slide Phylogenetic tree of α-proteobacteria based on CtrA. The tree was constructed with CtrA sequences from the genome of A. tumefaciens C58, S. meliloti 1021, B. abortus 2308, C. crescentus CB15, S. melonis TY, R. capsulatus SB 1003, D. shibae DFL 12, M. magneticum AMB-1, R. centenum SW ATCC 51521, R. prowazekii Rp22, Wolbachia wMel and E. chaffeensis Arkansas (https://biocyc.org/). Sequences were aligned with Clustal Omega (version 1.2.4; http://www.clustal.org/omega/) and curated manually with Jalview (http://www.jalview.org/download). The tree was created with PhyML (Guindon and Gascuel 2003) with the following settings: Starting tree = BioNJ; Tree Topology = SPR Move; Boostrap = 100 replicates; Substitution model = WAG. The final figure was formatted on iTol (https://itol.embl.de/), then on Inkscape. This tree is in agreement with previously published results (Williams, Sobral and Dickerman 2007; Greene et al.2012). Numbers correspond to bootstrap values. The unit of branch length are nucleotide substitutions per site (the number of changes divided by the length of the sequence). This figure suggests a possible timing, along evolution, of major events concerning genes that were taken under the control of CtrA. Note that cell cycle regulation seems to always coincide with a regulation of ftsZ and mismatch repair genes. Figure 4. View largeDownload slide Phylogenetic tree of α-proteobacteria based on CtrA. The tree was constructed with CtrA sequences from the genome of A. tumefaciens C58, S. meliloti 1021, B. abortus 2308, C. crescentus CB15, S. melonis TY, R. capsulatus SB 1003, D. shibae DFL 12, M. magneticum AMB-1, R. centenum SW ATCC 51521, R. prowazekii Rp22, Wolbachia wMel and E. chaffeensis Arkansas (https://biocyc.org/). Sequences were aligned with Clustal Omega (version 1.2.4; http://www.clustal.org/omega/) and curated manually with Jalview (http://www.jalview.org/download). The tree was created with PhyML (Guindon and Gascuel 2003) with the following settings: Starting tree = BioNJ; Tree Topology = SPR Move; Boostrap = 100 replicates; Substitution model = WAG. The final figure was formatted on iTol (https://itol.embl.de/), then on Inkscape. This tree is in agreement with previously published results (Williams, Sobral and Dickerman 2007; Greene et al.2012). Numbers correspond to bootstrap values. The unit of branch length are nucleotide substitutions per site (the number of changes divided by the length of the sequence). This figure suggests a possible timing, along evolution, of major events concerning genes that were taken under the control of CtrA. Note that cell cycle regulation seems to always coincide with a regulation of ftsZ and mismatch repair genes. In light of what can be observed in these three groups of bacteria, a hypothesis can be proposed about why B. abortus CtrA was not found to be important for infecting cells (Willett et al.2015; Francis et al.2017). It is possible that B. abortus does not rely solely on CtrA to regulate its cell cycle. Maybe another transcription factor, such as GcrA, directs this important function during the first phase of the infection, when B. abortus are in their non-replicative stage. Since the function of B. abortus CtrA during later times post-infection was not investigated, it would be interesting to test whether this transcription factor is required for reinfection, as in E. chaffeensis (Cheng et al.2011). It is also possible that CtrA is only necessary during infection when CckA responds to specific environmental stressors, similar to its role in C. crescentus (Heinrich, Sobetzko and Jonas 2016). These hypotheses are of course speculative and will need to be tested in the future. Could DNA repair regulation be an ancestral function of CtrA? Elucidation of the targets of M. magneticum CtrA suggests that motility is an ancestral trait of α-proteobacteria (Greene et al.2012). The authors of that study also proposed that the transition to the intracellular lifestyle of E. chaffeensis and R. prowazekii led to the loss of flagellar and chemotaxis genes and thus the loss of this regulation function (Greene et al.2012). Alternatively, the regulation of motility could have occurred on an evolutionary branch that is further away from the Rickettsiales (Fig. 4). Brucella was also considered as a non-motile and non-flagellated intracellular bacterium but it retains flagellar genes that appear to be important during infection (Halling 1998). Indeed, flagellin was shown to modulate the host response and bacterial proliferation in a mouse model of infection (Terwagne et al.2013). This is an interesting example of alteration of a given protein during evolution, from its initial function as a flagellin into a host protective factor (Shames and Finlay 2010; Terwagne et al.2013). The flagella of Bartonella bacilliformis, a close phylogenetic relative of Brucella, were also found to be required for entry into host erythrocytes (Scherer, DeBuron-Connors and Minnick 1993), illustrating that the function of these organelles diverged quickly during the course of evolution. In a transcriptomic study focusing on the M. magneticum CtrA regulon, the promoters of genes coding for proteins involved in motility were not predicted to be enriched amongst CtrA targets, even though the regulation of such genes by CtrA is proposed to be ancestral in α-proteobacteria (Greene et al.2012). Another functional category that could have been underestimated is DNA repair. Indeed, all α-proteobacteria studied in the context of CtrA regulation have at least one DNA repair gene predicted or effectively shown to be part of CtrA regulon. In the first and third groups of Fig. 3, one interesting observation is that promoters of genes coding for the Mismatch Repair (MR) system appear to be systematically targeted by CtrA. The MR system, composed of MutH, MutL and MutS proteins, is dependent on the Dam-related methylation status of E. coli DNA. Indeed, in this γ-proteobacterium, MutH is able to discern the parental DNA strand (that serves as template) from the newly synthesized one by recognizing the non-methylated state of the new DNA (Yamaguchi, Dao and Modrich 1998; Kunkel and Erie 2005). Thus, after MutS has detected the distortion in the helix caused by a base mismatch, MutL is recruited to allow the interaction between MutS and MutH. After MutH has excised the base, an exonuclease degrades a portion of DNA on the mutated and non-methylated strand, which is later repaired by the DNA polymerase III and a ligase (Kunkel and Erie 2005). In B. abortus and other α-proteobacteria, a homologous gene coding for MutH is missing (Martins-Pinheiro, Marques and Menck 2007; Guarne 2012). However, it has been proposed that in such a case, MutL is able to also perform the MutH function (Kadyrov et al.2006; Pillon et al.2010). In C. crescentus, mutS expression has been found to be cell cycle regulated (Laub et al.2000). Moreover, mutS was over-expressed in a CtrA depletion background, suggesting that it is under the control of CtrA (Laub et al.2000). As for mutL, it is not cell cycle regulated but its promoter has been found to be potentially linked to CtrA, based on a DNA microarray experiment (Laub, Chen and McAdams 2002). It should be noted that in C. crescentus, another gene is oriented opposite of mutL, so it is possible that this gene is the one being regulated by CtrA. However, the promoter of either mutL or mutS is consistently predicted to be bound by CtrA in other α-proteobacteria where CtrA probably regulates their cell cycle (Fig. 3, groups 1 and 3). One hypothesis would therefore be that the regulation of the MR system along the cell cycle would insure the correct timing for the utilization of this DNA repair mechanism. Indeed, the use of MR system is not always favourable, as it is known to result in enhanced mutagenesis in bacteria treated with e.g. alkylating agents (Nakano et al.2017). Note that in C. crescentus, CtrA directly activates the expression of the gene coding for S-adenosylmethionine (SAM) synthase, the enzyme responsible for the production of SAM (Laub, Chen and McAdams 2002). SAM is a methyl-donor for CcrM but it is also known to be a weak aspecific endogenous alkylating agent (Rydberg and Lindahl 1982). As for B. abortus, the promoter of tagA, which codes for a protein specifically involved in repairing alkylated DNA (Mielecki and Grzesiuk 2014), is directly bound by CtrA (Francis et al.2017). Incidentally, it would be interesting to know if TagA is required during B. abortus cellular infection, as it would suggest that alkylating stress is met by the bacterium inside its host. Knowing that several other genes involved in DNA repair are cell cycle regulated in C. crescentus, including the gene coding for the SOS repressor LexA (Laub et al.2000), it would also be interesting to investigate whether bacteria are more prone to DNA damage during certain stages of their cell cycle or not. In C. crescentus, both mutL and mutS, in addition to ctrA, are considered to be part of the GcrA regulon, as their promoters are all bound by GcrA in a ChIP-seq experiment and their expression changes in cells depleted in GcrA compared to wild-type cells (Haakonsen, Yuan and Laub 2015). As GcrA is able to sense CcrM-dependent methylation on DNA (Fioravanti et al.2013) and since this specific type of methylation is cell cycle regulated in C. crescentus (Stephens et al.1996) and probably also in B. abortus (Francis et al.2017), an interesting hypothesis would be that the regulation of the MR system by CtrA is a way to prevent it from functioning after full methylation has occurred and thus to avoid the cleavage of the wrong strand. However, CcrM-dependent methylation has been shown to be dispensable for C. crescentus to perform proper MR, as suggested by the frequency of rifampicin resistant mutants (Gonzalez et al.2014). This either means that the MR system is independent of CcrM methylation in C. crescentus (Gonzalez et al.2014), or alternatively that there are redundant DNA repair systems that prevent mismatches from occurring in C. crescentus, such as a robust base excision repair system (Martins-Pinheiro, Marques and Menck 2007). Two other genes that are supposedly involved in DNA repair could often be regulated by CtrA: dprA, also known as smf, and radC (Fig. 3). Both dprA and radC code for proteins with enigmatic functions. DprA interacts with RecA and is known to be essential for natural competence (Kidane et al.2012; Yadav et al.2013; Le et al.2017). However, the gene coding for this protein is conserved in many bacteria that are not naturally competent. In the α-proteobacterium R. capsulatus, DprA is involved in the regulation of Gene Transfer Agent (GTA) production in a CtrA-dependent manner (Brimacombe, Ding and Beatty 2014). A GTA system is analogous to phage transduction but it is unable to fully self-propagate and contains random segments of the host DNA that spread from one cell to another (Lang, Zhaxybayeva and Beatty 2012). As for RadC, and despite numerous efforts, its real function has remained elusive (Ogura et al.2002; Peterson et al.2004; Attaiech et al.2008). It is also known to be specifically expressed in naturally competent bacteria (Ogura et al.2002; Peterson et al.2004; Redfield et al.2005; Vickerman et al.2007). In the Rickettsiale pathogen Wolbachia, there exist three homologues of radC and all of them are associated with a cluster of genes that are distantly related to phage repressors (Wu et al.2004). Therefore, one hypothesis would be that RadC and DprA are not involved in competence in these bacteria but rather in coping with phage-derived DNA integration. Also, note that each α-proteobacterium seems to possess its own specific CtrA-dependent DNA repair targets. For example, D. shibae CtrA could regulate recA expression (Wang et al.2014), while it seems to be uvrB in the case of R. capsulatus (Mercer et al.2010). As for E. chaffeensis, it has a perfect 8-mer CtrA binding box in the promoter of its mfd gene, which codes for the Transcription Repair Coupling Factor that affects nucleotide excision repair (Selby and Sancar 1993). Thus, each bacterium probably optimized the different cellular functions regulated by CtrA through evolution according to its specific lifestyle. The clade of α-proteobacteria is composed of organisms with very different phenotypes and lifestyles. As proposed by others (Greene et al.2012), it seems that motility is an ancestral trait of CtrA regulon in these organisms. In addition to this observation, our review has proposed that DNA repair could also be a common target of this transcription factor. More precisely, the CtrA regulon seems to have evolved to couple the MR system to cell-cycle regulation in some bacteria and to modulate the levels of DprA and RadC in others. It also appears that each bacterium has selected the regulation of some specific DNA repair genes under the control of CtrA, which could reflect the kind of stresses that they meet in their respective environments. Yet, in B. abortus and most other α-proteobacteria, there is a gap in the literature on DNA repair. In the case of B. abortus, addressing this question would undoubtedly help to better understand what type of stresses are met by the bacterium inside its host cells and thus, to better understand the infectious process itself. In this regard, it would be very interesting to know if the blockage in G1 in HeLa cells and RAW 264.7 macrophages (Deghelt et al.2014) is linked to more efficient resistance to DNA damage. Concluding remarks One surprising conclusion about CtrA is that it is neither involved in the ability of B. abortus to enter inside its host cells, nor in its capacity to reach its replicative niche (Willett et al.2015; Francis et al.2017). As the survival of a B. abortus CtrA depletion strain decreases after 48 h post-infection (Willett et al.2015; Francis et al.2017), investigation of the impact of the protein at later times could lead to interesting discoveries and might provide answers to some open questions. For example, is the ability of CtrA to modify the bacterial envelope a way to modulate its pathogenicity? Is CtrA necessary for the cell-to-cell spreading of B. abortus, like in E. chaffeensis (Cheng et al.2011)? The fact remains that B. abortus does need to tightly regulate its cell cycle at early times post-infection, so that it is blocked in the G1 phase for up to 8 h after the entry inside HeLa cells and RAW 264.7 macrophages (Deghelt et al.2014). As this process is apparently independent of CtrA (Francis et al.2017), the question remains as to what is the molecular mechanism ensuring this G1 blockage. As B. abortus was suggested to face starvation while it is inside the eBCV, it is possible that the ppGpp-dependent starvation response is involved at that stage (Dozot et al.2006). In favour of this hypothesis, ppGpp has been found to regulate DnaA stability and initiation of DNA replication in carbon-starved C. crescentus (Lesley and Shapiro 2008), as well as to modulate the cell cycle when C. crescentus is unable to synthesize fatty acids (Stott et al.2015) or when it senses a decrease in intracellular glutamine concentration (Ronneau et al.2016). Another transcription factor that is supposed to be involved in cell-cycle regulation, such as GcrA, could also perform this function. Another important question to answer is to know whether this blockage occurs in all eukaryotic cell types, or if it is specific to infection of HeLa cells and RAW 264.7 macrophages. Conversely, one could wonder why B. abortus has evolved to favour a delay in its cell cycle progression during infection. One hypothesis would be that avoiding DNA replication as long as the bacterium is in the endosomal pathway is a way to prevent the fixation of mutations. Indeed, the passage through the eBCV is thought to be very stressful for the bacterium, which could cause DNA damage (Roop et al.2009). In addition, at that stage, most proteins—including those for DNA repair—are very weakly produced (Lamontagne et al.2009). It would therefore be more advantageous for B. abortus to wait for the storm to pass and repair its DNA when it is safe to do so. Otherwise, there is a risk that replication forks would stall and eventually collapse, which would lead to cell death (Cox et al.2000). As for the absence of growth itself during the first hours of the infection, it could be a way for B. abortus to limit its pathogen associated molecular pattern production and thus limit its recognition by host cells. Not growing could also be a way for the bacterium to avoid using too many resources while it still resides in the eBCV, which is usually considered a nutrient-poor environment (Roop et al.2009). The question of whether the G1 block is a common strategy to other intracellular pathogens also merits attention. Indeed, Legionella, Salmonella, Chlamydia and Francisella have also been reported to display a biphasic infection, with a relatively long non-proliferative period followed by a phase of massive proliferation (Salcedo and Holden 2005). There is a crucial need to address these fundamental issues in the future, as only a good knowledge of bacterial biology and infectious processes will allow us to combat pathogens that are becoming more and more resistant to antibiotic treatments. SUPPLEMENTARY DATA Supplementary data are available at FEMSRE online. Acknowledgements We thank J. Van Helden for uploading Rhodospirillum centenum genome on RSAT on request. We thank the members of the URBM for stimulating discussions. FUNDING We thank UNamur (https://www.unamur.be/) for financial and logistic support. This work was funded by The Fédération Wallonie-Bruxelles (ARC 17_22-087), and by the FRS-FNRS “Brucell-cycle” (PDR T.0060.15). K.P. and S.G. were supported by a FRIA PhD fellowship. Conflicts of interest. None declared. REFERENCES Archambaud C , Salcedo SP , Lelouard H et al. Contrasting roles of macrophages and dendritic cells in controlling initial pulmonary Brucella infection . Eur J Immunol 2010 ; 40 : 3458 – 71 . Attaiech L , Granadel C , Claverys JP et al. RadC, a misleading name? J Bacteriol 2008 ; 190 : 5729 – 32 . Ausmees N , Jacobs-Wagner C . Spatial and temporal control of differentiation and cell cycle progression in Caulobacter crescentus . Annu Rev Microbiol 2003 ; 57 : 225 – 47 . Barnett MJ , Hung DY , Reisenauer A et al. A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti . J Bacteriol 2001 ; 183 : 3204 – 10 . Bellaire BH , Roop RM 2nd , Cardelli JA . 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FEMS Microbiology ReviewsOxford University Press

Published: May 3, 2018

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