Molecular Microbiology

Dyall‐Smith, Mike

doi: 10.1111/j.1365-2958.2010.07451.xpmid: 21210528

The Cas/CRISPR system of prokaryotes is a recently recognized defence system that protects cells from invasion by foreign nucleic acids, such as plasmids or bacteriophages. Using the archaeon Sulfolobus, Gudbergsdottir et al. have demonstrated that when foreign DNA is both a target of the CRISPR system and also carries a gene that is essential for cell growth, surviving cells display mutations in the CRISPR system that eliminate the target directed spacer. This simulates natural scenarios where the invading DNA cannot be readily destroyed, such as a temperate bacteriophage that manages to evade CRISPR attack and integrate into the genome, so becoming ‘self’. CRISPR deletions ranged from precise removal of a single spacer up to elimination of an entire CRISPR array but the mechanism involved is not understood. Another surprising feature was that from one to two base mutations in the same target protospacer did not affect recognition by the Sulfolobus CRISPR system, something unexpected from studies in bacteria where spacer and target protospacer must match exactly. This study shows that even though the CRISPR system is a powerful weapon it can also be a dangerous one for the cell that carries it.

Gaji, Rajshekhar Y.; Behnke, Michael S.; Lehmann, Margaret M.; White, Michael W.; Carruthers, Vern B.

doi: 10.1111/j.1365-2958.2010.07441.xpmid: 21166903

Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signalling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short‐term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.

Gode‐Potratz, Cindy J.; Kustusch, Ryan J.; Breheny, Patrick J.; Weiss, David S.; McCarter, Linda L.

doi: 10.1111/j.1365-2958.2010.07445.xpmid: 21166906

Vibrio parahaemolyticus senses surfaces via impeded rotation of its polar flagellum. We have exploited this surface‐sensing mechanism to trick the organism into thinking it is on a surface when it is growing in liquid. This facilitated studies of global gene expression in a way that avoided many of the complications of surface‐to‐liquid comparisons, and illuminated ∼ 70 genes that respond to surface sensing per se. Almost all are surface‐induced (not repressed) and encode swarming motility proteins, virulence factors or sensory enzymes involved with chemoreception and c‐di‐GMP signalling. Follow‐up studies were performed to place the surface‐responsive genes in a regulatory hierarchy. Mapping the hierarchy revealed two surprises about LafK, a transcriptional activator that until now has been considered to be the master regulator for the lateral flagellar system. First, LafK controls a more diverse set of genes than previously appreciated. Second, some laf genes are not under LafK control, which means LafK is not the master regulator after all. Additional experiments motivated by the transcriptome analyses revealed that growth on a surface lowers c‐di‐GMP levels and enhances cytotoxicity. Thus, we demonstrate that V. parahaemolyticus can invoke a programme of gene control upon encountering a surface and the specific identities of the surface‐responsive genes are pertinent to colonization and pathogenesis.

Haanstra, Jurgen R.; Kerkhoven, Eduard J.; van Tuijl, Arjen; Blits, Marjolein; Wurst, Martin; van Nuland, Rick; Albert, Marie‐Astrid; Michels, Paul A. M.; Bouwman, Jildau; Clayton, Christine; Westerhoff, Hans V.; Bakker, Barbara M.

Reininger, Luc; Wilkes, Jonathan M.; Bourgade, Hélène; Miranda‐Saavedra, Diego; Doerig, Christian

doi: 10.1111/j.1365-2958.2010.07442.xpmid: 21166904

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora‐related kinases, Pfark‐1, ‐2, ‐3. Among these, Pfark‐1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark‐1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark‐1–GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti‐α‐tubulin antibody strongly suggests a recruitment of Pfark‐1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark‐1 gene with a knockout construct further indicate that Pfark‐1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.

Saini, Supreet; Floess, Emily; Aldridge, Christine; Brown, Jonathon; Aldridge, Phillip D.; Rao, Christopher V.

doi: 10.1111/j.1365-2958.2010.07444.xpmid: 21166907

The flagellar genes in Salmonella enterica are expressed in a temporal hierarchy that mirrors the assembly process itself. The σ28–FlgM regulatory circuit plays a key role in controlling this temporal hierarchy. This circuit ensures that the class 3 genes are expressed only when the hook‐basal body (HBB), a key intermediate in flagellar assembly, is complete. In this work, we investigated the role of the σ28–FlgM regulatory circuit in controlling the timing and magnitude of class 3 gene expression using a combination of mathematical modelling and experimental analysis. Analysis of the model predicted that this circuit continuously controls class 3 gene expression in response to HBB abundance. We experimentally validated these predictions by eliminating different components of the σ28–FlgM regulatory system and also by rewiring the transcriptional hierarchy. Based on these results, we conclude that the σ28–FlgM regulatory circuit continuously senses the HBB assembly process and regulates class 3 gene expression and possibly flagellar numbers in response.

Tan, Qian; Awano, Naoki; Inouye, Masayori

doi: 10.1111/j.1365-2958.2010.07433.xpmid: 21166897

Toxin–antitoxin (TA) systems of free‐living bacteria have recently demonstrated that these toxins inhibit cell growth by targeting essential functions of cellular metabolism. Here we show that YeeV toxin inhibits cell division, leads to a change in morphology and lysis of Escherichia coli cells. YeeV interacts with two essential cytoskeleton proteins, FtsZ and MreB. Purified YeeV inhibits both the GTPase activity and the GTP‐dependent polymerization of FtsZ. YeeV also inhibits ATP‐dependent polymerization of MreB. Truncated C‐terminal deletions of YeeV result in elongation of cells, and a deletion of the first 15 amino acids from the N‐terminus of YeeV caused lemon‐shaped cell formation. The YeeV toxin is distinct from other well‐studied toxins: it directs the binding of two cytoskeletal proteins and inhibits FtsZ and MreB simultaneously.

Tolla, Dean A.; Savageau, Michael A.

doi: 10.1111/j.1365-2958.2010.07437.xpmid: 21166900

The FNR protein in Escherichia coli is an O2 sensor that modifies global gene expression to adapt the cell to anaerobic growth. Regulation of FNR involves continuous cycling of the protein between its active and inactive states under aerobic conditions without apparent function. This raises the question of what benefit to the overall life cycle might compensate for the cost of cycling and reveals that the role of this process is poorly understood. To address this problem, we introduce the concept of a ‘system design space’, which provides a rigorous definition of phenotype at the molecular level and a means of visualizing the phenotypic repertoire of the system. Our analysis reveals undesirable and desirable phenotypes with an optimal constellation of parameter values for the system. To facilitate a more concrete understanding of what the design space represents, we analyse mutations that alter the apparent dimerization rate constant of FNR. We show that our estimated wild‐type value of this rate constant, which is difficult to measure in situ, is located within this constellation and that the behaviour of the system is compromised in mutants if the value of the apparent dimerization rate constant lies beyond the bounds of this optimal constellation.

Gudbergsdottir, Soley; Deng, Ling; Chen, Zhengjun; Jensen, Jaide V. K.; Jensen, Linda R.; She, Qunxin; Garrett, Roger A.

doi: 10.1111/j.1365-2958.2010.07452.xpmid: 21166892

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter‐genomically or intra‐genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single‐spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.

Zalila, Habib; González, Iveth J.; El‐Fadili, Amal Kuendig; Delgado, Maria Belen; Desponds, Chantal; Schaff, Cédric; Fasel, Nicolas

doi: 10.1111/j.1365-2958.2010.07443.xpmid: 21166905

Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.