Molecular Microbiology

Hochstrasser, Ramon; Hutter, Cedric A. J.; Arnold, Fabian M.; Bärlocher, Kevin; Seeger, Markus A.; Hilbi, Hubert

doi: 10.1111/mmi.14477pmid: 31997467

The water‐borne bacterium Legionella pneumophila replicates in environmental protozoa and upon inhalation destroys alveolar macrophages, thus causing a potentially fatal pneumonia termed ‘Legionnaires’ disease’. L. pneumophila employs the Legionella quorum sensing (Lqs) system to control its life cycle, pathogen–host cell interactions, motility and natural competence. Signaling through the Lqs system occurs through the α‐hydroxyketone compound Legionella autoinducer‐1 (LAI‐1) and converges on the prototypic response regulator LqsR, which dimerizes upon phosphorylation of the conserved aspartate, D108. In this study, we determine the high‐resolution structure of monomeric LqsR. The structure reveals a receiver domain adopting a canonical (βα)5 fold, which is connected through an additional sixth helix and an extended α5‐helix to a novel output domain. The two domains delineate a mainly positively charged groove, and the output domain adopts a five‐stranded antiparallel β‐sheet fold similar to nucleotide‐binding proteins. Structure‐based mutagenesis identified amino acids critical for LqsR phosphorylation and dimerization. Upon phosphorylation, the LqsRD172A and LqsRD302N/E303Q mutant proteins dimerized even more readily than wild‐type LqsR, and no evidence for semi‐phosphorylated heterodimers was obtained. Taken together, the high‐resolution structure of LqsR reveals functionally relevant amino acid residues implicated in signal transduction of the prototypic response regulator.

Hashiguchi, Yuichiro; Tezuka, Takeaki; Ohnishi, Yasuo

doi: 10.1111/mmi.14485pmid: 32052506

The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA‐family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1‐fliA2 double‐deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild‐type and mutant strains of fliA1 and/or fliA2. Comparative RNA‐Seq analyses among the wild‐type and gene deletion mutant strains showed probable targets of each FliA‐family sigma factor, indicating that FliA1‐ and FliA2‐dependent promoters are quite similar to each other, while the FliA3‐dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA‐family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence.

Gosavi, Ujwala; Srivastava, Ankita; Badjatia, Nitika; Günzl, Arthur

doi: 10.1111/mmi.14489pmid: 32068297

Trypanosoma brucei CRK9 is an essential cyclin‐dependent kinase for the parasite‐specific mode of pre‐mRNA processing. In trypanosomes, protein coding genes are arranged in directional arrays that are transcribed polycistronically, and individual mRNAs are generated by spliced leader trans‐splicing and polyadenylation, processes that are functionally linked. Since CRK9 silencing caused a decline of mRNAs, a concomitant increase of unspliced pre‐mRNAs and the disappearance of the trans‐splicing Y structure intermediate, CRK9 is essential for the first step of splicing. CRK9 depletion also caused a loss of phosphorylation in RPB1, the largest subunit of RNA polymerase (pol) II. Here, we established cell lines that exclusively express analog‐sensitive CRK9 (CRK9AS). Inhibition of CRK9AS in these cells by the ATP‐competitive inhibitor 1‐NM‐PP1 reproduced the splicing defects and proved that it is the CKR9 kinase activity that is required for pre‐mRNA processing. Since defective trans‐splicing was detected as early as 5 min after inhibitor addition, CRK9 presumably carries out reversible phosphorylation on the pre‐mRNA processing machinery. Loss of RPB1 phosphorylation, however, took 12–24 hr. Surprisingly, RNA pol II‐mediated RNA synthesis in 24 hr‐treated cells was upregulated, indicating that, in contrast to other eukaryotes, RPB1 phosphorylation is not a prerequisite for transcription in trypanosomes.

doi: 10.1111/mmi.14524pmid: 32542886

Zeden, Merve S.; Kviatkovski, Igor; Schuster, Christopher F.; Thomas, Vinai C.; Fey, Paul D.; Gründling, Angelika

doi: 10.1111/mmi.14479pmid: 31997474

A Staphylococcus aureus strain deleted for the c‐di‐AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine‐betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild‐type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c‐di‐AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c‐di‐AMP signalling in S. aureus.

Westerhausen, Sibel; Nowak, Melanie; Torres‐Vargas, Claudia E.; Bilitewski, Ursula; Bohn, Erwin; Grin, Iwan; Wagner, Samuel

doi: 10.1111/mmi.14490pmid: 32068313

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate–NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc‐based secretion assay features a very high signal‐to‐noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384‐well microplates. We further developed a split NanoLuc‐based assay that enables the real‐time monitoring of type III secretion‐dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT.

Märker, Ramona; Blank‐Landeshammer, Bernhard; Beier‐Rosberger, Anna; Sickmann, Albert; Kück, Ulrich

doi: 10.1111/mmi.14475pmid: 32022307

The highly conserved striatin‐interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification‐based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK‐dependent phosphorylated protein, we selected CLA4, a member of the conserved p21‐activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and ‐deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.

Zhang, Kai; He, Jun; Cantalano, Claudio; Guo, Youzhong; Liu, Jun; Li, Chunhao

doi: 10.1111/mmi.14482pmid: 32039533

The Lyme disease bacterium Borrelia burgdorferi has 7–11 periplasmic flagella (PF) that arise from the cell poles and extend toward the midcell as a flat‐ribbon, which is distinct from other bacteria. FlhF, a signal recognition particle (SRP)‐like GTPase, has been found to regulate the flagellar number and polarity; however, its role in B. burgdorferi remains unknown. B. burgdorferi has an FlhF homolog (BB0270). Structural and biochemical analyses show that BB0270 has a similar structure and enzymatic activity as its counterparts from other bacteria. Genetics and cryo‐electron tomography studies reveal that deletion of BB0270 leads to mutant cells that have less PF (4 ± 2 PF per cell tip) and fail to form a flat‐ribbon, indicative of a role of BB0270 in the control of PF number and configuration. Mechanistically, we demonstrate that BB0270 localizes at the cell poles and controls the number and position of PF via regulating the flagellar protein stability and the polar localization of the MS‐ring protein FliF. Our study not only provides the detailed characterizations of BB0270 and its profound impacts on flagellar assembly, morphology and motility in B. burgdorferi, but also unveils mechanistic insights into how spirochetes control their unique flagellar patterns.

Pérez‐Burgos, María; García‐Romero, Inmaculada; Valvano, Miguel A.; Søgaard Andersen, Lotte

doi: 10.1111/mmi.14486pmid: 32064693

The rod‐shaped cells of Myxococcus xanthus, a Gram‐negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA‐I proteins, some of which are part of a Wzx/Wzy‐dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl‐phosphate hexose‐1‐phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N‐acetylgalactosamine‐1‐P to undecaprenyl‐phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.

Springstein, Benjamin L.; Arévalo, Sergio; Helbig, Andreas O.; Herrero, Antonia; Stucken, Karina; Flores, Enrique; Dagan, Tal

doi: 10.1111/mmi.14483pmid: 32039534

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria‐specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z‐ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.