The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.

Clinically relevant enterococci are intrinsically resistant to antibiotics in the cephalosporin family. The CroS sensor kinase senses cephalosporin-induced cell wall stress to trigger signaling that drives cephalosporin resistance, but the mechanism by which CroS senses stress is unknown. We report the first functional characterization of the CroS extracellular segment, revealing that mutations in the extracellular segment did not prevent CroS from responding to cell wall stress but instead biased CroS to adopt a more active state during cephalosporin stress that led to an increase in CroR-dependent gene expression and hyper-resistance to ceftriaxone. Overall, our results suggest that the extracellular segment of CroS does not directly bind to a regulatory ligand but that it can modulate the magnitude of CroS signaling.

Plant pathogenic bacteria encounter a drastic increase in apoplastic pH during the early stages of plant immunity. The effects of alkalization on pathogen-host interactions have not been comprehensively characterized. Here, we used a global transcriptomic approach to assess the impact of environmental alkalization on Pseudomonas syringae pv. tomato DC3000 in vitro. In addition to the Type 3 Secretion System, we found expression of genes encoding other virulence factors such as iron uptake and coronatine biosynthesis to be strongly affected by environmental alkalization. We also found that the activity of AlgU, an important regulator of virulence gene expression, was induced at pH 5.5 and suppressed at pH 7.8, which are pH levels that this pathogen would likely experience before and during pattern-triggered immunity, respectively. This pH-dependent control requires the presence of periplasmic proteases, AlgW and MucP, that function as part of the environmental sensing system that activates AlgU in specific conditions. This is the first example of pH-dependency of AlgU activity, suggesting a regulatory pathway model where pH affects the proteolysis-dependent activation of AlgU. These results contribute to deeper understanding of the role apoplastic pH has on host-pathogen interactions.IMPORTANCEPlant pathogenic bacteria, like Pseudomonas syringae, encounter many environmental changes including oxidative stress and alkalization during plant immunity, but the ecological effects of the individual responses are not well understood. In this study, we found that transcription of many previously characterized virulence factors in P. syringae pv. tomato DC3000 is downregulated by the level of environmental alkalization these bacteria encounter during the early stages of plant immune activation. We also report for the first time the sigma factor AlgU is post-translationally activated by low environmental pH through its natural activation pathway, which partially accounts for the expression Type 3 Secretion System virulence genes at acidic pH. The results of this study demonstrate the importance of extracellular pH on global regulation of virulence-related gene transcription in plant pathogenic bacteria.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. Caulobacter crescentus adheres to surfaces using a polysaccharide adhesin called the holdfast. In C. crescentus, disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several C. crescentus genes involved in flagellar surface sensing. One of these, fssF, codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting fssF results in a severe motility defect, which we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that fssF promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of fssF or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (pleD, motB, and dgcB) contribute to both ∆flgH-dependent and ∆fssF-dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.IMPORTANCEBacterial biofilms pose a threat in clinical and industrial settings. Surface sensing is one of the first steps in biofilm formation. Studying surface sensing can improve our understanding of biofilm formation and develop preventative strategies. In this study, we use the freshwater bacterium Caulobacter crescentus to study surface sensing and the regulation of surface attachment. We characterize a previously unstudied gene, fssF, and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that fssF is required for chemotaxis behavior but dispensable for swimming motility. Lastly, our results indicate that deletion of fssF and other genes required for chemotaxis results in a hyperadhesive phenotype. These results support that surface sensing requires chemotaxis for a robust response to a surface.

In this issue (J Bacteriol. 206: e0014024, https://doi.org/10.1128/jb.00140-24), Ridone and Baker describe hybrids between two 5:2 heteroheptameric ion-powered motors. Chimeras were constructed between stator units of a bacterial flagellum and ExbBD of the Ton outer-membrane transport system. Only one of the 14 hybrids supported swimming in Escherichia coli. Three additional residue changes at sites distant from the hybrid region enhanced motility. This work suggests that flagellar stator units and ExbBD share an ancestor that diverged during evolution to perform different tasks.

Members of the kingdom Nanobdellati, previously known as DPANN archaea, are characterized by ultrasmall cell sizes and reduced genomes. They primarily thrive through ectosymbiotic interactions with specific hosts in diverse environments. Recent successful cultivations have emphasized the importance of adhesion to host cells for understanding the ecophysiology of Nanobdellati. Cell adhesion is often mediated by cell surface carbohydrates, and in archaea, this may be facilitated by the glycosylated S-layer protein that typically coats their cell surface. In this study, we conducted glycoproteomic analyses on two co-cultures of Nanobdellati with their host archaea, as well as on pure cultures of both host and non-host archaea. Nanobdellati exhibited various glycoproteins, including archaellins and hypothetical proteins, with glycans that were structurally distinct from those of their hosts. This indicated that Nanobdellati autonomously synthesize their glycans for protein modifications probably using host-derived substrates, despite the high energy cost. Glycan modifications on Nanobdellati proteins consistently occurred on asparagine residues within the N-X-S/T sequon, consistent with patterns observed across archaea, bacteria, and eukaryotes. In both host and non-host archaea, S-layer proteins were commonly modified with hexose, N-acetylhexosamine, and sulfonated deoxyhexose. However, the N-glycan structures of host archaea, characterized by distinct sugars such as deoxyhexose, nonulosonate sugar, and pentose at the nonreducing ends, were implicated in enabling Nanobdellati to differentiate between host and non-host cells. Interestingly, the specific sugar, xylose, was eliminated from the N-glycan in a host archaeon when co-cultured with Nanobdella. These findings enhance our understanding of the role of protein glycosylation in archaeal interactions.IMPORTANCENanobdellati archaea, formerly known as DPANN, are phylogenetically diverse, widely distributed, and obligately ectosymbiotic. The molecular mechanisms by which Nanobdellati recognize and adhere to their specific hosts remain largely unexplored. Protein glycosylation, a fundamental biological mechanism observed across all domains of life, is often crucial for various cell-cell interactions. This study provides the first insights into the glycoproteome of Nanobdellati and their host and non-host archaea. We discovered that Nanobdellati autonomously synthesize glycans for protein modifications, probably utilizing substrates derived from their hosts. Additionally, we identified distinctive glycosylation patterns that suggest mechanisms through which Nanobdellati differentiate between host and non-host cells. This research significantly advances our understanding of the molecular basis of microbial interactions in extreme environments.

Given the prevalence of antibiotic resistance, there is an increasing need to develop alternative approaches to managing pathogenic bacteria. In this work, we explore the feasibility of modulating the expression of various cellular systems in Vibrio cholerae using engineered regulatory sRNAs delivered into cells via DNA conjugation. These sRNAs are based on regulatory sRNAs found in V. cholerae and exploit its native regulatory machinery. By delivering these sRNAs conjugatively along with a real-time marker for DNA transfer, we found that complete knockdown of a targeted cellular system could be achieved within one cell division cycle after sRNA gene delivery. These results indicate that conjugative delivery of engineered regulatory sRNAs is a rapid and robust way of precisely targeting V. cholerae.

In almost all bacteria, the tubulin-like GTPase FtsZ polymerizes to form a "Z-ring" that marks the site of division. FtsZ recruits other proteins, collectively known as the divisome, that together remodel and constrict the envelope. Constriction is driven by peptidoglycan (PG) cell wall synthesis by the glycosyltransferase FtsW and the transpeptidase FtsI (FtsWI), but these enzymes require activation to function. How recruitment of FtsZ to the division site leads to FtsWI activation and constriction remains largely unknown. Previous work in our laboratory demonstrated that an FtsZ-binding protein, FzlA, is essential for activation of FtsWI in the alphaproteobacterium Caulobacter crescentus. Additionally, we found that FzlA binds to a DNA translocase called FtsK, suggesting that it may link constriction activation to chromosome segregation. FzlA is conserved throughout Alphaproteobacteria but has only been examined in detail in C. crescentus. Here, we explored whether FzlA function is conserved in diverse Alphaproteobacteria. We assessed FzlA homologs from Rickettsia parkeri and Agrobacterium tumefaciens, and found that, similar to C. crescentus FzlA, they bind directly to FtsZ and localize to midcell. The FtsZ-FzlA interaction interface is conserved, as we demonstrated that FzlA from each of the three species examined can bind to FtsZ from any of the three in vitro. Finally, we determined that A. tumefaciens FzlA can fulfill the essential function of FzlA when produced in C. crescentus, indicating conservation of function. These results suggest that FzlA serves as an important regulator that coordinates chromosome segregation with envelope constriction across diverse Alphaproteobacteria.IMPORTANCECell division is essential for bacterial replication and must be highly regulated to ensure robust remodeling of the cell wall in coordination with segregation of the genome to daughter cells. In Caulobacter crescentus, FzlA plays a major role in regulating this process by activating cell wall synthesis in a manner that couples constriction to chromosome segregation. FzlA is broadly conserved in Alphaproteobacteria, suggesting that it plays a similar function across this class of bacteria. Here, we have shown that, indeed, FzlA biochemical interactions and function are conserved in diverse Alphaproteobacteria. Because FzlA is conserved in Alphaproteobacterial human pathogens, understanding this protein and its interactome could present therapeutic benefits by identifying potential antibiotic targets to treat infections.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.