The vibriophage-encoded inhibitor OrbA abrogates BREX-mediated defense through the ATPase BrxC.

Bacteria and phages are locked in a co-evolutionary arms race where each entity evolves mechanisms to restrict the proliferation of the other. Phage-encoded defense inhibitors have proven powerful tools to interrogate how defense systems function. A relatively common defense system is BREX (Bacteriophage exclusion); however, how BREX functions to restrict phage infection remains poorly understood. A BREX system encoded by the SXT integrative and conjugative element, Vch Ind5, was recently identified in Vibrio cholerae , the causative agent of the diarrheal disease cholera. The lytic phage ICP1 that co-circulates with V. cholerae encodes the BREX inhibitor OrbA, but how OrbA inhibits BREX is unclear. Here, we determine that OrbA inhibits BREX using a unique mechanism from known BREX inhibitors by directly binding to the BREX component BrxC. BrxC has a functional ATPase domain that, when mutated, not only disrupts BrxC function but also alters how BrxC multimerizes. Furthermore, we find that OrbA binding disrupts BrxC-BrxC interactions. We determine that OrbA cannot bind BrxC encoded by the distantly related BREX system encoded by the SXT Vch Ban9, and thus fails to inhibit this BREX system that also circulates in epidemic V. cholerae . Lastly, we find that homologs of the Vch Ind5 BrxC are more diverse than the homologs of the Vch Ban9 BrxC. These data provide new insight into the function of the BrxC ATPase and highlight how phage-encoded inhibitors can disrupt phage defense systems using different mechanisms. Importance: With renewed interest in phage therapy to combat antibiotic-resistant pathogens, understanding the mechanisms bacteria use to defend themselves against phages and the counter-strategies phages evolve to inhibit defenses is paramount. Bacteriophage exclusion (BREX) is a common defense system with few known inhibitors. Here, we probe how the vibriophage-encoded inhibitor OrbA inhibits the BREX system of Vibrio cholerae , the causative agent of the diarrheal disease cholera. By interrogating OrbA function, we have begun to understand the importance and function of a BREX component. Our results demonstrate the importance of identifying inhibitors against defense systems, as they are powerful tools for dissecting defense activity and can inform strategies to increase the efficacy of some phage therapies.

The Division Defect of a Bacillus subtilis minD noc Double Mutant Can Be Suppressed by Spx-Dependent and Spx-Independent Mechanisms.

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic interchromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. In addition, an Spx-independent pathway reduced the length of the cytokinetic period, perhaps by increasing divisome activity. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation. IMPORTANCE Bacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In Bacillus subtilis, cell division site selection depends on the Min and Noc systems, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, due not to a defect in FtsZ localization but rather to a failure in the maturation of the cell division machinery. Suppressor mutations that restored growth were selected, and while some activated the expression of ZapA via the Spx stress response pathway, others appeared to directly enhance divisome activity.

NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA.

NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, ΔnusG, and NusA depletion ΔnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.

Contact with the CsrA Core Is Required for Allosteric Inhibition by FliW in Bacillus subtilis.

The RNA-binding protein CsrA is a posttranscriptional regulator encoded by genomes throughout the bacterial phylogeny. In the gammaproteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding. In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, which noncompetitively inhibits CsrA activity but for which the precise mechanism of antagonism is unclear. Here, we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition and these residues fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues surrounding the critical regulatory CsrA residue N55 and abolished interaction between the two proteins. Two loss-of-function alleles, however, mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of the FliW alleles reversed a residue charge to disrupt a salt bridge with the CsrA core, and a compensatory charge reversal in the CsrA partner residue restored both the salt bridge and antagonism. We propose a model in which the initial interaction between FliW and CsrA is necessary but not sufficient for antagonism, and for which salt bridge formation with, and deformation of, the CsrA core domain is likely required to allosterically abolish RNA-binding activity.IMPORTANCE CsrA is a small dimeric protein that binds RNA and is one of the few known examples of transcript-specific protein regulators of translation in bacteria. A protein called FliW binds to and antagonizes CsrA to govern flagellin homeostasis and flagellar assembly. Despite having a high-resolution three-dimensional structure of the FliW-CsrA complex, the mechanism of noncompetitive inhibition remains unresolved. Here, we identify FliW residues required for antagonism and we find that the residues make a linear connection in the complex from initial binding interaction with CsrA to a critical salt bridge near the core of the CsrA dimer. We propose that the salt bridge represents an allosteric contact that distorts the CsrA core to prevent RNA binding.

The hetZ gene indirectly regulates heterocyst development at the level of pattern formation in Anabaena sp. strain PCC 7120.

Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein-protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.

SwrD (YlzI) Promotes Swarming in Bacillus subtilis by Increasing Power to Flagellar Motors.

The bacterium Bacillus subtilis is capable of two kinds of flagellum-mediated motility: swimming, which occurs in liquid, and swarming, which occurs on a surface. Swarming is distinct from swimming in that it requires secretion of a surfactant, an increase in flagellar density, and perhaps additional factors. Here we report a new gene, swrD, located within the 32 gene fla-che operon dedicated to flagellar biosynthesis and chemotaxis, which when mutated abolished swarming motility. SwrD was not required for surfactant production, flagellar gene expression, or an increase in flagellar number. Instead, SwrD was required to increase flagellar power. Mutation of swrD reduced swimming speed and torque of tethered flagella, and all swrD-related phenotypes were restored when the stator subunits MotA and MotB were overexpressed either by spontaneous suppressor mutations or by artificial induction. We conclude that swarming motility requires flagellar power in excess of that which is needed to swim.IMPORTANCE Bacteria swim in liquid and swarm over surfaces by rotating flagella, but the difference between swimming and swarming is poorly understood. Here we report that SwrD of Bacillus subtilis is necessary for swarming because it increases flagellar torque and cells mutated for swrD swim with reduced speed. How flagellar motors generate power is primarily studied in Escherichia coli, and SwrD likely increases power in other organisms, like the Firmicutes, Clostridia, Spirochaetes, and the Deltaproteobacteria.

The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in Anabaena sp. strain PCC 7120.

The commitment of differentiating cells to a specialized fate is fundamental to the correct assembly of tissues within a multicellular organism. Because commitment is often irreversible, entry into and progression through this phase of development must be tightly regulated. Under nitrogen-limiting conditions, the multicellular cyanobacterium Anabaena sp. strain PCC 7120 terminally commits ∼10% of its cells to become specialized nitrogen-fixing heterocysts. Although commitment is known to occur 9-14 h after the induction of differentiation, the factors that regulate the initiation and duration of this phase have yet to be elucidated. Here, we report the identification of four genes that share a functional domain and modulate heterocyst commitment: hetP (alr2818), asl1930, alr2902, and alr3234 Epistatic relationships between all four genes relating to commitment were revealed by deleting them individually and in combination; asl1930 and alr3234 acted most upstream to delay commitment, alr2902 acted next in the pathway to inhibit development, and hetP acted most downstream to drive commitment forward. Possible protein-protein interactions between HetP, its homologs, and the heterocyst master regulator, HetR, were assessed, and interaction partners were defined. Finally, patterns of gene expression for each homolog, as determined by promoter fusions to gfp and reverse transcription-quantitative PCR, were distinct from that of hetP in both spatiotemporal organization and regulation. We posit that a dynamic succession of protein-protein interactions modulates the timing and efficiency of the commitment phase of development and note that this work highlights the utility of a multicellular cyanobacterium as a model for the study of developmental processes.

FliW antagonizes CsrA RNA binding by a noncompetitive allosteric mechanism.

CsrA (carbon storage regulator A) is a widely distributed bacterial RNA binding protein that regulates translation initiation and mRNA stability of target transcripts. In γ-proteobacteria, CsrA activity is competitively antagonized by one or more small RNAs (sRNAs) containing multiple CsrA binding sites, but CsrA in bacteria outside the γ-proteobacteria is antagonized by a protein called FliW. Here we show that FliW of Bacillus subtilis does not bind to the same residues of CsrA required for RNA binding. Instead, CsrA mutants resistant to FliW antagonism (crw) altered residues of CsrA on an allosteric surface of previously unattributed function. Some crw mutants abolished CsrA-FliW binding, but others did not, suggesting that FliW and RNA interaction is not mutually exclusive. We conclude that FliW inhibits CsrA by a noncompetitive mechanism that differs dramatically from the well-established sRNA inhibitors. FliW is highly conserved with CsrA in bacteria, appears to be the ancestral form of CsrA regulation, and represents a widespread noncompetitive mechanism of CsrA control.

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120.

UNLABELLED: To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. IMPORTANCE: Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program.

The trpE gene negatively regulates differentiation of heterocysts at the level of induction in Anabaena sp. strain PCC 7120.

Levels of 2-oxoglutarate (2-OG) reflect nitrogen status in many bacteria. In heterocystous cyanobacteria, a spike in the 2-OG level occurs shortly after the removal of combined nitrogen from cultures and is an integral part of the induction of heterocyst differentiation. In this work, deletion of one of the two annotated trpE genes in Anabaena sp. strain PCC 7120 resulted in a spike in the 2-OG level and subsequent differentiation of a wild-type pattern of heterocysts when filaments of the mutant were transferred from growth on ammonia to growth on nitrate. In contrast, 2-OG levels were unaffected in the wild type, which did not differentiate under the same conditions. An inverted-repeat sequence located upstream of trpE bound a central regulator of differentiation, HetR, in vitro and was necessary for HetR-dependent transcription of a reporter fusion and complementation of the mutant phenotype in vivo. Functional complementation of the mutant phenotype with the addition of tryptophan suggested that levels of tryptophan, rather than the demonstrated anthranilate synthase activity of TrpE, mediated the developmental response of the wild type to nitrate. A model is presented for the observed increase in 2-OG in the trpE mutant.

Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of 'new' (recently differentiated) and 'old' (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.