Biology subjects

Kearns, D. B.

Publications and source records attributed to Kearns, D. B..

At least 19 recordsLinked to original sources

bioRxiv2026

DegQ and SwrA differentially modulate protease production and flagellar motility in Bacillus subtilis

In Bacillus subtilis, the DegS-DegU two-component system modulates a range of adaptive responses, including extracellular protease production and the flagellar-surface motility called swarming. Two proteins, DegQ and SwrA, are thought to increase the amount of phosphorylated DegU and alter DegU DNA-binding abilities of DegU, respectively. Here we compare the relative contributions of DegQ and SwrA to DegU-related phenotypes in the B. subtilis ancestral strain that is functional for both proteins. We found that DegQ enhances the production of secreted proteases in stationary phase and inhibits swarming motility during exponential growth by a mechanism dependent on both DegS and DegU. Moreover, DegQ-mediated swarming inhibition was due, in part, to the attenuation of transcription levels within the long fla/che operon. SwrA by contrast, activates the promoter of the fla/che operon by a mechanism that depends on DegU but not DegS. Moreover, SwrA inhibits extracellular protease production by inhibiting the expression of DegQ. We conclude that DegQ and SwrA have opposing effects on DegU-dependent phenotypes and do so by different mechanisms. Thus, B. subtilis evolved third-party modulators to expand the conventional two-component system and achieve differential gene expression.

microbiology↗

bioRxiv2026

A two-step model of FtsZ-ring disassembly in Bacillus subtilis

Bacillus subtilis grows and divides by binary fission, directed by medial localization of cell division protein FtsZ. Disruption of either the Min system or EzrA results in aberrant FtsZ positioning. Here we compare FtsZ dynamics in cells disrupted for either MinD or EzrA when grown in microfluidic channels. Here we show that cells lacking MinD or EzrA appear to be similarly defective in Z-ring disassembly after septation, but play different roles as simultaneous disruption results in a synergistic defect in division. Moreover, we account for a low frequency of minicell formation in the absence of EzrA, as MinD but not EzrA is necessary for removal of ZapA from polar Z-rings. Finally, overexpression of MinCD results inhibits division through pervasive Z-ring disassembly but appears to concentrate ZapA through localized sequestration. Combined, our results indicate a closer relationship between MinCD and ZapA than previously recognized and show that Z-ring disassembly can be genetically separated into discrete steps. We propose a two-step model for Z-ring disassembly that mirrors the assembly process, such that after and/or during septation, the Z-ring separately decondenses and protofilaments are disassembled to monomers for recycling.

microbiology↗

bioRxiv2026

MinJ is a conserved nine-pass transmembrane protein that contains a putative transmembrane β-sheet

The Min system disassembles FtsZ-rings after septation in Bacillus subtilis and is localized to the nascent division plane and cell poles by the protein MinJ. The N-terminal region of MinJ contains transmembrane segments while the C-terminal region of MinJ contains a PDZ domain but its topology and functional domains are poorly understood. Here we empirically test MinJ topology based on a variety of transmembrane prediction models and find that the data is most consistent with Alphafold3, which predicts a 9-pass transmembrane protein with an external N-terminus and internal C-terminus. Deletion analysis indicates that all regions of the protein tested are required for function but deletion of the PDZ domain alone preserves polar localization and interaction with both MinD and DivIVA. Moreover, Alphafold predicts that transmembrane segments 6 and 7 comprise staves of an unusual transmembrane {beta}-sheet and deletion of the putative {beta}-sheet in the absence of MinD results in a minicell frequency that exceeds mutation of MinD alone. Bioinformatic analysis indicates that MinJ is highly conserved within Firmicutes and is co-conserved with MinD and DivIVA with which it interacts. Our data clarify the structure of MinJ and support models in which MinJ has functions in addition to restricting the activity of the Min system. IMPORTANCEFaithful positioning of the bacterial division site is important for cell growth and is coordinated by the conserved Min system. Although the Min system of Bacillus subtilis has been extensively studied, MinJ, the membrane protein that links the division inhibitor MinCD to the polar determinant DivIVA, remains the least well-understood. Here we experimentally define the membrane topology of MinJ and show that our data are most consistent with a nine-pass transmembrane architecture predicted by AlphaFold3. We further provide genetic, cell biological, and evolutionary evidence supporting that two of the staves form a highly conserved putative transmembrane {beta}-sheet, a structure normally excluded from the plasma membrane. Our findings refine MinJ structural organization and provide a framework for understanding its conserved functions in bacterial cell division.

microbiology↗

bioRxiv2026

Efficient septum formation is essential for chromosome segregation in Bacillus subtilis when SMC function is impaired

Structural maintenance of chromosomes (SMC) complexes play conserved roles in chromosome organization, segregation, and repair in all domains of life. In Bacillus subtilis, SMC is required for segregation of newly replicated origins. To investigate whether other proteins function with SMC in this process, we performed a synthetic lethal screen with an smc hypomorphic allele (smc*) that is mildly defective in chromosome segregation. In addition to recovering previously reported interactions of smc with parB and spoIIIE, our screen identified minJ and divIVA as essential in the smc* background. We show that the synthetic lethality between smc* and{Delta} minJ or{Delta} divIVA arises from defects in segregating the replication terminus. Importantly, deletion of minD, which suppresses the cell division defects of{Delta} minJ and{Delta} divIVA, restored terminus segregation and viability in the smc* background. These findings support a model in which proper septum formation promotes chromosome terminus resolution and segregation by enabling SpoIIIE-mediated DNA clearance from the division septum during cytokinesis. These findings highlight the interdependence between chromosome segregation and cell division. ImportanceThe SMC complex plays a central role in chromosome organization and segregation in Bacillus subtilis, but the cellular functions that become important when SMC activity is reduced are not well understood. Using a hypomorphic smc allele, we discovered that mutations affecting cell division become essential when chromosome organization and segregation are impaired. Our findings support a model in which efficient septum formation enables proper localization of the SpoIIIE DNA translocase, which in turn resolves and segregates the chromosome terminus region. These results highlight the critical role of cell division in supporting chromosome segregation.

microbiology↗

bioRxiv2026

The last bacterial common ancestor encoded a complex flagellum

Bacterial flagella are rotary nanomachines that enable motility in diverse environments. Although more than 40 genes are required to assemble, operate, and regulate a functional flagellum in model organisms, only 24 flagellar genes have previously been inferred to be conserved across bacteria. This discrepancy raises a fundamental question: did the last bacterial common ancestor encode a simpler, partial flagellum that was elaborated later in a lineage-specific manner, or does the apparent absence of conserved components reflect limitations in detecting highly diverged homologs? Here we combine large-scale profile- and sequence-based searches across a comprehensive bacterial genome set with conserved sequence signatures, gene-tree clustering and flagellar gene-neighborhood evidence to reconstruct the ancestral complexity of bacterial flagellar systems. We identify 28 additional flagellar gene families whose distributions and evolutionary histories support an origin before major bacterial diversification, yielding a 52-gene ancestral flagellum. The ancestral flagellum included all proteins of the secretion/export apparatus, basal body, axial components, motor-force generators and regulatory checkpoints required to build and operate a functional, contemporary flagellum. These findings revise models of early bacterial evolution and overturn the notion that the ancestral flagellum was genetically minimal. Instead, they suggest that the last bacterial common ancestor possessed a highly complex flagellar system comprising more components than are typically found in extant bacteria, many of whose flagella appear to have been shaped by lineage-specific gene loss.

microbiology↗

bioRxiv2026

Flagellar toxicity: flagellar synthesis is lytic for Bacillus subtilis in the absence of PBP1

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general. SIGNIFICANCEBacteria assemble nanomachines through the cell envelope but how the machines transit the peptidoglycan is poorly understood. Here we find that assembly of trans-envelope flagella results in cell lysis of Bacillus subtilis when the peptidoglycan repair protein PBP1 is absent. Lysis was due to multiple peptidoglycan lyases expressed as a consequence of flagellar assembly, and lytic activity required another PBP homolog, PBPX. Our work indicates that flagella, not normally thought to impact cell viability, can be lethal at the level of cell envelope integrity.

microbiology↗

bioRxiv2026

Constitutive, endogenous, fluorescent membrane reporters for dynamic cell cycle analysis in Bacillus subtilis

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.

microbiology↗

bioRxiv2025

Spx inhibits expression of the SwrA*DegU master flagellar activator in Bacillus subtilis.

Flagella are trans-envelope nanomachines expressed from genes organized in a complex regulatory hierarchy governed at the highest level by transcription factors called master activators. The master activator of flagellar biosynthesis in Bacillus subtilis is a hybrid of SwrA*DegU that is required to increase flagellar density to swarm over solid surfaces. Here we show that the ClpX unfoldase subunit of the ClpP protease is required for swarming motility, and that cells mutated for ClpX fail to swarm due to restricted levels of both SwrA and DegU. Suppressor mutations were found that increased expression of the fla/che operon under SwrA*DegU control, and mutation of the LonA protease elevates the levels of SwrA protein while mutation of the global transcriptional regulator Spx increases transcription of both the degU and swrA genes. We conclude that ClpX promotes swarming motility via degradation of Spx, which represses motility gene transcription including the Pfla/che, PdegU, and PswrA promoters each activated by DegU. The ClpX-dependent regulatory proteolysis of Spx is relieved upon stress conditions, and we infer that Spx may dampen DegU-mediated positive feedback to limit cell envelope stress caused by excessive flagellar biosynthesis.

microbiology↗

bioRxiv2025

The SinR*SlrR heteromer attenuates transcription of a long operon of flagellar genes in Bacillus subtilis

During growth, Bacillus subtilis differentiates into subpopulations of motile individuals and non-motile chains, associated with dispersal and biofilm formation respectively. The two cell types are dictated by the activity of the alternative sigma factor SigD encoded as the penultimate gene of the 27 kb long fla/che flagellar operon. The frequency of SigD-ON motile cells is increased by the heteromeric transcription factor SwrA*DegU that activates the fla/che promoter. Conversely, the frequency of motile cells is decreased by the heteromeric transcription factor SinR*SlrR, but the mechanism and location of inhibition is poorly understood. Here, using ChIP-Seq analysis, we determine the binding sites of the SinR*SlrR heteromer on the genome. We identified two sites within the fla/che operon that were both necessary and sufficient to attenuate transcript abundance by causing premature termination upstream of the gene that encodes SigD. Thus, cell motility and the transition to biofilm formation depend on the expression of a long operon governed by two opposing heteromeric transcription factors that operate at two different stages of the transcription cycle. More broadly, our study serves as a model for transcription factors that control transcriptional elongation and the regulation of long operons in bacteria.

microbiology↗

bioRxiv2024

Nascent flagellar basal bodies are immobilized by rod assembly in Bacillus subtilis

Flagella are complex, trans-envelope nanomachines that localize to species- specific cellular addresses. Here we study the localization dynamics of the earliest stage of basal body formation in Bacillus subtilis using a fluorescent fusion to the C-ring protein FliM. We find that B. subtilis basal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan. Rare basal bodies were observed to be mobile however, and the frequency of basal body mobility is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning and we propose that rod polymerization probes the peptidoglycan superstructure for pores of sufficient diameter that permit rod completion. Furthermore, mutation of the rod also disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that conformational changes in the basal body exchange information between rod synthesis and the cytoplasmic patterning proteins to restrict assembly at certain pores established by a grid-like pattern pre-existent in the peptidoglycan itself. IMPORTANCEBacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site. Bacillus subtilis assembles [~]15 flagella over the length of the cell body in a grid-like pattern and lacks all proteins associated with targeted assembly in polarly flagellated bacteria. Here we show that B. subtilis basal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan wall. Moreover, defects in the flagellar rod lead to an asymmetric distribution of flagella with respect to the midcell. We conclude that the patterning of flagella is different in B. subtilis, and we infer that the B. subtilis rod probes the peptidoglycan for holes that can accommodate the machine.

cell biology↗

bioRxiv2024

ComI inhibits transformation in Bacillus subtilis by selectively killing competent cells

Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, Bacillus subtilis, has a non-transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is preferentially expressed in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins and permeabilization activity is concentration dependent. Thus over time, the competent cells gradually producing ComI, are permeabilized and killed. Based on these observations we propose a new model for the mechanism of ComI, suggesting a response to competence activation that selectively eliminates the competent subpopulation. ImportanceNatural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of Bacillus subtilis into the "competent" state, such that cells with ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures the population can maintain unaltered plasmid and its predicted prophage.

microbiology↗

bioRxiv2023

SwrA extends DegU over an UP element to activate flagellar gene expression in Bacillus subtilis

SwrA activates flagellar gene expression in Bacillus subtilis to increase the frequency of motile cells in liquid and elevate flagellar density to enable swarming over solid surfaces. Here we use ChIP-seq to show that SwrA interacts with many sites on the chromosome in a manner that depends on the response regulator DegU. We identify a DegU-specific inverted repeat DNA sequence and show that SwrA synergizes with phosphorylation to increase DegU DNA binding affinity. We further show that SwrA increases the size of the DegU footprint expanding the region bound by DegU towards the promoter. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription more that could be explained by deactivation. We conclude that SwrA/DegU forms a heteromeric complex that enables both remote binding and interaction between the activator and RNA polymerase in the context of an interceding UP element. We speculate that multimeric activators that resolve cis-element spatial conflicts are common in bacteria and likely act on flagellar biosynthesis loci and other long operons of other multi-subunit complexes. IMPORTANCEIn Bacteria, the sigma subunit of RNA polymerase recognizes specific DNA sequences called promoters that determine where gene transcription begins. Some promoters also have sequences immediately upstream called an UP element that is bound by the alpha subunit of RNA polymerase and is often necessary for transcription. Finally, promoters may be activated by transcription factors that bind DNA specific sequences and help recruit RNA polymerase to weak promoter elements. Here we show that the promoter for the 32 gene long flagellar operon in Bacillus subtilis requires an UP element and is activated by a heteromeric transcription factor of DegU and SwrA. Our evidence suggests that SwrA oligomerizes DegU over the DNA to allow RNA polymerase to interact with DegU and the UP element simultaneously. Heteromeric activator complexes are known but poorly-understood in bacteria and we speculate they may be needed to resolve spatial conflicts in the DNA sequence.

microbiology↗

bioRxiv2023

The alternative sigma factor SigN of Bacillus subtilis is intrinsically toxic

Sigma factors bind and direct the RNA polymerase core to specific promoter sequences and alternative sigma factors direct transcription of different regulons of genes. Here, we study the pBS32 plasmid-encoded sigma factor SigN of Bacillus subtilis to determine how it contributes to DNA damage-induced cell death. We find that SigN causes cell death when expressed at high level and does so in the absence of its regulon suggesting it is intrinsically toxic. One way toxicity was relieved was by curing the pBS32 plasmid, which eliminated a positive feedback loop that lead to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally-encoded transcriptional repressor protein AbrB and derepressing a potent antisense transcript that antagonized SigN expression. We note that SigN exhibits a relatively high affinity for the RNA polymerase core, efficiently competing with the vegetative sigma factor SigA, suggesting that toxicity was due to the competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a potentially toxic sigma factor is unclear but SigN may be related to phage-like genes also encoded on pBS32. SIGNIFICANCEAlternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded SigN of Bacillus subtilis is activated by the DNA damage response and leads to cellular demise. Here we find that SigN impairs viability by hyper-accumulating and outcompeting the vegetative sigma factor for the RNA polymerase core. Why B. subtilis retains a plasmid with a deleterious alternative sigma factor is unknown.

microbiology↗

bioRxiv2023

Penicillin-binding protein redundancy in Bacillus subtilis enables growth during alkaline shock

Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Seemingly redundant proteins can be important for enabling an organism to cope with environmental stressors. We sought to evaluate the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of B. subtilis PBPs change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are preferred for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly redundant periplasmic enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/533529v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@bf28c7org.highwire.dtl.DTLVardef@e2e88org.highwire.dtl.DTLVardef@1112259org.highwire.dtl.DTLVardef@1e604ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Active bulging promotes biofilm formation in a bacterial swarm

Microbial communities such as biofilms are commonly found at interfaces. However, it is unclear how the physical environment of interfaces may contribute to the development and behavior of surface-associated microbial communities. Combining multi-mode imaging, single-cell tracking and numerical simulations, here we discovered that an interfacial process denoted as "active bulging" promotes biofilm formation. During this process, an initially two-dimensional layer of swarming bacteria spontaneously develops scattered liquid bulges; the bulges have a higher propensity to transit from motile to sessile biofilm state, presumably due to the enrichment of pre-existing immotile cells in the colony. We further demonstrate that the formation of liquid bulges can be controlled reversibly by manipulating the speed and local density of cells with light. Our findings reveal a unique physical mechanism of biofilm formation and provide a new strategy for biofilm patterning in engineered living materials as well as for directed self-assembly in active fluids.

biophysics↗

bioRxiv2022

Fatty acid synthesis knockdown promotes biofilm wrinkling and inhibits sporulation in Bacillus subtilis

Many bacterial species typically live in complex three-dimensional biofilms, yet little is known about systematic changes to gene function between non-biofilm and biofilm lifestyles. Here, we created a CRISPRi library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain 3610. We show that gene essentiality is largely conserved between liquid and surface growth and between two media. We developed an image analysis algorithm to quantify biofilm colony wrinkling, which identified strains with high or low levels of wrinkling that were uncorrelated with extracellular matrix gene expression. We also designed a high-throughput screen for sensitive quantification of sporulation efficiency and performed the first screens of sporulation during essential gene knockdown. We found that all basal knockdowns of essential genes were competent for sporulation in a sporulation-inducing medium, but certain strains exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. Knockdown of fatty acid synthesis increased wrinkling and inhibited sporulation. These results highlight the importance of essential genes in biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. Abstract ImportanceFor many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles and are held together by extracellular matrix. To examine how gene function varies between non-biofilm and biofilm growth, we created a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the model species Bacillus subtilis 3610, which can develop into a wrinkled biofilm structure or a spore capable of surviving harsh environments. This library enabled us to determine when gene essentiality depends on growth conditions. We also developed high-throughput assays and computational algorithms to identify essential genes involved in biofilm wrinkling and sporulation. Knockdown of fatty acid synthesis increased the density of wrinkles, and also inhibited sporulation in a medium with generally lower sporulation levels. These findings indicate that essential processes such as fatty acid synthesis can play important and multifaceted roles in bacterial development.

microbiology↗

bioRxiv2022

Identification of genes required for swarming motility in Bacillus subtilis using transposon mutagenesis and high-throughput sequencing (TnSeq)

Bacillus subtilis exhibits swarming motility, a flagellar-mediated form of surface motility. Here we use high-throughput transposon mutagenesis and sequencing (TnSeq) to identify candidate genes required for swarming. The TnSeq approach identified all of the known genes required for flagellar biosynthesis as well as nearly all of the previously reported regulators that promote swarming. Moreover, we identified an additional 36 genes that improve swarming and validated them individually. Among these, two mutants with severe defects were recovered including fliT, required for flagellar biosynthesis, and a gene of unknown function yolB, whose defect could not be attributed to a lack of flagella. Our work indicates that TnSeq is a powerful approach for the identification of genes required for motility that drives outward expansion of a colony on plates. IMPORTANCEIn TnSeq, transposons are randomly inserted throughout the chromosome at a population level, but insertions that disrupt genes of essential function cause strains that carry them to fall out of the population and appear under-represented at the sequence level. Here we apply TnSeq to the non-essential phenotype of motility in B. subtilis and spatially select for cells proficient in swarming. We find that insertions in nearly all genes previously identified as required for swarming are under-represented in TnSeq analysis, and we identify 36 additional genes that enhance swarming. We suggest that TnSeq is a powerful tool for the genetic analysis of motility and likely other non-lethal screens for which enrichment is strong.

microbiology↗

bioRxiv2021

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, an intense 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 inter-chromosome 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. Additionally, an Spx-independent pathway increased the activity of the divisome to reduce the length of the cytokinetic period. 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. IMPORTANCEBacteria 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 B. subtilis, cell division site selection is thought to depend on two systems called Min and Noc, 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, not due to a defect in FtsZ localization, but rather a failure in the maturation of the cell division machinery. To understand what happens when the division machinery fails to mature, suppressor mutations that bypass the need for Min, Noc, or both were selected. Some of the mutants activated the Spx stress response pathway while others appeared to directly enhance divisome activity.

microbiology↗