Biology subjects

Kearns, D. B.

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

29 records · Page 2Linked to original sources

bioRxiv2021

Molecular and cell biological analysis of SwrB in Bacillus subtilis

Swarming motility is flagellar-mediated movement over a solid surface and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion but the mechanism by which it might perform this function is unknown. Here we show that SwrB likely acts sub-stoichiometrically as it localizes as puncta at the membrane in numbers fewer than that of flagellar basal bodies. Moreover the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely co-localized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs. IMPORTANCEType III secretion systems (T3SS) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moroever, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here we explore a poorly understood activator the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.

microbiology↗

bioRxiv2021

CwlQ is required for swarming motility but not flagellar assembly in Bacillus subtilis

Hydrolytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan (PG) integrity must be unfailingly stable to preserve cell integrity but must also be dynamically remodeled for the cell grow, divide and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG and the insertion of which is aided by localized activity of a dedicated PG hydrolase in Gram-negative bacteria. To date, there is no known dedicated hydrolase in Gram-positive bacteria for insertion of flagella and here we take a reverse-genetic candidate-gene approach to find that cells mutated for the lytic transglycosylase CwlQ exhibited a severe defect in flagellar dependent swarming motility. We show that CwlQ required its active site to promote swarming, was expressed by the motility sigma factor SigD, and was secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells mutated for CwlQ remained proficient for flagellar biosynthesis even when mutated in combination with four other hydrolases related to motility (LytC, LytD, LytF, and CwlO). The PG hydrolase essential for flagellar synthesis in B. subtilis, if any, remains unknown.

microbiology↗

bioRxiv2020

Noc corrals migration of FtsZ protofilaments during cytokinesis in Bacillus subtilis

Bacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In B. subtilis, the DNA- and membrane-binding Noc protein is thought to mediate nucleoid occlusion to prevent FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent FtsZ formation over the chromosome or control cell division site selection. Instead, Noc inhibits migration of FtsZ protofilaments from one FtsZ structure to the next. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the Noc mutant phenotype can be suppressed by ZapA overexpression. Thus, Noc maintains a high local concentration of FtsZ to stabilize FtsZ rings during cytokinesis. IMPORTANCEIn bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery, and Z-ring formation is inhibited over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis, nucleoid occlusion has been reported to be mediated by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid.

microbiology↗

bioRxiv2020

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

NusA and NusG are transcription elongation factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor, the role of NusG in this process had not been explored. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, {Delta}nusG, and NusA depletion {Delta}nusG 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 1,400 identified intrinsic terminators. The loss of both proteins leads to global misregulation of gene expression. 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. Moreover, the loss of NusG results in flagella and swimming motility defects.

microbiology↗

bioRxiv2020

Contact with the CsrA core is required for allosteric inhibition by FliW in Bacillus subtilis

The RNA-binding protein CsrA is a post-transcriptional regulator that is encoded in genomes throughout the bacterial phylogeny. In the gamma-proteobacteria, 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, that non-competitively inhibits CsrA activity but the precise mechanism of antagonism is unclear. Here we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition that fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues that surround the critical regulatory CsrA residue N55 and abolished CsrA interaction. Two loss-of-function alleles however mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of these two alleles reversed charge at what appeared to be a salt bridge with the CsrA core region, charge reversal of the CsrA partner residue phenocopied the FliW allele, and charge reversal of both residues simultaneously restored antagonism. We propose a model in which initial interaction between FliW and CsrA is necessary but not sufficient for antagonism which also requires salt bridge formation with, and deformation of, the CsrA core domain to allosterically abolish RNA binding activity. SummaryCsrA is a small dimeric protein that binds RNA and is one of the few known examples of transcript-specific translational regulators in bacteria. A protein called FliW binds to and antagonizes CsrA; despite having a high-resolution three-dimensional structure of the FliW-CsrA complex, the mechanism of non-competitive 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.

microbiology↗

bioRxiv2019

Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 {beta}-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for {beta}-lactams and speculated to be involved in resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

microbiology↗

bioRxiv2019

The Min system disassembles FtsZ foci and inhibits polar peptidoglycan remodeling in Bacillus subtilis

A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis. Consistent with previous reports, we find that after division FtsZ rings remain at the cell pole, and FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in maintenance of multiple Z-rings simultaneously, that are restricted by competition for newly synthesized FtsZ. Whereas the parameters of FtsZ dynamics change in the minD mutant, the overall cell cycle remains the same, albeit with elongated cells necessary to accumulate a threshold concentration of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant suggesting that not only is the Min system important for recycling FtsZ but also may have a secondary role in the regulation of peptidoglycan remodeling. IMPORTANCEMany bacteria grow and divide by binary fission in which a mothercell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyze FtsZ dynamics during growth and find that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the "inert" property of cell walls at the poles.

microbiology↗

bioRxiv2019

TnFLX: a third-generation mariner-based transposon system for Bacillus subtilis

Random transposon mutagenesis is a powerful genetic tool to answer fundamental biological questions in an unbiased approach. Here, we introduce an improved mariner-based transposon system with higher stability, and with versatile applications. We take advantage of the lower frequency of unintended recombination during vector construction and propagation in a low copy number system in E. coli to improve construct integrity. We generated a variety of transposons allowing for gene disruption or artificial overexpression each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance flexibility of future transposon modification and application in Bacillus and other organisms.\n\nImportanceThe optimization of transposase encoding vectors in terms of stability during cloning and propagation is crucial for the reliable application of this system in any host organism. With an increased number of antibiotic resistance markers and the possibility to detect translational activity, the TnFLX transposon system will significantly help the implication of forward genetic methods in the field of cellular biology.

microbiology↗

bioRxiv2019

Bacterial Hyperswarming as a Protective Response to Intestinal Stress

Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. Here, we report for the first time that bacterial swarmers are associated with protection against intestinal inflammation. We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers contrast to their respective swarming-deficient, but swimming-competent isogenic strains abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family, Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal metatranscriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants conjecturing that bacterial swarming in vivo might influence SM3s access to S24-7 in the intestines. Overall, our work identifies a new paradigm in which intestinal stress allows for the emergence of swarming bacteria, which can counterintuitively heal intestinal inflammation.

microbiology↗

bioRxiv2019

Biosurfactant production maintains viability in anoxic conditions by depolarizing the membrane in Bacillus subtilis

The presence or absence of oxygen in the environment is a strong effector of cellular metabolism and physiology. Like many eukaryotes and some bacteria, Bacillus subtilis is an obligate aerobe that primarily utilizes oxygen during respiration to generate ATP. Despite the importance of oxygen for B. subtilis survival, we know little about how oxygen is consumed during growth and how populations respond to shifts in oxygen availability. Here, we find that when oxygen was depleted from stationary phase cultures [~]90% of B. subtilis 3610 cells died and lysed due to autolysin activity; the remaining cells maintained colony-forming ability. Interestingly, the domesticated 168 strain maintained a higher optical density than 3610 during oxygen depletion due to the formation of cell-wall-less protoplasts, but the remaining, rod-shaped cells were >100-fold less viable than 3610. We discovered that the higher viability in 3610 was due to its ability to produce the antibacterial compound surfactin, as surfactin addition rescued 168 viability and also increased yield in aerobic growth. We further demonstrate that surfactin strongly depolarizes the B. subtilis membrane, and that other known membrane-potential disruptors restore viability to 168. These findings highlight the importance of surfactin for survival during oxygen-depleted conditions and demonstrate that antimicrobials normally considered harmful can instead benefit cells in stressful conditions when the terminal electron acceptor in respiration is limiting.

microbiology↗

bioRxiv2019

Transcriptional Regulation and Mechanism of SigN (ZpdN), a pBS32 encoded Sigma Factor

Laboratory strains of Bacillus subtilis encodes as many as 16 alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility. The ancestral strain of B. subtilis also encodes an additional sigma factor homolog, ZpdN, not found in lab strains due to it being encoded on the large, low copy number plasmid pBS32 that was lost during domestication. DNA damage triggers pBS32 hyper-replication and cell death in a manner that depends on ZpdN but how ZpdN mediates these effects was unknown. Here we show that ZpdN is a bona fide sigma factor that can direct RNA polymerase to transcribe ZpdN-dependent genes and we rename ZpdN to SigN accordingly. Rend-seq analysis was used to determine the SigN regulon on pBS32, and the 5 ends of transcripts were used to predict the SigN consensus sequence. Finally, we characterize the regulation of SigN itself, and show that it is transcribed by at least three promoters: PsigN1, a strong SigA-dependent LexA-repressed promoter, PsigN2, a weak SigA-dependent constitutive promoter, and PsigN3, a SigN-dependent promoter. Thus, in response to DNA damage LexA is derepressed, SigN is expressed and then experiences positive feedback. How cells die in a pBS32-dependent manner remains unknown, but we predict that death is the product of expressing one or more genes in the SigN regulon.\n\nIMPORTANCESigma factors are utilized by bacteria to control and regulate gene expression. Extra cytoplasmic function sigma factors are activated during times of stress to ensure the survival of the bacterium. Here, we report the presence of a sigma factor that is encoded on a plasmid that leads to cellular death after DNA damage.

microbiology↗