Active interface bulging in Bacillus subtilis swarms promotes self-assembly and biofilm formation.

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 multimode imaging, single-cell tracking, and numerical simulations, here, we found that activity-induced interface bulging promotes colony biofilm formation in Bacillus subtilis swarms presumably via segregation and enrichment of sessile cells in the bulging area. Specifically, the diffusivity of passive particles is ~50% lower inside the bulging area than elsewhere, which enables a diffusion-trapping mechanism for self-assembly and may account for the enrichment of sessile cells. We also uncovered a quasilinear relation between cell speed and surface-packing density that underlies the process of active interface bulging. Guided by the speed-density relation, we demonstrated reversible formation of liquid bulges by manipulating the speed and local density of cells with light. Over the course of development, the active bulges turned into striped biofilm structures, which eventually give rise to a large-scale ridge pattern. Our findings reveal a unique physical mechanism of biofilm formation at air-solid interface, which is pertinent to engineering living materials and directed self-assembly in active fluids.

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 poorly 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 expressed at higher levels 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. Time-lapse microscopy reveals that competent cells producing ComI are first permeabilized and then die. Based on these observations, we propose a new model for the mechanism of ComI in which response to competence activation leads to selective elimination of the competent subpopulation. IMPORTANCE: Natural 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 the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.

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 maintenance, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Apparently-redundant proteins can be important for enabling an organism to cope with environmental stressors. In this study, we evaluated the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of PBPs in B. subtilis 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 favored 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.IMPORTANCEMicrobes adapt to ever-changing environments and thrive over a vast range of conditions. While bacterial genomes are relatively small, significant portions encode for "redundant" functions. Apparent redundancy is especially pervasive in bacterial proteins that reside outside of the inner membrane. While conditions within the cytoplasm are carefully controlled, those of the periplasmic space are largely determined by the cell's exterior environment. As a result, proteins within this environmentally exposed region must be capable of functioning under a vast array of conditions, and/or there must be several similar proteins that have evolved to function under a variety of conditions. This study examines the activity of a class of enzymes that is essential in cell wall construction to determine if individual proteins might be adapted for activity under particular growth conditions. Our results indicate that a subset of these proteins are preferred for growth under alkaline conditions, while others are readily dispensable.

Functional Regulators of Bacterial Flagella.

Bacteria move by a variety of mechanisms, but the best understood types of motility are powered by flagella (72). Flagella are complex machines embedded in the cell envelope that rotate a long extracellular helical filament like a propeller to push cells through the environment. The flagellum is one of relatively few biological machines that experience continuous 360° rotation, and it is driven by one of the most powerful motors, relative to its size, on earth. The rotational force (torque) generated at the base of the flagellum is essential for motility, niche colonization, and pathogenesis. This review describes regulatory proteins that control motility at the level of torque generation.

Concordance between ER, PR, Ki67, and HER2-low expression in breast cancer by MammaTyper RT-qPCR and immunohistochemistry: implications for the practising pathologist.

BACKGROUND: There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). METHODS: We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. RESULTS: RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). CONCLUSION: RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation.

Morphological and molecular changes of oestrogen receptor-positive breast cancer following bridging endocrine therapy: a United Kingdom multicentre study.

AIMS: Standard neoadjuvant endocrine therapy (NAET) is used for 6-9 months to downstage hormone-receptor-positive breast cancer. Bridging ET was introduced during the COVID-19 pandemic to delay surgical intervention. There are no data in the literature on the effect of short course therapy on tumour response. We aimed to analyse the effect of bridging ET and validate the previously proposed neoadjuvant ET pathological reporting criteria. METHODS AND RESULTS: This was a multicentre cohort of 256 patients who received bridging ET between March and October 2020. Assessment of paired pre- and post-NAET hormone receptors and HER2 and posttherapy Ki67 expression was done. The median duration of NAET was 45 days. In all, 86% of cases achieved partial pathological response and 9% showed minimal residual disease. Histological response to ET was observed from as early as day 6 posttherapy. Central scarring was noted in 32.8% of cases and lymphocytic infiltrate was seen in 43.4% of cases. Significant changes associated with the duration of ET were observed in tumour grade (21%), with downgrading identified in 12% of tumours (P < 0.001), progesterone receptor (PR) expression with switch to PR-negative status in 26% of cases (P < 0.001), and HER2 status with a switch from HER2-low to HER2-negative status in 32% of cases (P < 0.001). The median patient survival was 475 days, with an overall survival rate of 99.6%. CONCLUSIONS: Changes characteristic of tumour regression and significant changes in PR and HER2 occurred following a short course of NAET. The findings support biomarker testing on pretreatment core biopsies and retesting following therapy.

Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence.

Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we examined immune cell infiltration in 230 breast cancer samples, emphasizing diverse ethnic populations. Leveraging tissue microarrays (TMAs) and core samples, we applied multiplex immunofluorescence (mIF) to dissect immune cell subtypes across TME regions. Our analysis revealed distinct immune cell distribution patterns, particularly enriched in aggressive molecular subtypes triple-negative and HER2-positive tumors. We observed significant correlations between immune cell abundance and key clinicopathological parameters, including tumor size, lymph node involvement, and patient overall survival. Notably, immune cell location within different TME regions showed varying correlations with clinicopathologic parameters. Additionally, ethnicities exhibited diverse distributions of cells, with certain ethnicities showing higher abundance compared to others. In TMA samples, patients of Chinese and Caribbean origin displayed significantly lower numbers of B cells, TAMs, and FOXP3-positive cells. These findings highlight the intricate interplay between immune cells and breast cancer progression, with implications for personalized treatment strategies. Moving forward, integrating advanced imaging techniques, and exploring immune cell heterogeneity in diverse ethnic cohorts can uncover novel immune signatures and guide tailored immunotherapeutic interventions, ultimately improving breast cancer management.

Profiling of Tumour-Infiltrating Lymphocytes and Tumour-Associated Macrophages in Ovarian Epithelial Cancer-Relation to Tumour Characteristics and Impact on Prognosis.

Early evidence suggests a strong impact of tumour-infiltrating lymphocytes (TILs) on both the prognosis and clinical behaviour of ovarian cancer. Proven associations, however, have not yet translated to successful immunotherapies and further work in the field is urgently needed. We aimed to analyse the tumour microenvironment of a well-characterised cohort of ovarian cancer samples. Tumour markers were selected owing to their comparative underrepresentation in the current literature. Paraffin-embedded, formalin-fixed tumour tissue blocks of 138 patients representative of the population and including early stage disease were identified, stained for CD3, CD20, CD68 and CD163 and analysed for both the stromal and intertumoral components. Data were statistically analysed in relation to clinical details, histological subtype, borderline vs. malignant status, survival and management received. Mean stromal CD3, total CD3 count, mean stromal CD20 and total CD20 count all correlated negatively with survival. Malignant ovarian tumours consistently demonstrated significantly higher infiltration of all analysed immune cells than borderline tumours. Assessment of the stromal compartment produced a considerably higher proportion of significant results when compared to the intra-tumoural infiltrates. Customary assessment of solely intra-tumoural cells in advanced stage disease patients undergoing primary debulking surgery should be challenged, with recommendations for future scoring systems provided.

Pathological Changes Following Neoadjuvant Endocrine Therapy (NAET): A Multicentre Study of 391 Breast Cancers.

Oestrogen receptor (ER)-positive breast cancer (BC) is generally well responsive to endocrine therapy. Neoadjuvant endocrine therapy (NAET) is increasingly being used for downstaging ER-positive tumours. This study aims to analyse the effect of NAET on a well-characterised cohort of ER-positive BC with particular emphasis on receptor expression. This is a retrospective United Kingdom (UK) multicentre study of 391 patients who received NAET between October 2012 and October 2020. Detailed analyses of the paired pre- and post-NAET morphological changes and hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression were performed. The median duration of NAET was 86 days, with median survival and overall survival rates of 380 days and 93.4%, respectively. A total of 90.3% of cases achieved a pathological partial response, with a significantly higher rate of response in the HER2-low cancers. Following NAET, BC displayed some pathological changes involving the tumour stroma including central scarring and an increase in tumour infiltrating lymphocytes (TILs) and tumour cell morphology. Significant changes associated with the duration of NAET were observed in tumour grade (30.6% of cases), with downgrading identified in 19.3% of tumours (p < 0.001). The conversion of ER status from positive to low or negative was insignificant. The conversion of progesterone receptor (PR) and HER2 status to negative status was observed in 31.3% and 38.1% of cases, respectively (p < 0.001). HER2-low breast cancer decreased from 63% to 37% following NAET in the paired samples. Significant morphological and biomarker changes involving PR and HER2 expression occurred following NAET. The findings support biomarker testing on pre-treatment core biopsies and post-treatment residual carcinoma.

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 levels 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 led to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally encoded transcriptional repressor protein AbrB, thereby derepressing a potent antisense transcript that antagonized SigN expression. SigN efficiently competed with the vegetative sigma factor SigA in vitro, and SigN accumulation in the absence of positive feedback reduced SigA-dependent transcription suggesting that toxicity may be due to competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a toxic sigma factor is unclear but SigN may function in host-inhibition during lytic conversion, as phage lysogen genes are also encoded on pBS32. IMPORTANCE Alternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded alternative sigma factor SigN of Bacillus subtilis however, 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.

SmiA is a hybrid priming/scaffolding adaptor for the LonA protease in Bacillus subtilis.

Regulatory proteolysis targets properly folded clients via a combination of cis-encoded degron sequences and trans-expressed specificity factors called adaptors. SmiA of Bacillus subtilis was identified as the first adaptor protein for the Lon family of proteases, but the mechanism of SmiA-dependent proteolysis is unknown. Here, we develop a fluorescence-based assay to measure the kinetics of SmiA-dependent degradation of its client SwrA and show that SmiA-SwrA interaction and the SwrA degron were both necessary, but not sufficient, for proteolysis. Consistent with a scaffolding adaptor mechanism, we found that stoichiometric excess of SmiA caused substrate-independent inhibition of LonA-dependent turnover. Furthermore, SmiA was strictly required even when SwrA levels were high suggesting that a local increase in substrate concentration mediated by the scaffold was not sufficient for proteolysis. Moreover, SmiA function could not be substituted by thermal denaturation of the substrate, consistent with a priming adaptor mechanism. Taken together, we conclude that SmiA functions via a mechanism that is a hybrid between scaffolding and priming models.

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 transposon mutagenesis and sequencing (TnSeq) to perform a high-throughput screen for candidate genes required for swarming. The TnSeq approach identified all of the known genes required for flagellar biosynthesis and 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. In addition to discovering additional genes required for B. subtilis swarming, our work validates TnSeq as a powerful approach for comprehensively identifying genes important for nonessential processes such as colony expansion on plates. IMPORTANCE In 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 underrepresented at the sequence level. Here, we apply TnSeq to the nonessential 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 underrepresented in TnSeq analysis, and we identify 36 additional genes that enhance swarming. We demonstrate that TnSeq is a powerful tool for the genetic analysis of motility and likely other nonlethal screens for which enrichment is strong.

RnhP is a plasmid-borne RNase HI that contributes to genome maintenance in the ancestral strain Bacillus subtilis NCIB 3610.

RNA-DNA hybrids form throughout the chromosome during normal growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, and increase mutagenesis. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for the loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore, resulting in SOS induction and inhibition of cell division. We conclude that all three functional RNase H enzymes are present in B. subtilis NCIB 3610 and that the plasmid-encoded RNase HI contributes to chromosome stability, while the chromosomally encoded RNase HIII is important for chromosome stability and plasmid hyper-replication.

Bacterial Swarmers Enriched During Intestinal Stress Ameliorate Damage.

BACKGROUND AND AIMS: Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. This study aims to define a role for bacterial swarmers in amelioration of intestinal stress. METHODS: We developed a polymicrobial plate agar assay to detect swarming and screened mice and humans with intestinal stress and inflammation. From chemically induced colitis in mice, as well as humans with inflammatory bowel disease, we developed techniques to isolate the dominant swarmers. We developed swarm-deficient but growth and swim-competent mutant bacteria as isogenic controls. We performed bacterial reinoculation studies in mice with colitis, fecal 16S, and meta-transcriptomic analyses, as well as in vitro microbial interaction studies. RESULTS: 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, in contrast to their mutant strains, abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family of Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal meta-transcriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants; however, because SM3, but not mutants, induced S24-7 in vivo, we concluded that swarming plays an essential role in disseminating SM3 in vivo. CONCLUSIONS: Overall, our work identified a new but counterintuitive paradigm in which intestinal stress allows for the emergence of swarming bacteria; however, these bacteria act to heal intestinal inflammation.

The structure and regulation of flagella in Bacillus subtilis.

Bacterial flagellar motility is among the most extensively studied physiological systems in biology, but most research has been restricted to using the highly similar Gram-negative species Escherichia coli and Salmonella enterica. Here, we review the recent advances in the study of flagellar structure and regulation of the distantly related and genetically tractable Gram-positive bacterium Bacillus subtilis. B. subtilis has a thicker layer of peptidoglycan and lacks the outer membrane of the Gram-negative bacteria; thus, not only phylogenetic separation but also differences in fundamental cell architecture contribute to deviations in flagellar structure and regulation. We speculate that a large number of flagella and the absence of a periplasm make B. subtilis a premier organism for the study of the earliest events in flagellar morphogenesis and the type III secretion system. Furthermore, B. subtilis has been instrumental in the study of heterogeneous gene transcription in subpopulations and of flagellar regulation at the translational and functional level.

Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P.

Translation elongation factor P (EF-P) alleviates ribosome pausing at a subset of motifs encoding consecutive proline residues, and is required for growth in many organisms. Here we show that Bacillus subtilis EF-P also alleviates ribosome pausing at sequences encoding tandem prolines and ribosomes paused within several essential genes without a corresponding growth defect in an efp mutant. The B. subtilis efp mutant is instead impaired for flagellar biosynthesis which results in the abrogation of a form of motility called swarming. We isolate swarming suppressors of efp and identify mutations in 8 genes that suppressed the efp mutant swarming defect, many of which encode conserved ribosomal proteins or ribosome-associated factors. One mutation abolished a translational pause site within the flagellar C-ring component FliY to increase flagellar number and restore swarming motility in the absence of EF-P. Our data support a model wherein EF-P-alleviation of ribosome pausing may be particularly important for macromolecular assemblies like the flagellum that require precise protein stoichiometries.

CwlQ Is Required for Swarming Motility but Not Flagellar Assembly in Bacillus subtilis.

Lytic 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 must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown.IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.

Molecular and Cell Biological Analysis of SwrB in Bacillus subtilis.

Swarming motility is flagellum-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 substoichiometrically as it localizes as puncta at the membrane in numbers fewer than those 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 colocalized 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. IMPORTANCE Type III secretion systems (T3SSs) 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. Moreover, 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 of 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.

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.

The Large pBS32/pLS32 Plasmid of Ancestral Bacillus subtilis.

The ancestral strain of Bacillus subtilis NCIB3610 (3610) bears a large, low-copy-number plasmid, called pBS32, that was lost during the domestication of laboratory strain derivatives. Selection against pBS32 may have been because it encodes a potent inhibitor of natural genetic competence (ComI), as laboratory strains were selected for high-frequency transformation. Previous studies have shown that pBS32 and its sibling, pLS32 in Bacillus subtilis subsp. natto, encode a replication initiation protein (RepN), a plasmid partitioning system (AlfAB), a biofilm inhibitor (RapP), and an alternative sigma factor (SigN) that can induce plasmid-mediated cell death in response to DNA damage. Here, we review the literature on pBS32/pLS32, the genes found on it, and their associated phenotypes.