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.

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.

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.

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.

Thrombectomy of the Profunda Femoral Vein in Iliofemoral Deep Venous Thrombosis Using an Antegrade Popliteal Approach.

PURPOSE: Residual or undertreated inflow disease is a major cause of stent occlusion following endovascular thrombectomy for iliofemoral deep venous thrombosis (DVT). The profunda femoral vein (PFV) is an important inflow vessel alongside the femoral vein but is traditionally challenging to treat via an antegrade popliteal approach. This technical note describes a novel approach for PFV clearance in iliofemoral thrombectomy via the popliteal vein. MATERIALS AND METHODS: Eight patients underwent PFV clearance as part of iliofemoral DVT thrombectomy via an antegrade popliteal approach. In seven patients, a popliteal-profunda communicating vessel was identified permitting PFV access and thrombectomy. In one patient, a popliteal-profunda communicator was not identified and an 'up and over' approach via the femoral bifurcation from the same popliteal access was utilised. Thrombectomy was performed using the Inari ClotTriever device or Penumbra's Indigo system. RESULTS: Technical success in PFV thrombectomy was 100%. Six patients (75%) underwent stenting for an iliac stenotic lesion or May Thurner compression point. At the four-week ultrasound follow-up, the pelvic iliofemoral segment was patent in 7 patients (87.5%). The PFV was patent in 7 patients (87.5%) whereas the FV was only patent in 4 patients (50%). One patient underwent reintervention for iliofemoral stent occlusion. No PFV injury occurred and no post-procedure profunda reflux was identified. CONCLUSION: PFV clearance can be achieved via an antegrade popliteal approach in iliofemoral thrombectomy to optimise inflow, negating the need for alternative or additional venous access. PFV may maintain upstream iliofemoral vein patency even with an occluded femoral vein. LEVEL OF EVIDENCE: Level 4, Case Series.

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.

SwrA-mediated Multimerization of DegU and an Upstream Activation Sequence Enhance Flagellar Gene Expression in Bacillus subtilis.

The earliest genes in bacterial flagellar assembly are activated by narrowly-conserved proteins called master regulators that often act as heteromeric complexes. A complex of SwrA and the response-regulator transcription factor DegU is thought to form the master flagellar regulator in Bacillus subtilis but how the two proteins co-operate to activate gene expression is poorly-understood. Here we find using ChIP-Seq that SwrA interacts with a subset of DegU binding sites in the chromosome and does so in a DegU-dependent manner. Using this information, we identify a DegU-specific inverted repeat DNA sequence in the Pflache promoter region and show that SwrA synergizes with DegU phosphorylation to increase binding affinity. We further demonstrate that the SwrA/DegU footprint extends from the DegU binding site towards the promoter, likely through SwrA-induced DegU multimerization. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription by disrupting a previously-unrecognized upstream activation sequence (UAS). Thus, the SwrA-DegU heteromeric complex likely enables both remote binding and interaction between the activator and RNA polymerase. Small co-activator proteins like SwrA may allow selective activation of subsets of genes where activator multimerization is needed. Why some promoters require activator multimerization and some require UAS sequences is unknown.

ProGlide entrapment of the occlusive balloon during repair of an iatrogenic subclavian artery injury.

The insertion of any central venous catheter (CVC) is associated with a risk of damage to neurovascular structures, pneumothorax, cardiac arrhythmias, and infection1. Unintentional arterial puncture remains rare, occurring in 6.3-9.4% of attempted internal jugular vein (IJV) catheterisation and 3.1-4.9% of attempted subclavian vein catheterisation2. We present a previously undocumented complication encountered while utilising the Perclose ProGlide device in the case of a 59-year-old male who underwent right subclavian artery closure following the accidental insertion of a 14Fr Vascath into the right subclavian artery. This was performed using two ProGlide devices and one Angio-Seal device. Following deployment of the ProGlide devices, an uninflated balloon passed into the subclavian artery as a precaution, but not used, was removed. One of the ProGlide devices became dislodged having been deployed into the balloon, threatening haemostasis.

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.

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. IMPORTANCE: In 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.

Immunohistochemical profiling of the tumour microenvironment in borderline and malignant ovarian tumours in young women.

Ovarian cancer is a major cause of cancer-related deaths in women. Our previous study highlighted the interaction between cancer cells and the host immune response in solid cancers. The present study aimed to analyse the proportion, density and distribution of T and B lymphocytes within the tumour and surrounding stroma, and their prognostic significance in young women with borderline and malignant ovarian surface epithelial tumours. Full clinicopathological and outcome data were collected for 57 women aged <50 years diagnosed between January 2010 and December 2015. Representative tumour sections were stained for CD3 (T cells) and CD20 (B cells) and tumour-infiltrating lymphocytes (TILs) were scored following the TILs Working Group Recommendations and described as stromal, intra-tumoural, lymphoid aggregates and touching lymphocytes. Data were statistically analysed and the association with clinicopathological variables was assessed. The median age was 41 years and the most common histological type was serous carcinoma (n=21). The risk of malignancy index was a significant predictor of ovarian cancer diagnosis (P<0.05). A total of 15 out of 34 patients with cancer died. There was significantly greater stromal infiltration of CD3 and CD20 TILs (P=0.01 and P=0.03, respectively) and higher intratumoral CD20 expression in ovarian epithelial cancers compared with borderline tumours. The highest CD3 stroma count and density were observed in serous carcinoma, which also exhibited the highest numbers of CD3 and CD20 aggregates. There was no statistically significant difference between touching lymphocytes and tumour histological subtype. There was no significant association between TIL expression and patient survival. The count, distribution and density of T and B lymphocytes in ovarian tumours varied depending on tumour type and invasiveness. Their topographic distribution within the tumour and surrounding stroma did not impact prognosis in young women with ovarian cancer. TIL analysis in an older age group of women with ovarian tumours is ongoing to determine its potential prognostic significance.

A rare case of disseminated genitourinary tract tuberculosis complicated by emphysematous prostatitis and seminal vesicle abscess.

Urogenital tuberculosis (UGTB) can affect the entire urinary tract including the kidneys, ureters (strictures), urinary bladder, prostate in addition to involving reproductive tracts. In modern day practice, both ultrasound and cross-sectional imaging play an important role in the radiological diagnosis of UGTB. The sequalae of untreated UGTB is morbid and can lead to end-stage renal failure, infertility, and life-threatening systemic infection. UGTB is less commonly observed in developed countries and may mimic other pathologies including malignancy. Thus, it is important that radiologists consider the differential diagnosis early, particularly individuals with risk factors such as travel to endemic regions, to allow optimal treatment and ensure best prognostic outcomes. UGTB can typically be managed by Infectious Disease clinicians with multidrug chemotherapy. We have presented a case of microbiologically proven extrapulmonary tuberculosis (TB) predominantly involving the genitourinary tract. The response to TB agents and lack of evidence of co-infection with another organism, might suggest this as the first published case of emphysematous tuberculous prostatitis. Emphysematous prostatitis is indicative of a gas-forming infection of the prostate, and is associated with abscess formation in the vast majority of case and is an easily identified radiological feature on CT. It is not a well-recognised feature of Mycobacterium tuberculosis infection and thus microbiological diagnosis should be sought to confirm the diagnosis.

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. SIGNIFICANCE: Alternative 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.

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.

Corner Flows Induced by Surfactant-Producing Bacteria Bacillus subtilis and Pseudomonas fluorescens.

A mechanistic understanding of bacterial spreading in soil, which has both air and water in angular pore spaces, is critical to control pathogenic contamination of soil and to design bioremediation projects. A recent study (J. Q. Yang, J. E. Sanfilippo, N. Abbasi, Z. Gitai, et al., Proc Natl Acad Sci U S A 118:e2111060118, 2021, https://doi.org/10.1073/pnas.2111060118) shows that Pseudomonas aeruginosa can self-generate flows along sharp corners by producing rhamnolipids, a type of biosurfactants that change the hydrophobicity of solid surfaces. We hypothesize that other types of biosurfactants and biosurfactant-producing bacteria can also generate corner flows. Here, we first demonstrate that rhamnolipids and surfactin, biosurfactants with different chemical structures, can generate corner flows. We identify the critical concentrations of these two biosurfactants to generate corner flow. Second, we demonstrate that two common soil bacteria, Pseudomonas fluorescens and Bacillus subtilis (which produce rhamnolipids and surfactin, respectively), can generate corner flows along sharp corners at the speed of several millimeters per hour. We further show that a surfactin-deficient mutant of B. subtilis cannot generate corner flow. Third, we show that, similar to the finding for P. aeruginosa, the critical corner angle for P. fluorescens and B. subtilis to generate corner flows can be predicted from classic corner flow theories. Finally, we show that the height of corner flows is limited by the roundness of corners. Our results suggest that biosurfactant-induced corner flows are prevalent in soil and should be considered in the modeling and prediction of bacterial spreading in soil. The critical biosurfactant concentrations we identify and the mathematical models we propose will provide a theoretical foundation for future predictions of bacterial spreading in soil. IMPORTANCE The spread of bacteria in soil is critical in soil biogeochemical cycles, soil and groundwater contamination, and the efficiency of soil-based bioremediation projects. However, the mechanistic understanding of bacterial spreading in soil remains incomplete due to a lack of direct observations. Here, we simulate confined spaces of hydrocarbon-covered soil using a transparent material with similar hydrophobicity and visualize the spread of two common soil bacteria, Pseudomonas fluorescens and Bacillus subtilis. We show that both bacteria can generate corner flows at the velocity of several millimeters per hour by producing biosurfactants, soap-like chemicals. We provide quantitative equations to predict the critical corner angle for bacterial corner flow and the maximum distance of the corner spreading. We anticipate that bacterial corner flow is prevalent because biosurfactant-producing bacteria and angular pores are common in soil. Our results will help improve predictions of bacterial spreading in soil and facilitate the design of soil-related bioremediation projects.

Fatty Acid Synthesis Knockdown Promotes Biofilm Wrinkling and Inhibits Sporulation in Bacillus subtilis.

Many bacterial species typically live in complex three-dimensional biofilms, yet much remains unknown about differences in essential processes between nonbiofilm and biofilm lifestyles. Here, we created a CRISPR interference (CRISPRi) library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain NCIB 3610 and investigated growth, biofilm colony wrinkling, and sporulation phenotypes of the knockdown library. First, we showed that gene essentiality is largely conserved between liquid and surface growth and between two media. Second, we quantified biofilm colony wrinkling using a custom image analysis algorithm and found that fatty acid synthesis and DNA gyrase knockdown strains exhibited increased wrinkling independent of biofilm matrix gene expression. Third, we designed a high-throughput screen to quantify sporulation efficiency after essential gene knockdown; we found that partial knockdowns of essential genes remained competent for sporulation in a sporulation-inducing medium, but knockdown of essential genes involved in fatty acid synthesis exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. We conclude that a subset of essential genes are particularly important for biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. IMPORTANCE For many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles held together by extracellular matrix. To examine how essential gene function varies between vegetative growth and the developmental states of biofilm formation and sporulation, we created and screened a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the biofilm-capable Bacillus subtilis strain 3610. High-throughput assays and computational algorithms identified a subset of essential genes involved in biofilm wrinkling and sporulation and indicated that fatty acid synthesis plays important and multifaceted roles in bacterial development.

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.

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.

TnFLXopen: Markerless Transposons for Functional Fluorescent Fusion Proteins and Protein Interaction Prediction.

Fluorescence microscopy of cells expressing proteins translationally linked to a fluorophore can be a powerful tool to investigate protein localization dynamics in vivo. One major obstacle to reliably analyze biologically relevant localization is the construction of a fusion protein that is both fluorescent and functional. Here, we develop a strategy to construct fluorescent fusions at theoretically any location in the protein by using TnFLXopen random transposon mutagenesis to randomly insert a gene encoding a fluorescent protein. Moreover, insertions within a target gene are enriched by an inducible gene-trap strategy and selection by fluorescence activated cell sorting. Using this approach, we isolate a variety of fluorescent fusions to FtsZ that exhibit ring-like localization and a fusion to the flagellar stator protein that both is functional for supporting motility and localizes as fluorescent puncta. Finally, we further modify TnFLXopen to insert the coding sequence for the C-terminal half of mVenus for use in bimolecular fluorescence complementation (BiFC) and the in vivo detection of protein-protein interaction candidates. As proof-of-concept, the DivIVA polar scaffolding protein was fused to the N terminus of mVenus, the C terminus of mVenus was delivered by transposition, and a combination of fluorescence activated cell sorter (FACS) sorting and whole-genome sequencing identified the known self-interaction of DivIVA as well as other possible candidate interactors. We suggest that the FACS selection is a viable alternative to antibiotic selection in transposon mutagenesis that can generate new fluorescent tools for in vivo protein characterization. IMPORTANCE Transposon mutagenesis is a powerful tool for random mutagenesis, as insertion of a transposon and accompanying antibiotic resistance cassette often disrupt gene function. Here, we present a series of transposons with fluorescent protein genes which, when integrated in frame, may be selected with a fluorescence activated cell sorter (FACS). An open reading frame runs continuously through the transposon such that fluorescent protein fusions may be inserted theoretically anywhere in the primary sequence and potentially preserve function of the target protein. Finally, the transposons were further modified to randomly insert a partial fluorescent protein compatible with bimolecular fluorescence complementation (BiFC) to identify protein interaction candidates.

Structural and functional characterization of the bacterial biofilm activator RemA.

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain.