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The type VIIb secretion system (T7SSb) is a multisubunit protein export machine found in Gram-positive Firmicute bacteria which plays a key role in interbacterial competition. The T7SSb secretes a variety of toxic effector proteins targeting closely related strains, however the mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by theStaphylococcus aureusT7SSb, which forms a pre-secretion complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins of unknown function, EsxB, EsxC and EsxD. Here we show that these three proteins bind to EsaD and function as EsaD export factors and we report the first structural information for a complete T7SSb substrate pre-secretion complex. Cryo-EM of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers an elongated conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export.Significance StatementStaphylococcus aureusis an opportunistic human pathogen associated with severe infections and antimicrobial resistance.S. aureusstrains utilize a Type VII secretion system to secrete toxins targeting competitor bacteria, which likely facilitates colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains ofS. aureusas well as other related bacterial species. Here we have identified three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.

The Type VIIb protein secretion system (T7SSb) is found in Bacillota (firmicute) bacteria and has been shown to mediate interbacterial competition. EssC is a membrane-bound ATPase that is a critical component of the T7SSb and plays a key role in substrate recognition. Prior analysis of available genome sequences of the foodborne bacterial pathogenListeria monocytogeneshas shown that although the T7SSb was encoded as part of the core genome, EssC could be found as one of seven different sequence variants. While each sequence variant was associated with a specific suite of candidate substrate proteins encoded immediately downstream ofessC, many LXG-domain proteins were encoded across multipleessCsequence variants. Here we have extended this analysis using a diverse collection of 37,930L. monocytogenesgenomes. We have identified a rare eighth variant of EssC present in tenL. monocytogenesLineage III genomes. These genomes also encode a large toxin of the rearrangement hotspot (Rhs) repeat family adjacent toessC8, along with a probable immunity protein and three small accessory proteins. We have further identified nine novel LXG-domain proteins, and four additional chromosomal hotspots acrossL. monocytogenesgenomes where LXG proteins can be encoded. The eightL. monocytogenesEssC variants were also found in otherListeriaspecies, with additional novel EssC types also identified. Across the genus, species frequently encoded multiple EssC types, indicating that T7SSb diversity is a primary feature of the genusListeria.DATA SUMMARYAll genome sequences used in this study are available via Genbank, and the assembly accession numbers are provided in Table S1. This file also lists relevant metadata (name, source category, country, year and clonal complex).IMPACT STATEMENTListeria monocytogenesis a soil-borne saprophytic bacterium and a food-borne pathogen of humans. Decomposing plant matter and the human GI tract are rich in diverse microbial species and to colonise these nichesL. monocytogenesmust be able to compete with other bacteria. The type VII secretion system (T7SS) of Bacillota has been shown to secrete protein toxins that target other bacteria. In this study we have analysed a diverse collection ofL. monocytogenesgenome sequences to study the diversity of theListeriaT7SS and its putative effector proteins. We show that the EssC component of theL. monocytogenesT7SS is highly diverse, clustering into one of eight sequence variants. Each EssC variant is associated with a specific toxin candidate, and the EssC8 variant T7SS likely secretes a novel rearrangement hotspot (Rhs) repeat toxin. We also identify multiple new LXG-families of T7SS toxins and describe genomic hotspots where they are encoded. We find no link between EssC variants and clinical outcome. In agreement with this, analysis of EssC variability in available genomes of otherListeriaspecies showed that all eightL. monocytogenesEssC variants are present in non-monocytogenesListeriaspecies.

The twin arginine transport (Tat) pathway exports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of chloroplasts. In Escherichia coli and other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organization and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A ‘polar’ cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6, is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. Characterization of three stably produced TatC variants, P221R, M222R and L225P, each of which is inactive for protein transport, demonstrated that the substitutions did not affect assembly of the Tat receptor. Moreover, the substitutions that we analysed did not abolish TatA or TatB binding to either binding site. Using targeted mutagenesis we introduced bulky substitutions into the TatA binding site. Molecular dynamics simulations and crosslinking analysis indicated that TatA binding at this site was substantially reduced by these amino acid changes, but TatC retained function. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicates that this region of the protein has a critical function.

IntroductionOne third of people with CF in the UK are co-infected by bothStaphylococcus aureusandPseudomonas aeruginosa. Chronic bacterial infection in CF contributes to the gradual destruction of lung tissue, and eventually respiratory failure in this group.Gap StatementThe contribution ofS. aureusto cystic fibrosis (CF) lung decline in the presence or absence ofP. aeruginosais unclear. Defining the molecular and phenotypic characteristics of a range ofS. aureusclinical isolates will help further understand its pathogenic capabilities.AimOur objective was to use molecular and phenotypic tools to characterise twenty-five clinicalS. aureusisolates collected from mono- and coinfection withP. aeruginosafrom people with CF at the Royal Victoria Infirmary, Newcastle upon Tyne.MethodologyGenomic DNA was extracted and sequenced. Multilocus sequence typing was used to construct phylogeny from the seven housekeeping genes. A pangenome was calculated using Roary. and cluster of Orthologous groups were assigned using eggNOG-mapper which were used to determine differences within core, accessory, and unique genomes. Characterisation of sequence type, clonal complex,agrandspatypes was carried out using PubMLST, eBURST, AgrVATE and spaTyper, respectively. Antibiotic resistance was determined using Kirby Bauer disk diffusion tests. Phenotypic testing of haemolysis was carried out using ovine red blood cell agar plates and mucoid phenotypes visualised using Congo red agar.ResultsClinical strains clustered closely based onagrtype, sequence type and clonal complex. COG analysis revealed statistically significant enrichment of COG families between core, accessory and unique pangenome groups. The unique genome was significantly enriched for replication, recombination and repair, and defence mechanisms. The presence of known virulence genes and toxins were high within this group, and unique genes were identified in 11 strains. Strains which were isolated from the same patient all surpassed average nucleotide identity thresholds, however, differed in phenotypic traits. Antimicrobial resistance to macrolides was significantly higher in the coinfection group.ConclusionThere is huge variation in genetic and phenotypic capabilities ofS. aureusstrains. Further studies on how these may differ in relation to other species in the CF lung may give insight into inter-species interactions.Data summaryThe assembled GenBank (gbk) files for all clinical isolates in this study have been deposited in ENA under the study accession PRJEB56184, accession numbers for each of the twenty-five clinical isolates have been provided in Table S1. The reference strains were collected from the NCBI BioSample database (www.ncbi.nlm.nih.gov/biosample): MRSA_252 (NC_002952.2), HO 5096 0412 (NC_017763.1), ST398 (NC_017333.1) and NCTC8325 (NC_007795.1).

The twin arginine transport (Tat) pathway exports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of chloroplasts. InEscherichia coliand other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organisation and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A ‘polar’ cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6 is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. Characterisation of three stably produced TatC variants, P221R, M222R and L225P, each of which is inactive for protein transport, demonstrated that the substitutions did not affect assembly of the Tat receptor. Moreover, the substitutions that we analysed did not abolish TatA or TatB binding to either binding site. Using targeted mutagenesis we introduced bulky substitutions into the TatA binding site. Molecular dynamics simulations and crosslinking analysis indicated that TatA binding at this site was substantially reduced by these amino acid changes, however TatC retrained function. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicate that this region of the protein has a critical function.

The type VII protein secretion system (T7SS) of Staphylococcus aureus is encoded at the ess locus. T7 substrate recognition and protein transport are mediated by EssC, a membrane-bound multidomain ATPase. Four EssC sequence variants have been identified across S. aureus strains, each accompanied by a specific suite of substrate proteins. The ess genes are upregulated during persistent infection, and the secretion system contributes to virulence in disease models. It also plays a key role in intraspecies competition, secreting nuclease and membrane-depolarizing toxins that inhibit the growth of strains lacking neutralizing immunity proteins. A genomic survey indicates that the T7SS is widely conserved across staphylococci and is encoded in clusters that contain diverse arrays of toxin and immunity genes. The presence of genomic islands encoding multiple immunity proteins in species such as Staphylococcus warneri that lack the T7SS points to a major role for the secretion system in bacterial antagonism.

RN6390 is a commonly used laboratory strain of Staphylococcus aureus derived from NCTC8325. In this study, we sequenced the RN6390 genome and compared it to available genome sequences for NCTC8325. We confirmed that three prophages, F 11, F 12 and F 13, which are present in NCTC8325 are absent from the genome of RN6390, consistent with the successive curing events leading to the generation of this strain. However, we noted that a separate prophage is present in RN6390 that is not found in NCTC8325. Two separate genome sequences have been deposited for the parental strain, NCTC8325. Analysis revealed several differences between these sequences, in particular, between the copy number of esaG genes, which encode immunity proteins to the type VII secreted anti-bacterial toxin, EsaD. Single nucleotide polymorphisms were also detected in ribosomal RNA genes and genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM) between the two NCTC8325 sequences. Comparing each NCTC8325 sequence to other strains in the RN6390 lineage confirmed that sequence assembly errors in the earlier NCTC8325 sequence are the most likely explanation for most of the differences observed.

The Type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified inStaphylococcus aureus, EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identicalesaGandtsaIimmunity genes are encoded in allS. aureusstrains. To investigate the evolution ofesaGrepertoires, we analysed the sequences of the tandemesaGgenes and their encoded proteins. We identified three blocks of high sequence similarity shared by allesaGgenes and identified evidence of extensive recombination events betweenesaGparalogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss and expansion ofesaGgenes inS. aureusgenomes and we identified evidence of such events during evolution of strains in clonal complex 8. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence similarity locate to the 5’ and 3’ end oftipCgenes, and we found strong evidence for recombination betweentipCparalogues encoded byStreptococcus mitisBCC08. By contrast, we found only a single homology block acrosstsaIgenes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.DATA SUMMARYAll sequence data for strains used in this study are available on NCBI under BioProject PRJNA789916. Sequences from the NCTC3000 project are available on NCBI under BioProject PRJEB6403. Supplementary data 2 and all custom scripts are available on Github:https://github.com/GM110Z/Garret-et-al.-recombination-paper.IMPACT STATEMENTThe type VII secretion system (T7SS) in firmicutes secretes polymorphic toxins that target other bacteria. To protect from the action of these toxins, bacteria carry multiple paralogous copies of immunity protein-encoding genes that are sequence-related but non-identical. To date, little is known about how T7 immunity gene families evolve. In this study we analysed a cluster of EsaG-encoding genes inStaphylococcus aureuswhich are found at theess/T7secretion locus and provide immunity against the T7 secreted nuclease toxin, EsaD. We identified three homology blocks coveringesaGgenes and their downstream intergenic regions, which are separated by two variable regions. We have shown that recombination can occur between these homology blocks, leading to loss or expansion ofesaGgenes at this locus. Using a historical dataset of closely relatedS. aureusstrains from clonal complex 8, we identified several independent recombination events leading to changes in theesaGrepertoire. We further showed that similar events are observed for an immunity protein encoded by Group BStreptococcusspp. suggesting that recombination plays a broader role in the evolution of T7SS immunity-encoding genes. We speculate that gain and loss of T7 immunity genes is weighed in response to environmental pressure and metabolic burden.

The discovery of penicillin by Alexander Fleming marked a new era for modern medicine, allowing not only the treatment of infectious diseases, but also the safe performance of life-saving interventions, like surgery and chemotherapy. Unfortunately, resistance against penicillin, as well as more complex β-lactam antibiotics, has rapidly emerged since the introduction of these drugs in the clinic, and is largely driven by a single type of extra-cytoplasmic proteins, hydrolytic enzymes called β-lactamases. While the structures, biochemistry and epidemiology of these resistance determinants have been extensively characterized, their biogenesis, a complex process including multiple steps and involving several fundamental biochemical pathways, is rarely discussed. In this review, we provide a comprehensive overview of the journey of β-lactamases, from the moment they exit the ribosomal channel until they reach their final cellular destination as folded and active enzymes.

RN6390 is a commonly used laboratory strain of Staphylococcus aureus derived from NCTC8325. In this study, we sequenced the RN6390 genome and compared it to available genome sequences for NCTC8325. We confirmed that three prophages, Φ11, Φ12 and Φ13, which are present in NCTC8325 are absent from the genome of RN6390, consistent with the successive curing events leading to the generation of this strain. However, we noted that a separate prophage is present in RN6390 that is not found in NCTC8325. Two separate genome sequences have been deposited for the parental strain, NCTC8325. Analysis revealed several differences between these sequences, in particular between the copy number of esaG genes, which encode immunity proteins to the type VII secreted anti-bacterial toxin, EsaD. Single nucleotide polymorphisms were also detected in ribosomal RNA genes and genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM) between the two NCTC8325 sequences. Comparing each NCTC8325 sequence to other strains in the RN6390 lineage confirmed that sequence assembly errors in the earlier NCTC8325 sequence are the most likely explanation for most of the differences observed.

Maturation of [NiFe]-hydrogenases often involves specific proteases responsible for cleavage of the catalytic subunits. Escherichia coli HycI is the protease dedicated to maturation of the Hydrogenase-3 isoenzyme, a component of formate hydrogenlyase-1. In this work, it is demonstrated that a Pectobacterium atrosepticum HycI homologue, HyfK, is required for hydrogenase-4 activity, a component of formate hydrogenlyase-2, in that bacterium. The P. atrosepticum ΔhyfK mutant phenotype could be rescued by either P. atrosepticum hyfK or E. coli hycI on a plasmid. Conversely, an E. coli ΔhycI mutant was complemented by either E. coli hycI or P. atrosepticum hyfK in trans. E. coli is a rare example of a bacterium containing both hydrogenase-3 and hydrogenase-4, however the operon encoding hydrogenase-4 has no maturation protease gene. This work suggests HycI should be sufficient for maturation of both E. coli formate hydrogenlyases, however no formate hydrogenlyase-2 activity was detected in any E. coli strains tested here.

The twin‐arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N‐terminal signal sequence containing a highly conserved twin‐arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation., The twin‐arginine protein transport (Tat) pathway transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N‐terminal signal sequence containing a highly conserved twin‐arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. This review will summarise recent advances in our understanding of Tat transport.

Serratia marcescens is a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein ‘secretome’ comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the ‘chitinolytic machinery’, which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon source. Secretion of the chitinases across the outer membrane is governed by the chiWXYZ operon encoding a holin/endopeptidase pair. Expression of the chiWXYZ operon is co-ordinated with the chitinase genes and is also bimodal, as normally only 1% of the population expresses the chitinolytic machinery. In this study, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation of chiWXYZ with both chiA and chiC. Moreover, overexpression of chiR alone was able to increase the proportion of the cell population expressing chitinase genes to >60 %. In addition, quantitative label-free proteomic analysis of cells overexpressing chiR established that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression in S. marcescens , and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. β-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of β-lactam drugs is to populate the periplasmic space with β-lactamases. Resistance to β-lactam drugs is spread by lateral transfer of genes encoding β-lactamases from one species of bacteria to another. However, the resistance phenotype depends in turn on these "alien" protein sequences being recognized and exported across the cytoplasmic membrane by either the Sec or Tat protein translocation machinery of the new bacterial host. Here, we examine BKC-1, a carbapenemase from an unknown bacterial source that has been identified in a single clinical isolate of Klebsiella pneumoniae. BKC-1 was shown to be located in the periplasm, and functional in both K. pneumoniae and Escherichia coli. Sequence analysis revealed the presence of an unusual signal peptide with a twin arginine motif and a duplicated hydrophobic region. Biochemical assays showed this signal peptide directs BKC-1 for translocation by both Sec and Tat translocons. This is one of the few descriptions of a periplasmic protein that is functionally translocated by both export pathways in the same organism, and we suggest it represents a snapshot of evolution for a β-lactamase adapting to functionality in a new host. IMPORTANCE Bacteria can readily acquire plasmids via lateral gene transfer (LGT). These plasmids can carry genes for virulence and antimicrobial resistance (AMR). Of growing concern are LGT events that spread β-lactamases, particularly carbapenemases, and it is important to understand what limits this spread. This study provides insight into the sequence features of BKC-1 that exemplify the limitations on the successful biogenesis of β-lactamases, which is one factor limiting the spread of AMR phenotypes by LGT. With a very simple evolutionary adaptation, BKC-1 could become a more effective carbapenemase, underscoring the need to understand the evolution, adaptability, and functional assessment of newly reported β-lactamases rapidly and thoroughly.

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus , is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

Located at the tip of cell surface glycoconjugates, sialic acids are at the forefront of host–microbe interactions and, being easily liberated by sialidase enzymes, are used as metabolites by numerous bacteria, particularly by pathogens and commensals living on or near diverse mucosal surfaces. These bacteria rely on specific transporters for the acquisition of host-derived sialic acids. Here, we present the first comprehensive genomic and phylogenetic analysis of bacterial sialic acid transporters, leading to the identification of multiple new families and subfamilies. Our phylogenetic analysis suggests that sialic acid-specific transport has evolved independently at least eight times during the evolution of bacteria, from within four of the major families/superfamilies of bacterial transporters, and we propose a robust classification scheme to bring together a myriad of different nomenclatures that exist to date. The new transporters discovered occur in diverse bacteria, including Spirochaetes , Bacteroidetes , Planctomycetes and Verrucomicrobia , many of which are species that have not been previously recognized to have sialometabolic capacities. Two subfamilies of transporters stand out in being fused to the sialic acid mutarotase enzyme, NanM, and these transporter fusions are enriched in bacteria present in gut microbial communities. Our analysis supports the increasing experimental evidence that competition for host-derived sialic acid is a key phenotype for successful colonization of complex mucosal microbiomes, such that a strong evolutionary selection has occurred for the emergence of sialic acid specificity within existing transporter architectures.

The Type VII protein secretion system (T7SS) has been characterised in members of the actinobacteria and firmicutes phyla. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus, is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG-domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG-domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system which uses a holin membrane protein in tandem with a cell wall editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall editing enzymes employed was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS). Potential mechanisms for secretion across the outer membrane are discussed. Special Issue: Protein Secretion and Transport

Significance Staphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a type VII secretion system that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a type VII substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo., The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.

In Escherichia coli, citrate-mediated iron transport is a key nonheme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasm- and periplasm-orientated moieties at the N and C termini. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and the substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, the fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin-arginine signal sequence. IMPORTANCE Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli. The interactions of FecR with the outer membrane protein FecA and cytoplasmic anti-sigma factor FecI have been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that the targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin-arginine signal sequence.

Youth with problem gambling behaviors are susceptible to serious academic, behavioral, and mental health consequences including school failure, criminal involvement, and depression. Coupled with increased exposure to gambling formats, issues related to youth gambling have been deemed a serious public health issue requiring increased prevention efforts. However, the literature is limited in terms of evidence-based gambling prevention programs warranting the development of The Maryland Smart Choices Program (MD-Smart Choices), a gambling prevention program for middle and high school youth. This 3-session, 45-min program was developed for implementation in Baltimore City Public Schools, an urban and predominately African American district with specific aims to engage students, encourage positive behavior, and facilitate learning related to gambling disorder. Pre-post program participation assessments were collected from 72 students across 5 different schools. Results yielded significant increases in student awareness and knowledge following participation in MD-Smart Choices. Focus group data collected from program facilitators suggested high student engagement and participation, program feasibility, and ease of implementation. Study implications and future directions are discussed.

The Type VII protein secretion system (T7SS) is found in actinobacteria and firmicutes, and plays important roles in virulence and interbacterial competition. A membrane-bound ATPase protein, EssC in Staphylococcus aureus, lies at the heart of the secretion machinery. The EssC protein from S. aureus strains can be grouped into four variants (EssC1-EssC4) that display sequence variability in the C-terminal region. Here we show that the EssC2, EssC3 and EssC4 variants can be produced in a strain deleted for essC1 and that they are able to mediate secretion of EsxA, an essential component of the secretion apparatus. They are, however, unable to support secretion of the substrate protein EsxC, which is encoded only in essC1-specific strains. This finding indicates that EssC is a specificity determinant for T7 protein secretion. Our results support a model where the C-terminal domain of EssC interacts with substrate proteins whereas EsxA interacts elsewhere.

Serratia marcescensis a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein ‘secretome’ comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the ‘chitinolytic machinery’, which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon and nitrogen source. Secretion of the chitinases across the outer membrane is governed by thechiWXYZoperon encoding a holin/endopeptidase pair. Expression of thechiWXYZoperon is co-ordinated with the chitinase genes and is also bimodal, since normally only 1% of the population expresses the chitinolytic machinery. In this work, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation ofchiWXYZwith bothchiAandchiC. Moreover, overexpression ofchiRalone was able to increase the proportion of the cell population expressing chitinase genes to >60%. In addition, quantitative label-free proteomic analysis of cells overexpressingchiRestablished that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression inS. marcescens, and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.IMPORTANCEThe opportunistic pathogenSerratia marcescenssecretes chitinases through the action of thechiWXYZoperon, which encodes a holin/endopeptidase pair. Expression ofchiWXYZis normally bimodal, with only 1% of the population transcribing these genes. In this work, it is demonstrated that overexpression ofchiRinduces exquisitely co-ordinated holin/endopeptidase and chitinase gene expression in the majority of the population of cells, establishing that ChiR is a key player in biomodal gene expression. This work also reveals for the first time that co-operating pathways are induced by ChiR, including enzymes involved in ammonia, nitrite and nitrate metabolism. This work expands knowledge of basic bacterial physiology and could have applications in the biomedical and biotechnological research fields.

Antiretrovirals (ARVs) have transformed HIV prognosis; most individuals will take treatment lifelong and will enjoy a normal life expectancy. Based on 2015/6 ARV expenditure of £429 million in England, the HIV Clinical Reference Group (CRG) and NHS England (NHSE) developed an improving value (IV)

The twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.

Structural dissection of EssC, a membrane-bound component of the bacterial Type VII secretion system, reveals two β-sandwich domains at the N-terminus and two ATPase domains at the C-terminus. A structure for the potential pore of the secretion system is proposed., The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.

Organophosphate hydrolase (OPH), encoded by the organophosphate degradation (opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents. OPH is targeted to the inner membrane of Brevundimonas diminuta in a pre-folded conformation by the twin arginine transport (Tat) pathway. The OPH signal peptide contains an invariant cysteine residue at the junction of the signal peptidase (Spase) cleavage site along with a well conserved lipobox motif. Treatment of cells producing native OPH with the signal peptidase II inhibitor globomycin resulted in accumulation of most of the pre-OPH in the cytoplasm with negligible processed OPH detected in the membrane. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. Membrane-bound OPH was shown to interact with the outer membrane efflux protein TolC and with PstS, the periplasmic component of the ABC transporter complex (PstSACB) involved in phosphate transport. Interaction of OPH with PstS appears to facilitate transport of Pi generated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Consistent with this model, opd null mutants of B. diminuta failed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate.

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues.Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

The article Enhancing the Relevance and Effectiveness of a Youth Gambling Prevention Program for Urban, Minority Youth: A Pilot Study of Maryland Smart Choices, written by Brittany R. Parham, Carl Robertson, Nancy Lever, Sharon Hoover, Tracy Palmer, Phyllis Lee, Kelly Willis and Joanna Prout, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 18 August 2018 with open access. With the author(s)' decision to step back from Open Choice, the copyright of the article changed on 10 September 2018 to © Springer Science+Business Media, LLC, part of Springer Nature 2018 and the article is forthwith distributed under the terms of copyright.The original article has been corrected.

The Escherichia coli formate hydrogenlyase (FHL) complex is produced under fermentative conditions and couples formate oxidation to hydrogen production. In this work, the architecture of FHL has been probed by analysing affinity-tagged complexes from various genetic backgrounds. In a successful attempt to stabilize the complex, a strain encoding a fusion between FdhF and HycB has been engineered and characterised. Finally, site-directed mutagenesis of the hycG gene was performed, which is predicted to encode a hydrogenase subunit important for regulating sensitivity to oxygen. This work helps to define the core components of FHL and provides solutions to improving the stability of the enzyme.

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being an essential component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. Quantitative western blotting indicates that EsaB is present at very low levels in cells. Substitution of a highly conserved threonine for alanine or arginine resulted in a loss of EsaB activity and destabilisation of the protein. Taken together our findings show that EsaB is essential for T7SS activity in RN6390.

The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in cis to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway., IMPORTANCE Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.