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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.

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.