The accessory protein TagV is required for full Type VI secretion system activity in Serratia marcescens.

The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter- and intra-species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well-characterised core proteins necessary for effector delivery (TssA-M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS-dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ-independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins.

Acoustic component programming in children with cochlear implants using electrocochleography.

OBJECTIVE: Objective measurements improve reliability and effectiveness of hearing assessment and cochlear implant (CI) programming in young children. In CI recipients with acoustic hearing in the implanted ear, electrocochleography (ECochG) can be conducted using intracochlear electrodes. The cochlear microphonic (CM) portion of ECochG has been shown to correlate with pure-tone audiometric thresholds in adult and paediatric CI recipients. Our goal was to determine if ECochG thresholds can be used to appropriately fit the acoustic component to the implanted ear in children. DESIGN: Prospective. STUDY SAMPLE: Eight children (aged 3.5-15.5 years, 10 ears) implanted with Advanced Bionics HiFocus Mid-Scala electrode array were recruited. CI ear acoustic thresholds were measured behaviourally and via ECochG. Two acoustic component enabled CI programs were created using the two sets of thresholds. Age and language appropriate speech outcomes and subjective feedback were obtained. RESULTS: Speech scores were equivalent with the behavioural and ECochG thresholds programs. Subjectively, the ECochG thresholds program was preferred by 7/8 subjects. One subject preferred to use an electric only program. CONCLUSION: Our data suggest that ECochG thresholds can be used to supplement the behavioural clinical methods and aid the reliable fitting of the acoustic component in paediatric CI recipients.

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness.

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram-negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.

The plant pathogen Pectobacterium atrosepticum contains a functional formate hydrogenlyase-2 complex.

Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram-negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane-bound enzyme termed formate hydrogenlyase-2 (FHL-2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL-2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL-2 complex was shown to comprise a rare example of an active [NiFe]-hydrogenase-4 (Hyd-4) isoenzyme, itself linked to an unusual selenium-free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL-2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL-2 function and structure in particular.

Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases.

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.

Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system.

The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. 'Defensive' T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are 'offensive', firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of Serratia marcescens to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in S. marcescens. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.

Comparison of Pure-Tone Thresholds and Cochlear Microphonics Thresholds in Pediatric Cochlear Implant Patients.

OBJECTIVES: In adult cochlear implant patients, conventional audiometry is used to measure postoperative residual hearing which requires active listening and patient feedback. However, audiological measurements in pediatric cochlear implant patients are both challenging as well as time consuming. Intracochlear electrocochleography (ECOG) offers an objective and a time-efficient method to measure frequency-specific cochlear microphonic or difference thresholds (CM/DIF) thresholds that closely approximate auditory thresholds in adult cochlear implant patients. The correlation between CM/DIF and behavioral thresholds has not been established in pediatric cochlear implant patients. In the present study, CM/DIF thresholds were compared with audiometric thresholds in pediatric cochlear implant patients with postoperative residual hearing. DESIGN: Thirteen (11 unilateral and 2 bilateral) pediatric cochlear implant patients (mean age = 9.2 years ± 5.1) participated in this study. Audiometric thresholds were estimated using conventional, condition play, or visual reinforcement audiometry. A warble tone stimulus was used to measure audiometric thresholds at 125, 250, 500, 1000, and 2000 Hz. ECOG waveforms were elicited using 50-msec acoustic tone-bursts. The most apical intracochlear electrode was used as the recording electrode with an extra-cochlear ground electrode. The ECOG waveforms were analyzed to determine CM/DIF thresholds that were compared with pediatric cochlear implant patients' audiometric thresholds. RESULTS: The results show a significant correlation (r = 0.77, p < 0.01) between audiometric and CM/DIF thresholds over a frequency range of 125 to 2000 Hz in pediatric cochlear implant patients. Frequency-specific comparisons revealed a correlation of 0.82, 0.74, 0.69, 0.41, and 0.32 between the audiometric thresholds and CM/DIF thresholds measured at 125, 250, 500, 1000, and 2000 Hz, respectively. An average difference of 0.4 dB (±14 dB) was measured between the audiometric and CM/DIF thresholds. CONCLUSIONS: Intracochlear ECOG can be used to measure CM/DIF thresholds in pediatric cochlear implant patients with residual hearing in the implanted ear. The CM/DIF thresholds are similar to the audiometric thresholds at lower test frequencies and offer an objective method to monitor residual hearing in difficult-to-test pediatric cochlear implant patients.

The Type VI secretion system: a versatile bacterial weapon.

The Type VI secretion system (T6SS) is a protein nanomachine that is widespread in Gram-negative bacteria and is used to translocate effector proteins directly into neighbouring cells. It represents a versatile bacterial weapon that can deliver effectors into distinct classes of target cells, playing key roles in inter-bacterial competition and bacterial interactions with eukaryotic cells. This versatility is underpinned by the ability of the T6SS to deliver a vast array of effector proteins, with many distinct activities and modes of interaction with the secretion machinery. Recent work has highlighted the importance and diversity of interactions mediated by T6SSs within polymicrobial communities, and offers new molecular insights into effector delivery and action in target cells.

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System.

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery.

Molecular weaponry: diverse effectors delivered by the Type VI secretion system.

The Type VI secretion system is a widespread bacterial nanomachine, used to deliver toxins directly into eukaryotic or prokaryotic target cells. These secreted toxins, or effectors, act on diverse cellular targets, and their action provides the attacking bacterial cell with a significant fitness advantage, either against rival bacteria or eukaryotic host organisms. In this review, we discuss the delivery of diverse effectors by the Type VI secretion system, the modes of action of the so-called 'anti-bacterial' and 'anti-eukaryotic' effectors, the mechanism of self-resistance against anti-bacterial effectors and the evolutionary implications of horizontal transfer of Type VI secretion system-associated toxins. Whilst it is likely that many more effectors remain to be identified, it is already clear that toxins delivered by this secretion system represent efficient weapons against both bacteria and eukaryotes.

Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein.

UNLABELLED: The type VI secretion system (T6SS) is widespread in Gram-negative bacteria and can deliver toxic effector proteins into eukaryotic cells or competitor bacteria. Antibacterial T6SSs are increasingly recognized as key mediators of interbacterial competition and may contribute to the outcome of many polymicrobial infections. Multiple antibacterial effectors can be delivered by these systems, with diverse activities against target cells and distinct modes of secretion. Polymorphic toxins containing Rhs repeat domains represent a recently identified and as-yet poorly characterized class of T6SS-dependent effectors. Previous work had revealed that the potent antibacterial T6SS of the opportunistic pathogen Serratia marcescens promotes intraspecies as well as interspecies competition (S. L. Murdoch, K. Trunk, G. English, M. J. Fritsch, E. Pourkarimi, and S. J. Coulthurst, J Bacteriol 193:6057-6069, 2011, http://dx.doi.org/10.1128/JB.05671-11). In this study, two new Rhs family antibacterial effectors delivered by this T6SS have been identified. One of these was shown to act as a DNase toxin, while the other contains a novel, cytoplasmic-acting toxin domain. Importantly, using S. marcescens, it has been demonstrated for the first time that Rhs proteins, rather than other T6SS-secreted effectors, can be the primary determinant of intraspecies competition. Furthermore, a new family of accessory proteins associated with T6SS effectors has been identified, exemplified by S. marcescens EagR1, which is specifically required for deployment of its associated Rhs effector. Together, these findings provide new insight into how bacteria can use the T6SS to deploy Rhs-family effectors and mediate different types of interbacterial interactions. IMPORTANCE: Infectious diseases caused by bacterial pathogens represent a continuing threat to health and economic prosperity. To counter this threat, we must understand how such organisms survive and prosper. The type VI secretion system is a weapon that many pathogens deploy to compete against rival bacterial cells by injecting multiple antibacterial toxins into them. The ability to compete is vital considering that bacteria generally live in mixed communities. We aimed to identify new toxins and understand their deployment and role in interbacterial competition. We describe two new type VI secretion system-delivered toxins of the Rhs class, demonstrate that this class can play a primary role in competition between closely related bacteria, and identify a new accessory factor needed for their delivery.

Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system.

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. "Antibacterial" T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell-cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG subcomplex.

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system.

Communication, cooperation, and social interactions: a report from the third Young Microbiologists Symposium on microbe signalling, organisation, and pathogenesis.

The third Young Microbiologists Symposium took place on the vibrant campus of the University of Dundee, Scotland, from the 2nd to 3rd of June 2014. The symposium attracted over 150 microbiologists from 17 different countries. The significant characteristic of this meeting was that it was specifically aimed at providing a forum for junior scientists to present their work. The meeting was supported by the Society for General Microbiology and the American Society for Microbiology, with further sponsorship from the European Molecular Biology Organization, the Federation of European Microbiological Societies, and The Royal Society of Edinburgh. In this report, we highlight some themes that emerged from the many exciting talks and poster presentations given by the young and talented microbiologists in the area of microbial gene expression, regulation, biogenesis, pathogenicity, and host interaction.

Signal peptide etiquette during assembly of a complex respiratory enzyme.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen capable of respiration with a number of terminal electron acceptors. Tetrathionate reductase is important for the infection process and is encoded by the ttrBCA operon where TtrA and TtrB are metallocofactor-containing proteins targeted to the periplasmic side of the membrane by two different Tat targeting peptides. In this work, the inter-relationship between these two signal peptides has been explored. Molecular genetics and biochemical approaches reveal that the processing of the TtrB Tat signal peptide is dependent on the successful assembly of its partner protein, TtrA. Inactivation of either the TtrA or the TtrB Tat targeting peptides individually was observed to have limited overall effects on assembly of the enzyme or on cellular tetrathionate reductase activity. However, inactivation of both signal peptides simultaneously was found to completely abolish physiological tetrathionate reductase activity. These data suggest both signals are normally active during assembly of the enzyme, and imply a code of conduct exists between the signal peptides where one can compensate for inactivity in the other. Since it appears likely that tetrathionate reductase presents itself for export as a multi-signal complex, these observations also have implications for the mechanism of the bacterial Tat translocase.

Role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by Serratia marcescens Db10.

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites.

Stable-isotope labeling with amino acids in nematodes.

We describe an approach for accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted stable-isotope labeling with amino acids in cell culture (SILAC) for nematodes by feeding worms a heavy lysine- and heavy arginine-labeled Escherichia coli strain and report a genetic solution to elminate the problem of arginine-to-proline conversion. Combining our approach with quantitative proteomics methods, we characterized the heat-shock response in worms.

Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure-activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1-Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.