Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane ...toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.
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•Type VI secretion effector Tse6 acts by depleting bacteria of NAD+ and NADP+•Entry of Tse6 into target cells requires its binding to elongation factor Tu•Tse6 is a membrane protein that requires a chaperone for intercellular transport•EM structures reveal the mechanism for Tse6 deployment to recipient cells
To protect its niche within a microbial community, Pseudomonas secretes a toxin that depletes competing bacterial cells of NAD+ and NADP+. Structures of the toxin and accessory secretory proteins reveal a surprising requirement for the housekeeping protein EF-Tu in toxin delivery and provide mechanistic insight into intercellular protein transport between bacteria.
Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the ...extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.
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•Bacterial T6SS divides into three phylogenetically distinct subtypes (T6SSi–iii)•T6SSiii is restricted to Bacteroidetes and is composed of unique components•T6SSiii targets toxic effectors to competing Proteobacteria and other Bacteroidetes•Bacteroides fragilis T6SSiii targets B. thetaiotaomicron and is expressed in vivo
While many Bacteroidetes occupy densely populated niches, including the human gut, cell contact-dependent antagonism of competing bacteria has not been described in this phylum. Russell et al. describe three type VI secretion system subgroups and demonstrate that a subgroup encoded by Bacteroidetes can mediate competition between prominent commensals.
Bacteria have evolved sophisticated mechanisms to inhibit the growth of competitors
. One such mechanism involves type VI secretion systems, which bacteria can use to inject antibacterial toxins ...directly into neighbouring cells. Many of these toxins target the integrity of the cell envelope, but the full range of growth inhibitory mechanisms remains unknown
. Here we identify a type VI secretion effector, Tas1, in the opportunistic pathogen Pseudomonas aeruginosa. The crystal structure of Tas1 shows that it is similar to enzymes that synthesize (p)ppGpp, a broadly conserved signalling molecule in bacteria that modulates cell growth rate, particularly in response to nutritional stress
. However, Tas1 does not synthesize (p)ppGpp; instead, it pyrophosphorylates adenosine nucleotides to produce (p)ppApp at rates of nearly 180,000 molecules per minute. Consequently, the delivery of Tas1 into competitor cells drives rapid accumulation of (p)ppApp, depletion of ATP, and widespread dysregulation of essential metabolic pathways, thereby resulting in target cell death. Our findings reveal a previously undescribed mechanism for interbacterial antagonism and demonstrate a physiological role for the metabolite (p)ppApp in bacteria.
The plant‐sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due ...to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2‐pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl‐CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP‐citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl‐CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12‐ACL1‐ACC1 using fed‐batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl‐CoA‐derived polyketides from low‐cost carbon sources.
Triacetic acid lactone (TAL) is a promising platform chemical. Cao et al. overexpressed 2‐pyrone synthase in oleaginous yeast Rhodotorula toruloides to produce TAL. They systematically evaluated various metabolic gene targets to increase acetyl‐CoA and malonyl‐CoA levels for TAL production and found that overexpression of both ACL1 and ACC1 led to 28 or 23 g/L of TAL from glucose or oilcane juice with acetate supplementation, respectively, in fed‐batch fermentation.
SUMMARY
Schrenkiella parvula
, a leading extremophyte model in Brassicaceae, can grow and complete its lifecycle under multiple environmental stresses, including high salinity. Yet, the key ...physiological and structural traits underlying its stress‐adapted lifestyle are unknown along with trade‐offs when surviving salt stress at the expense of growth and reproduction. We aimed to identify the influential adaptive trait responses that lead to stress‐resilient and uncompromised growth across developmental stages when treated with salt at levels known to inhibit growth in Arabidopsis and most crops. Its resilient growth was promoted by traits that synergistically allowed primary root growth in seedlings, the expansion of xylem vessels across the root‐shoot continuum, and a high capacity to maintain tissue water levels by developing thicker succulent leaves while enabling photosynthesis during salt stress. A successful transition from vegetative to reproductive phase was initiated by salt‐induced early flowering, resulting in viable seeds. Self‐fertilization in salt‐induced early flowering was dependent upon filament elongation in flowers otherwise aborted in the absence of salt during comparable plant ages. The maintenance of leaf water status promoting growth, and early flowering to ensure reproductive success in a changing environment, were among the most influential traits that contributed to the extremophytic lifestyle of
S. parvula
.
The apelinergic system is a mammalian peptide hormone network with key physiological roles. Apelin isoforms and analogues are believed to be promising therapeutics for cardiovascular disease. Despite ...extensive studies on apelin-13 degradation patterns, only one protease, angiotensin-converting enzyme 2 (ACE2), had been implicated in its physiological regulation. Through use of a peptide-based fluorescent probe, we identified the metalloprotease neprilysin (NEP, a target for Entresto used in treatment of heart failure) as an enzyme that cleaves apelin isoforms. In vitro NEP proteolysis generated fragments that lacked the ability to bind to the apelin receptor, thereby making NEP the first protease to fully inactivate apelin. The involvement of NEP in the apelinergic system contributes to the understanding of its role in cardiovascular physiology.
A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved ...digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
Reliable size-based protein separation is an invaluable biological technique. Unfortunately, size separation in solution is underutilized, owing perhaps to the poor resolution of conventional ...techniques. Here, we report an enhanced multiplexed GELFrEE (gel-eluted liquid fraction entrapment electrophoresis) device which incorporates eight independent separation channels, operating with high repeatability. This enables simultaneous size separation of independent proteome samples, each into 16 well resolved liquid fractions, covering 10−150 kDa in 1.5 h. A novel strategy to increase sample loads while maintaining electrophoretic resolution is presented by distributing the sample among the eight channels with subsequent pooling of collected fractions. Liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis of the S. cerevisiae proteome following GELFrEE separation and sodium dodecyl sulfate (SDS) removal demonstrates the resolution and high correlation achieved between molecular weight and fraction number for the identified proteins. This device is highly orthogonal to solution isoelectric focusing, enabling our disclosure of a fully multiplexed high-throughput two-dimensional liquid electrophoretic (2D LE) platform that separates analogously to 2D polyacrylamide gel electrophoresis (PAGE). With 2D LE, a total of 128 well-resolved liquid fractions are obtained from 1 mg of S. cerevisiae proteins covering ranges 3.8 < pI < 7.8 and 10 kDa < MW < 150 kDa in an unprecedented 3.25 h total separation time.
The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only ...partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes—one of two major phyla in the gut—also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.
Type VII secretion systems (T7SSs) are poorly understood protein export apparatuses found in mycobacteria and many species of Gram‐positive bacteria. To date, this pathway has predominantly been ...studied in Mycobacterium tuberculosis, where it has been shown to play an essential role in virulence; however, much less studied is an evolutionarily divergent subfamily of T7SSs referred to as the T7SSb. The T7SSb is found in the major Gram‐positive phylum Firmicutes where it was recently shown to target both eukaryotic and prokaryotic cells, suggesting a dual role for this pathway in host‐microbe and microbe‐microbe interactions. In this review, we compare the current understanding of the molecular architectures and substrate repertoires of the well‐studied mycobacterial T7SSa systems to that of recently characterized T7SSb pathways and highlight how these differences may explain the observed biological functions of this understudied protein export machine.
The bacterial type VII secretion system (T7SS) is a membrane‐embedded nanomachine found in Firmicutes and Actinobacteria that mediates pathogenesis, nutrient acquisition, and bacterial competition. Over the past two decades, our understanding of this protein secretion system has increased significantly but there are many important discoveries still to be made. This MicroReview is intended to be a T7SS “beginner’s guide” that we hope serves as a comprehensive introduction to newcomers in the field.
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