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PubMed2026

A novel reverse lipase toxin substrate of the Staphylococcus aureus type VII secretion system.

The type VII secretion system (T7SS) is found in many Gram-positive bacteria and secretes toxins with antibacterial activity. Most characterized substrates have an N-terminal LXG domain that interacts with other helical partner proteins to form a composite T7SS targeting signal. Here we describe only the second substrate family to have a reverse domain arrangement. We show that TslM has a C-terminal LXG-like domain and an N-terminal lipase domain that has phospholipase activity. Secretion of TslM requires a single helical partner protein that binds to the TslM C-terminus, and its toxic activity is neutralized by a distinct family of membrane proteins. Genome analysis reveals that Staphylococcus aureus strains have the capacity to encode up to seven paralogous copies of this toxin family. Taken together our findings show that lipases are an important component of the staphylococcal T7SS toxin arsenal, and that toxins with a reverse domain arrangement are more widespread than previously appreciated.

Staphylococcus aureus↗

PubMed2026

Experimental evolution reveals genetic routes for adaptive loss of the antibacterial type VI secretion system.

The type VI secretion system (T6SS) is a contractile nanomachine used by Gram-negative bacteria to deliver effector proteins into target cells, contributing to both interbacterial competition and pathogenesis. Although T6SS gene clusters are present in recently isolated commensal and pathogenic Escherichia coli strains, they are absent from classical laboratory strains that have been propagated for decades in pure cultures, suggesting that T6SS can be lost in the absence of competition. Here, we combined experimental evolution with whole-genome sequencing to track the fate of the enteroaggregative Escherichia coli (EAEC) Sci1 T6SS during competition with either T6SS-susceptible or T6SS-immune bacteria. After ∼640 generations, T6SS activity was largely maintained during competition with T6SS-susceptible bacteria, whereas ∼90% of clones evolved with T6SS-immune bacteria lost or attenuated T6SS activity through diverse mutations within the sci1 promoter, essential T6SS structural genes, or the rfaH transcriptional antiterminator. We identified two RfaH-binding ops elements within the sci1 cluster, revealing antitermination as a regulatory element of EAEC T6SS transcription, which is conserved among Enterobacteriaceae. Our findings highlight how experimental evolution can reveal the selective forces shaping T6SS maintenance and identify new regulatory components controlling its activity.

Journal Article↗

PubMed2025

Effectidor II: a pan-genomic AI-based algorithm for the prediction of type III secretion system effectors.

MOTIVATION: Type III secretion systems are used by many Gram-negative bacteria to inject type 3 effectors (T3Es) directly into eukaryotic cells, promoting disease or provoking immune response. Because of these opposing evolutionary forces, T3E repertoires often vary within taxonomic groups. Identifying the full effector gene repertoire in genomes of related individuals is crucial for determining core and specialized effectors, understanding the disease dynamics, and developing appropriate management strategies against pathogens. It can also help uncover novel T3Es that have recently emerged in a population. Our previously published Effectidor web server successfully addressed the challenge of identifying T3Es in a single bacterial genome. Here, we enriched the web server with various novel capabilities, including the identification of T3Es from multiple genome sequences simultaneously. RESULTS: We present Effectidor II, a web server that relies on machine learning to predict T3E-encoding genes within bacterial pan-genomes. We demonstrate the benefit of learning based on features extracted from the entire sequences comprising the pan-genome and report a novel T3E discovered by it in Xanthomonas euroxanthea. AVAILABILITY AND IMPLEMENTATION: Effectidor II is available at: https://effectidor.tau.ac.il and the source code is available at: https://github.com/naamawagner/Effectidor. A stand-alone version of Effectidor II is available at: https://github.com/naamawagner/Effectidor/tree/StandAlone. The source code for the standalone version and the data used in this work are also provided in https://doi.org/10.5281/zenodo.15081636.

Type III Secretion Systems↗

PubMed2025

Assembly of the Mycobacterium tuberculosis type VII ESX-1 secretion system in Mycobacterium smegmatis identifies a new transcriptional activator of esx-1 genes and a novel TB vaccine.

Mycobacterium tuberculosis (M. tb) uses its type VII secretion system (T7SS) ESX-1 to export immunogenic, virulence-mediating protein effectors. In this study, the fast-growing, non-pathogenic model mycobacteria Mycobacterium smegmatis mc2-155 was engineered to express the M. tb T7SS ESX-1 system. We found that M. smegmatis transformed with M. tb esx-1 locus genes only, as well as M. smegmatis transformed with M. tb esx-1 and espACD operon genes (designated MSX-1), produces and secretes the M. tb ESX-1 protein effectors EsxA, EsxB, and EspB. However, the abundance of these proteins was higher inside the cell and culture filtrate of the MSX-1 strain. Although ESX-1 is critical for M. tb pathogenesis, expression of M. tb ESX-1 did not make the recombinant M. smegmatis strains virulent in macrophages. Serendipitously, transformation of M. smegmatis with a modified esx-1 locus in this study revealed rv3860, a gene of previously unknown function, to be required for the transcription of pe35, ppe68, esxB, and esxA genes. Finally, mice vaccinated with MSX-1 were found to be as protected as mice vaccinated with Mycobacterium bovis BCG against M. tb infection, without becoming sensitized to tuberculin. These results show that a functional M. tb ESX-1 system can be assembled in M. smegmatis to uncover novel facets of the secretion machinery and that the modified M. smegmatis strain can function as a tuberculosis (TB) vaccine. Unlike BCG, however, its deployment may be compatible with tests currently used to diagnose TB.IMPORTANCEIn this study, we modified Mycobacterium smegmatis, which is often used as a surrogate model organism in mycobacterial research, to produce and assemble a functional Mycobacterium tuberculosis (M. tb) ESX-1 protein secretion system. One such M. smegmatis strain named MSX-1 was found to make a functional M. tb ESX-1 system without becoming virulent. And in using M. smegmatis as a chassis to study the ESX-1 system, we found that rv3860, an M. tb gene of previously unknown function, is needed for the production of key ESX-1 proteins. Finally, mice vaccinated with MSX-1 were as protected from tuberculosis (TB) as mice given BCG, the only approved TB vaccine. Notably, we found that unlike BCG, MSX-1 does not sensitize mice to the antigens used in existing TB diagnostic tests. These observations, taken together, highlight the utility of M. smegmatis as a chassis to study the M. tb ESX-1 secretion machinery.

Mycobacterium smegmatis↗

PubMed2025

Type VI secretion system activity at lethal antibiotic concentrations leads to overestimation of weapon potency.

Competition assays are a mainstay of modern microbiology, offering a simple and cost-effective means to quantify microbe-microbe interactions in vitro. Here, we demonstrate a key weakness of this method that arises when competing microbes interact via toxins, such as those secreted via the type VI secretion system (T6SS). Time-lapse microscopy reveals that T6SS-armed Acinetobacter baylyi bacteria can maintain lethal T6SS activity against E. coli target cells, even under selective conditions intended to eliminate A. baylyi. Further, this residual killing creates a density- and T6SS-dependent bias in the apparent recovery of E. coli, leading to a misreporting of competition outcomes where target survival is low. We also show that incubating A. baylyi/E. coli co-cultures in liquid antibiotic prior to selective plating can substantially correct this bias. Our findings demonstrate the need for caution when using selective plating as part of T6SS competition assays, or assays involving other toxin-producing bacteria.

Type VI Secretion Systems↗

PubMed2025

The Pseudomonas aeruginosa Type VI secretion system toxin Tse8 evolved from a novel N-carbamoylputrescine amidohydrolase.

The polyamine putrescine is synthesized primarily from L-arginine via agmatine in bacteria. There are currently three known routes from agmatine to putrescine, including direct conversion by agmatinase. The other two routes use agmatine deiminase to produce N-carbamoylputrescine from agmatine, then one of two nonhomologous enzymes, putrescine transcarbamylase or N-carbamoylputrescine amidohydrolase (NCPAH), converts N-carbamoylputrescine to putrescine. Here, we functionally identify enzymes from phylogenetically distant bacteria, the ɣ-proteobacterium Shewanella oneidensis, and the actinomycetota species Microterricola gilva, that are novel alternative, nonhomologous, noncanonical NCPAHs that we term AguY, which have emerged by convergent evolution. Kinetic analysis indicates that the AguY enzymes are as efficient as the canonical NCPAH from Pseudomonas aeruginosa in converting N-carbamoylputrescine to putrescine. Genomic evidence suggests that the AguY enzymes may participate in putrescine biosynthetic or agmatine catabolic pathways and are occasionally encoded in genomes that also encode agmatinase. We show that the Type VI secretion system toxin Tse8 from P. aeruginosa has evolved from AguY. It is formally possible that AguY evolved directly or indirectly from the ancient glutamine amidohydrolase GatA, a component of the transamidosome, an RNA/protein complex required for the production of glutamine-charged tRNA. Our study provides a further example of the prevalence of convergent evolution and horizontal gene transfer in polyamine biosynthesis, suggesting pervasive selective pressure to evolve polyamine metabolism in bacteria.

Pseudomonas aeruginosa↗

PubMed2026

Genome sequences of pirAB+ and pirAB- Vibrio campbellii strains isolated from shrimp ponds with mortality outbreaks carry type VI secretion systems.

Vibrio campbellii strains PH1401 and PH1409 were isolated from shrimp ponds with documented mortality outbreaks in the Philippines. PH1401 and PH1409 share identity with V. campbellii strain BoB-53. Whole-genome analysis reveals full-length pirAB binary toxin genes in PH1401. Both strains carry three type VI secretion systems.

Vibrio campbellii↗

PubMed2026

Systematic analysis of the type VII secretion system in Streptococcus gallolyticus subsp. gallolyticus reveals genomic diversity and functional associations.

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathobiont associated with bacteremia, infective endocarditis, and colorectal cancer. However, the genomic diversity of this subspecies and the distribution of key virulence determinants, particularly the type VII secretion system (T7SS), remain poorly characterized. Here, we performed genomic analyses of 76 Sgg strains from diverse geographic and host origins. Core- and pan-genome analyses, multi locus sequence typing, and phylogenetic reconstruction revealed dominant sequence types (STs) that correlate with geographic origin or source of isolation. Furthermore, systematic characterization of the T7SS locus identified five new T7SS subtypes and demonstrated a strong association between T7SS subtype and ST. We further expanded the known repertoire of T7SS LXG domain-containing polymorphic toxins (LXG toxins) in Sgg substantially through genome-wide searches. Distinct distribution patterns were observed for the LXG toxins across the strains. Lastly, our data indicated that T7SS subtype was significantly associated with biofilm formation capacity of Sgg strains. Together, these findings advance our understanding of Sgg genomic diversity, reveal substantial lineage-associated variation in T7SS architecture and effector repertoires, and suggest a previously unrecognized connection between T7SS and biofilm formation in Sgg.

LXG toxins↗

PubMed2026

Unveiling a missing component of the atypical type IV secretion system required for natural transformation of Helicobacter pylori.

Exchange of genetic information by natural transformation shapes bacterial evolution. In Helicobacter pylori it is thought to drive its unusually high recombination rate, which has a crucial role in the evolution of virulence and the propagation of antibiotics resistance genes. While in most cases uptake of the incoming DNA into the periplasm is mediated by type IV pili, in H. pylori this initial step of natural transformation requires ComB, a unique competence-specific type IV secretion system (T4SS). The mechanisms by which ComB mediates DNA uptake are still poorly understood, since T4SS are usually involved in an opposite process of DNA export. Here, we identify a gene (hp1421) that is absolutely required for uptake of the transforming DNA into the periplasm, although distant from the comB operons. We show that hp1421 codes for a hexameric ATPase from the VirB11 family. HP1421 is present in the cytoplasm and interacts with ComB4, another ATPase of the T4SS inner membrane subcomplex. The structural modelling and functional analysis of HP1421 and its interaction with ComB4 indicate that HP1421 is a missing component of the ComB inner-membrane subcomplex that we propose to name ComB11. Phylogenetic analyses show that comB11 is a H. pylori core gene and suggest that the competence-dedicated ComB T4SS was a recent acquisition within Helicobacteraceae. Hence, co-option of the T4SS for DNA transformation requires nearly all the proteins that were previously essential for DNA conjugation.

Helicobacter pylori↗

PubMed2026

Unveiling a missing component of the atypical type IV secretion system required for natural transformation of Helicobacter pylori.

Journal Article↗

PubMed2025

WhiB6 Transduces Contact-Dependent Signaling in Mycobacterium smegmatis and Coordinately Induces Both ESX-1 and ESX-4.

Bacteria have evolved complex conditional pathways that respond to environmental stresses and signals. We use conjugation in Mycobacterium smegmatis to identify contact-recognition and response pathways that mediate interactions between donor and recipient cells. Contact with a compatible donor cell initiates a response in the recipient that requires the ESX-1 secretion system and subsequently activates the dormant ESX-4 secretion system. The links of this signal transduction pathway, the mechanism of coordination and dependency between ESX-1 and ESX-4 secretion systems, are unknown. Previous studies identified SigM as a cell-contact responsive sigma factor dedicated to activating ESX-4. WhiB proteins are iron-sulfur-binding stress-response transcription factors exclusively found in Actinobacteria. WhiB6 has been shown to regulate ESX-1 associated gene expression in other mycobacteria. Here, we show that WhiB6 is required both for conjugation and for transducing cell-contact dependent signaling in the recipient cell. Our RNA-seq, ChIP-seq, and proteomic profiling data define a WhiB6 regulon that supports conjugative cell-cell interaction. The WhiB6 regulon includes genes encoding ESX-1, ESX-4, SigM, as well as dispersed operons that likely support ESX secretion. Our data demonstrate that WhiB6 is epistatic to SigM and ESX-4 in this signal transduction pathway. This work shows that WhiB6 functions as a signal transduction node in recipient cells: it coordinates the expression of two ESX systems and it also induces uncharacterized proteins that collectively constitute a complete secretion response to recipient contact with a donor cell.

Mycobacterium smegmatis↗

PubMed2026

Metagenomic insights into mechanisms of coral larval settlement induction and inhibition by marine biofilms.

BACKGROUND: Biofilms are essential to larval settlement in many marine invertebrates, yet the mechanisms driving settlement induction or inhibition in corals remain poorly resolved. This challenge lies in the vast taxonomic and functional diversity of marine biofilms, making it difficult to identify cues associated with settlement. To address this, we analysed the metagenomes of biofilms used to induce settlement (attachment and metamorphosis) of four broadcast-spawning non-acroporid coral species: Dipsastrea favus, Platygyra sinensis, Echinophyllia aspera and Porites lobata. Biofilms were developed for one or two months, under light or dark treatments, with light biofilms inducing significantly higher settlement than dark biofilms. RESULTS: Gene composition varied strongly among treatments, with light biofilms enriched in genes encoding carotenoid biosynthesis and nitrate reduction, while dark biofilms encoded more genes for denitrification and nitric oxide production. Modelling revealed the abundance of genes encoding GABA biosynthesis and the type III secretion system (SS) were positively associated with settlement, while genes encoding the type II secretion system, flagellar and lipopolysaccharides were negatively associated. Genes predicted to promote settlement were concentrated in metagenome assembled genomes (MAGs) assigned to Flavobacteriaceae, Rhodobacteraceae and Pirellulaceae, consistent with previous research identifying these lineages as potential inducers. While we detected homologues of some biosynthesis genes for the settlement-inducing compounds cycloprodigiosin and tetrabromopyrrole in the MAGs, pathways were incomplete suggesting additional compounds promote settlement on these biofilms. CONCLUSIONS: These findings link biofilm metagenomics to coral larval settlement for the first time, suggesting carotenoids may attract larvae to biofilm surfaces, while GABA may promote searching and attachment. Additional compounds, for example cycloprodigiosin, tetrabromopyrrole or effector proteins, may be required to complete metamorphosis, however the specific compounds responsible likely vary across biofilm communities and suggest multiple mechanisms can lead to settlement. Simultaneously, elevated levels of nitric oxide, type II SS exudates or an abundance of flagellar potentially inhibit the settlement process. This study advances our understanding of the complex microbial processes underpinning coral larval settlement.

Biofilm↗

PubMed2025

Sucrose and malic acid in the tobacco plant induce hrp regulon in a phytopathogen Ralstonia pseudosolanacearum.

Genes encoding a type III secretion system in Ralstonia pseudosolanacearum are regulated by HrpB as an hrp regulon and induced only in plants. This study aimed to identify the plant signals that induce the hrp regulon and confirm the signal recognition mechanism. Signaling molecules that induce hrpB expression were screened using resting cells of the hrpB-lacZ reporter strain. Only the soluble fraction of smashed tobacco seedlings induced hrpB expression. The heated soluble fraction retained its hrpB-inducing activity, indicating that the signaling molecules were not proteins. When the soluble fraction was fractionated into acidic, neutral, and basic components, both the acidic and neutral fractions induced hrpB expression. As neutral compounds, sucrose, glucose, and fructose have been found to induce hrpB expression. Sucrose-induced hrpB expression was greatly reduced in the prhA mutant, indicating that the TonB-dependent receptor PrhA perceives sugars. Among the organic acids found in the acidic fractions, malic acid most efficiently induced hrpB expression, which was reduced by the mutation of a hybrid histidine kinase gene of a two-component system, rsc1598, indicating that Rsc1598 may sense malic acid. We demonstrated direct binding of Rsc1598 to malic acid using isothermal titration calorimetry.IMPORTANCESimilar to other Gram-negative plant pathogens, the type III secretion system (T3SS) is the most important virulence factor in Ralstonia pseudosolanacearum. The genes for the T3SS are regulated as an hrp regulon, activated only when the pathogen encounters the plants, indicating that the pathogen must sense plant signals. For the first time, we identified two signaling compounds, sucrose and malic acid, that are abundantly found in tobacco roots. The hrp operon was induced even in non-host plants, possibly because sucrose and malic acid are common in plants. We also found that R. pseudosolanacearum membrane proteins received sucrose and malic acid independently. As a next step, antagonists of signaling molecules can be screened.

Malates↗

PubMed2026

Metagenomic polymorphic toxin effector and immunity profiling predicts microbiome development and disease-related dysbiosis.

Bacteria use antagonistic interbacterial weapons, such as polymorphic toxin secretion systems (TSS), to compete for niches in the human gut microbiome. We hypothesized that TSS influence gut microbiome development and disease-related dysbiosis. We developed a bioinformatic marker gene approach (PolyProf) to quantify TSS including ~200 effector and immunity genes and applied it to ~15,000 publicly available human metagenomes. PolyProf alpha and beta diversity readily distinguished 12 different human disease states and enabled the construction of highly accurate linear regression classifier machine learning models. Elastic net machine learning models integrating bacterial taxonomy with PolyProf had strong predictive value for 12 disease states, outperforming models utilizing taxonomy alone. During microbiome development in the first year of life, PolyProf alpha diversity increases, and beta diversity becomes increasingly like the maternal microbiome, influenced by vertical transfer, delivery mode, and breastfeeding. PolyProf is related to strain sharing among adults through social interactions. In summary, TSS genes strongly correlate with microbiome development and interpersonal strain sharing, suggesting roles for interbacterial antagonism. Since PolyProf distinguishes diverse adult disease statuses, these dynamics may contribute to non-genetic inheritance.IMPORTANCEPrevious research has demonstrated that bacteria compete within the gut microbiome using toxin secretion systems (TSS). How TSS contribute to human microbiome development and the microbiome alterations observed in human diseases is not known. This study develops a new bioinformatic tool for profiling TSS-related genes in metagenomic data. Application of this approach to large-scale human fecal metagenomic data demonstrates the dynamic association of TSS during microbiome development, including the exchange of strains among social contacts. TSS gene abundance patterns are highly predictive of 12 disease states. This study advances the field by enabling TSS profiling in metagenomes and by identifying disease and microbiome development biomarkers that provide hypotheses for future mechanistic studies and may be useful for disease diagnosis.

Dysbiosis↗

PubMed2026

Perfluorooctane sulfonate drives the synergistic dissemination of antimicrobial resistance and pathogenicity during sludge anaerobic digestion.

Per- and polyfluoroalkyl substances, one of the most prevalent and persistent emerging contaminants in sludge, may drive the dissemination of antimicrobial resistance and pathogenicity during sludge treatment. However, the mechanisms underlying perfluorooctane sulfonate (PFOS)-mediated propagation of antibiotic resistance genes (ARGs) and virulence factors (VFs) remain poorly understood. This study investigated the effects of PFOS (1 and 10 μg/g-dw) on ARGs dynamics and virulence risks. Quantitative PCR and metagenomic analysis revealed that PFOS stress led to the widespread enrichment of ARGs, the total abundance of mobile genetic elements (MGEs) and VFs also increased by 33.22-37.62% and 6.71-8.41%, respectively. Metagenomic binning results demonstrated that most metagenome-assembled genomes carrying ARGs or VFs simultaneously harbored MGEs. Mechanistically, excessive reactive oxygen species production and enhanced substrate-level phosphorylation for ATP generation may contribute to the increased horizontal transfer potential of ARGs under PFOS stress, which further facilitated the convergence of antimicrobial resistance and virulence traits within pathogens. Furthermore, PFOS may have hindered the negative regulation of the RhlI/RhlR quorum sensing system on the Type III secretion system, stimulating the secretion of VFs. This study elucidates the mechanisms by which PFOS promotes the dissemination of ARGs and pathogenicity during anaerobic digestion, highlighting the potentially overlooked environmental health risks of PFOS during sludge disposal.

Alkanesulfonic Acids↗

PubMed2026

Deletion of the Salmonella pathogenicity island 2 gene, spiC, in attenuated Salmonella Typhimurium VNP20009 optimizes its potential for bacterial schwannoma therapy.

UNLABELLED: Recent advances in systems biology and immunotherapy have spurred the investigation of bacteria as therapeutic vehicles for cancer treatment. Currently, Bacillus Calmette-Guérin remains the only FDA-approved bacterial cancer therapy; it is a live attenuated mycobacterium that is indicated for the treatment and prophylaxis of carcinoma in situ of the urinary bladder and for the prophylaxis of primary or recurrent papillary tumors following transurethral resection. Although safety concerns have been raised, attenuated Salmonella Typhimurium strains such as VNP20009 have advanced to clinical trials targeting fast-growing human tumors. Notably, this strain induces robust immunological control of slow-growing tumors such as NF2-related schwannomatosis (NF2-SWN) in preclinical murine models. Here, we genetically characterize VNP20009 with the goal of constructing genetically defined attenuated strains that retain its promising therapeutic features while improving safety. Specifically, we investigated the contribution of the Salmonella pathogenicity island I (SPI-1) and SPI-2 type III secretion systems to antitumor efficacy and biosafety. Mutation of the SPI-1 gene sipB, a key structural component required for SPI-1 type III secretion system function, partially reduced tumor control in NF2-SWN murine schwannoma models, suggesting that bacterial invasion alone does not fully account for antitumor activity. In contrast, deletion of the SPI-2 gene spiC, a key effector required for intracellular survival, preserved robust tumor regression in NF2-SWN murine schwannoma models while improving safety and reducing systemic toxicity. To create a genetically defined and tractable platform, we generated two attenuated strains-AST101 and AST101-ΔspiC-which retain key mutations present in VNP20009 but lack ill-characterized background mutations. In the syngeneic NF2-SWN mouse schwannoma model, both strains significantly suppressed tumor growth compared to PBS. Collectively, these findings support the development of rationally engineered Salmonella Typhimurium strains with enhanced safety and preserved antitumor efficacy. IMPORTANCE: Given long-standing safety concerns surrounding the therapeutic use of live bacteria, we constructed a ΔspiC mutant of VNP20009 and demonstrated that it provides a markedly improved safety profile while retaining antitumor efficacy in NF2-related schwannomatosis mouse schwannoma models. In addition, we created two genetically defined Salmonella Typhimurium strains, AST01 and AST01-ΔspiC, which incorporate the key-targeted mutations found in VNP20009 and VNP20009-ΔspiC, respectively. These engineered strains offer a well-defined genetic background, enabling precise investigation of the bacterial traits responsible for Salmonella Typhimurium-mediated tumor control and thus further improvement of attenuated strains optimized for bacteriotherapy of neoplasms.

Salmonella typhimurium↗

PubMed2026

Genomic analysis of Neisseria gonorrhoeae strains from disseminated, urogenital, and rectal infections reveals differences in genes for iron acquisition and type IV secretion.

Neisseria gonorrhoeae, an obligate human pathogen, causes the sexually transmitted infection gonorrhea. Delayed treatment or immunodeficiencies can result in a higher risk of disseminated gonococcal infection (DGI), where the infection spreads to normally sterile anatomic sites, including the bloodstream. The gonococcus produces TonB-dependent transporters (TdTs) to sequester metals from host nutritional immunity proteins. These transporters are critical for survival and are important virulence factors. We characterized genes encoding iron-regulated TdTs in the genomes of strains from disseminated (n = 47), urogenital (n = 86), and rectal (n = 12) infections. We found that gonococcal strains isolated from DGI infections were more likely to express a functional hemoglobin-iron utilization operon (hpuAB wild type and phase on [32%, n = 15/47]), to harbor nonsense mutations in tdfF (57%, n = 27/47), and were predicted to express low levels of fetA (43%, n = 20/47) when compared to strains from urogenital or rectal infections. In contrast, gonococcal strains from localized urogenital infections were associated with a higher frequency of functional tdfF genes (66%, n = 57/86) and high fetA expression (30%, n = 26/86). Additionally, strains from rectal infections were more frequently found to have high fetA expression (33%, n = 4/12) and a functional tdfF (75%, n = 9/12). Furthermore, we identified associations between the presence and absence of tdfF and the gonococcal genetic island, which encodes a type IV secretion system. These findings inform our evolving understanding of the molecular mechanisms underlying disseminated gonococcal infections.IMPORTANCEGonorrhea is an emerging, global health threat, with over 100 million new cases each year. Delayed treatment, immunosuppressive medications, and immunodeficiencies can contribute to the spread of infection to the blood, presenting as a serious disseminated manifestation. With rising antimicrobial resistance and no licensed preventative vaccine, untreatable gonorrhea is a possibility in the near future. TonB-dependent transporters are highly conserved and vital for metal acquisition and survival, making them promising targets for therapeutic or preventative strategies. Associated surface-exposed lipoproteins display greater sequence variation but contribute to metal acquisition. We analyzed the genes encoding TonB-dependent transporters and associated lipoproteins from strains associated with distinct disease manifestations. We conclude that gonococci isolated from disseminated, urogenital, and rectal sites demonstrate different phase and allelic variations in genes encoding iron transport systems and the gonococcal genetic island.

Humans↗

PubMed2026

Improving a Tn7-based luciferase reporter system for promoter activity studies.

Single-copy chromosomal integration systems are essential tools for stable gene expression in bacteria, minimizing variability associated with plasmid-based systems. The Tn7 transposon-based system is widely used for this purpose, and one important application is the generation of reporter systems, such as the bioluminescent luxCDABE operon (lux). However, current Tn7-lux vectors exhibit undesirable background expression due to cryptic promoter activity near the antibiotic resistance cassette. Here, we report the construction of an improved vector, pTn7-lux-B0015, incorporating a strong synthetic terminator upstream of the lux operon. This modification effectively eliminated basal luminescence in the absence of a promoter and enhanced the dynamic range and responsiveness of the reporter. Using a Xanthomonas citri type III secretion system promoter as a model, we demonstrate that pTn7-lux-B0015 enables more accurate detection of gene expression under relevant growth conditions. This vector provides a valuable tool for the development of precise and tunable bioluminescent reporters in bacterial systems.

Promoter Regions, Genetic↗