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Bacteriophages are the most abundant biological entities on Earth, but our understanding of many aspects of their lifecycles is still incomplete. Here, we have structurally analysed the infection cycle of the siphophage Casadabanvirus JBD30. Using its baseplate, JBD30 attaches to Pseudomonas aeruginosavia the bacterial type IV pilus, whose subsequent retraction brings the phage to the bacterial cell surface. Cryo-electron microscopy structures of the baseplate-pilus complex show that the tripod of baseplate receptor-binding proteins attaches to the outer bacterial membrane. The tripod and baseplate then open to release three copies of the tape-measure protein, an event that is followed by DNA ejection. JBD30 major capsid proteins assemble into procapsids, which expand by 7% in diameter upon filling with phage dsDNA. The DNA-filled heads are finally joined with 180-nm-long tails, which bend easily because flexible loops mediate contacts between the successive discs of major tail proteins. It is likely that the structural features and replication mechanisms described here are conserved among siphophages that utilize the type IV pili for initial cell attachment. Synopsis: To date, available structural insights into architecture and infection cycles of various bacteriophages remain fragmented. Here, several cryo-electron microscopy structures reveal the complete lifecycle of the siphophage Casadabanvirus JBD30 during infection of its host Pseudomonas aeruginosa. JBD30 uses its baseplate to bind to the bacterial type IV pilus, whose retraction brings JBD30 to the bacterial cell surface. The structure of the baseplate-pilus complex reveals a trimer of baseplate receptor-binding proteins that attach to the outer bacterial membrane. JBD30 major capsid proteins assemble into procapsids that undergo conformational changes to allow expansion upon filling with phage dsDNA. The tail of JBD30 bends easily due to flexible loops that mediate contacts between the successive discs of major tail proteins. Cryo-electron microscopy structures of the siphophage Casadabanvirus JBD30 reveal its cell attachment, genome delivery, and virion assembly steps. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, which emerged in early 2020, has had a profound and lasting impact on global health, resulting in over 7.0 million deaths and persistent challenges. In addition to acute concerns, there is growing attention being given to the long COVID health consequences for survivors of COVID-19 with documented cases of cardiovascular abnormalities, liver disturbances, lung complications, kidney issues, and noticeable cognitive deficits. Recent studies have investigated the physiological changes in various organs following prolonged exposure to murine hepatitis virus-1 (MHV-1), a coronavirus, in mouse models. One significant finding relates to the effects on the gastrointestinal tract, an area previously understudied regarding the long-lasting effects of COVID-19. This research sheds light on important observations in the intestines during both the acute and the prolonged phases following MHV-1 infection, which parallel specific changes seen in humans after exposure to SARS-CoV-2. Our study investigates the histopathological alterations in the small intestine following MHV-1 infection in murine models, revealing significant changes reminiscent of inflammatory bowel disease (IBD), celiac disease. Notable findings include mucosal inflammation, lymphoid hyperplasia, goblet cell hyperplasia, and immune cell infiltration, mirroring pathological features observed in IBD. Additionally, MHV-1 infection induces villous atrophy, altered epithelial integrity, and inflammatory responses akin to celiac disease and IBD. SPIKENET (SPK) treatment effectively mitigates intestinal damage caused by MHV-1 infection, restoring tissue architecture and ameliorating inflammatory responses. Furthermore, investigation into long COVID reveals intricate inflammatory profiles, highlighting the potential of SPK to modulate intestinal responses and restore tissue homeostasis. Understanding these histopathological alterations provides valuable insights into the pathogenesis of COVID-induced gastrointestinal complications and informs the development of targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

Microorganisms are key players in the global biogeochemical sulfur cycle. Among them, some have garnered particular attention due to their electrical activity and ability to perform extracellular electron transfer. A growing body of research has highlighted their extensive phylogenetic and metabolic diversity, revealing their crucial roles in ecological processes. In this review, we delve into the electron transfer process between sulfate-reducing bacteria and anaerobic alkane-oxidizing archaea, which facilitates growth within syntrophic communities. Furthermore, we review the phenomenon of long-distance electron transfer and potential extracellular electron transfer in multicellular filamentous sulfur-oxidizing bacteria. These bacteria, with their vast application prospects and ecological significance, play a pivotal role in various ecological processes. Subsequently, we discuss the important role of the pili/cytochrome for electron transfer and presented cutting-edge approaches for exploring and studying electroactive microorganisms. This review provides a comprehensive overview of electroactive microorganisms participating in the biogeochemical sulfur cycle. By examining their electron transfer mechanisms, and the potential ecological and applied implications, we offer novel insights into microbial sulfur metabolism, thereby advancing applications in the development of sustainable bioelectronics materials and bioremediation technologies. [ABSTRACT FROM AUTHOR]

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Geobacter sulfurreducens profoundly shapes Earth's biogeochemistry by discharging respiratory electrons to minerals and other microbes through filaments of a two-decades-long debated identity. Cryogenic electron microscopy has revealed filaments of redox-active cytochromes, but the same filaments have exhibited hallmarks of organic metal-like conductivity under cytochrome denaturing/inhibiting conditions. Prior structure-based calculations and kinetic analyses on multi-heme proteins are synthesized herein to propose that a minimum of ~7 cytochrome 'nanowires' can carry the respiratory flux of a Geobacter cell, which is known to express somewhat more (=20) filaments to increase the likelihood of productive contacts. By contrast, prior electrical and spectroscopic structural characterizations are argued to be physiologically irrelevant or physically implausible for the known cytochrome filaments because of experimental artifacts and sample impurities. This perspective clarifies our mechanistic understanding of physiological metalmicrobe interactions and advances synthetic biology efforts to optimize those interactions for bioremediation and energy or chemical production. [ABSTRACT FROM AUTHOR]

To advance probiotic research, a comprehensive understanding of bacterial interactions with human physiology at the molecular and cellular levels is fundamental. Lacticaseibacillus rhamnosus LGG® is a bacterial strain that has long been recognized for its beneficial effects on human health. Probiotic effector molecules derived from LGG®, including secreted proteins, surface-anchored proteins, polysaccharides, and lipoteichoic acids, which interact with host physiological processes have been identified. In vitro and animal studies have revealed that specific LGG® effector molecules stimulate epithelial cell survival, preserve intestinal barrier integrity, reduce oxidative stress, mitigate excessive mucosal inflammation, enhance IgA secretion, and provide long-term protection through epigenetic imprinting. Pili on the cell surface of LGG® promote adhesion to the intestinal mucosa and ensure close contact to host cells. Extracellular vesicles produced by LGG® recapitulate many of these effects through their cargo of effector molecules. Collectively, the effector molecules of LGG® exert a significant influence on both the gut mucosa and immune system, which promotes intestinal homeostasis and immune tolerance. [ABSTRACT FROM AUTHOR]

The Gram-positive human pathogen Group A Streptococcus (GAS, Streptococcus pyogenes) employs an arsenal of virulence factors that contribute to its pathogenesis. The pilus is an important factor that enables the pathogen to adhere to and colonize host tissues. Emerging research in pilus function shows that pili's involvement in establishing infection extends beyond host adhesion. The diversity of GAS pilus types reflect the varying characteristics identified in different pili. With the development of new experimental systems and animal models, a wider range of biological functions have been explored. This brief review summarizes recent reports of new functions in different GAS pilus types and the methodologies that contributed to the findings. The established importance of the pilus in GAS pathogenesis makes this surface structure a promising vaccine target. This article also reviews recent advancements in pilus-based vaccine strategies and discusses certain aspects that should be considered in vaccine development according to the newly defined properties of pili. [ABSTRACT FROM AUTHOR]

ABSTRACTAmong genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as “pilin types.” Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

The human pathogen Streptococcus pyogenes, or Group A Streptococcus (GAS), is associated with a variety of diseases ranging from mild skin and soft tissue infections to invasive diseases and immune sequelae such as rheumatic heart disease. We have recently reported that one of the virulence factors of this pathogen, the pilus, has inflammatory properties and strongly stimulates the innate immune system. Here we used a range of nonpathogenic Lactococcus lactis gain‐of‐function mutants, each expressing one of the major pilus types of GAS, to compare the immune responses generated by various types of fully assembled pili. In vitro assays indicated variability in the inflammatory response induced by different pili, with the fibronectin‐binding, collagen‐binding, T antigen (FCT)‐1‐type pilus from GAS serotype M6/T6 inducing significantly stronger cytokine secretion than other pili. Furthermore, we established that the same trend of pili‐mediated immune response could be modeled in Galleria mellonella larvae, which possess a similar innate immune system to vertebrates. Counterintuitively, across the panel of pili types examined in this study, we observed a negative correlation between the intensity of the immune response demonstrated in our experiments and the disease severity observed clinically in the GAS strains associated with each pilus type. This observation suggests that pili‐mediated inflammation is more likely to promote bacterial clearance instead of causing disruptive damages that intensify pathogenesis. This also indicates that pili may not be the main contributor to the inflammatory symptoms seen in GAS diseases. Rather, the immune‐potentiating properties of the pilus components could potentially be exploited as a vaccine adjuvant. [ABSTRACT FROM AUTHOR]

Geobacter sulfurreducens profoundly shapes Earth’s biogeochemistry by discharging respiratory electrons to minerals and other microbes through filaments of a two-decades-long debated identity. Cryogenic electron microscopy has revealed filaments of redox-active cytochromes, but the same filaments have exhibited hallmarks of organic metal-like conductivity under cytochrome denaturing/inhibiting conditions. Prior structure-based calculations and kinetic analyses on multi-heme proteins are synthesized herein to propose that a minimum of ~7 cytochrome ‘nanowires’ can carry the respiratory flux of a Geobacter cell, which is known to express somewhat more (≥20) filaments to increase the likelihood of productive contacts. By contrast, prior electrical and spectroscopic structural characterizations are argued to be physiologically irrelevant or physically implausible for the known cytochrome filaments because of experimental artifacts and sample impurities. This perspective clarifies our mechanistic understanding of physiological metal-microbe interactions and advances synthetic biology efforts to optimize those interactions for bioremediation and energy or chemical production.

ABSTRACTThe ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems regularly utilized as DNA-editing tools. While Neisseria gonorrhoeae does not have an endogenous CRISPR, the commensal species Neisseria lactamica encodes a functional Type I-C CRISPR-Cas system. We have established an isopropyl β-d-1-thiogalactopyranoside added (IPTG)-inducible, CRISPR interference (CRISPRi) platform based on the N. lactamica Type I-C CRISPR missing the Cas3 nuclease to allow locus-specific transcriptional repression. As proof of principle, we targeted a non-phase-variable version of the opaD gene. We show that CRISPRi can downregulate opaD gene and protein expression, resulting in bacterial inability to stimulate neutrophil oxidative responses and to bind to an N-terminal fragment of CEACAM1. Importantly, we used CRISPRi to effectively knockdown all the transcripts of all 11 opa genes using a five-spacer CRISPR array, allowing control of the entire phase-variable opa family in strain FA1090. We also report that repression is reversible following IPTG removal. Finally, we showed that the Type I-C CRISPRi system can conditionally reduce the expression of two essential genes. This CRISPRi system will allow the interrogation of every Gc gene, essential and non-essential, to study physiology and pathogenesis and aid in antimicrobial development.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have proven instrumental in genetically manipulating many eukaryotic and prokaryotic organisms. Despite its usefulness, a CRISPR system had yet to be developed for use in Neisseria gonorrhoeae (Gc), a bacterium that is the main etiological agent of gonorrhea infection. Here, we developed a programmable and IPTG-inducible Type I-C CRISPR interference (CRISPRi) system derived from the commensal species Neisseria lactamica as a gene repression system in Gc. As opposed to generating genetic knockouts, the Type I-C CRISPRi system allows us to block transcription of specific genes without generating deletions in the DNA. We explored the properties of this system and found that a minimal spacer array is sufficient for gene repression while also facilitating efficient spacer reprogramming. Importantly, we also show that we can use CRISPRi to knockdown genes that are essential to Gc that cannot normally be knocked out under laboratory settings. Gc encodes ~800 essential genes, many of which have no predicted function. We predict that this Type I-C CRISPRi system can be used to help categorize gene functions and perhaps contribute to the development of novel therapeutics for gonorrhea.

Microorganisms are key players in the global biogeochemical sulfur cycle. Among them, some have garnered particular attention due to their electrical activity and ability to perform extracellular electron transfer. A growing body of research has highlighted their extensive phylogenetic and metabolic diversity, revealing their crucial roles in ecological processes. In this review, we delve into the electron transfer process between sulfate-reducing bacteria and anaerobic alkane-oxidizing archaea, which facilitates growth within syntrophic communities. Furthermore, we review the phenomenon of long-distance electron transfer and potential extracellular electron transfer in multicellular filamentous sulfur-oxidizing bacteria. These bacteria, with their vast application prospects and ecological significance, play a pivotal role in various ecological processes. Subsequently, we discuss the important role of the pili/cytochrome for electron transfer and presented cutting-edge approaches for exploring and studying electroactive microorganisms. This review provides a comprehensive overview of electroactive microorganisms participating in the biogeochemical sulfur cycle. By examining their electron transfer mechanisms, and the potential ecological and applied implications, we offer novel insights into microbial sulfur metabolism, thereby advancing applications in the development of sustainable bioelectronics materials and bioremediation technologies.

Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine-isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.

Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes - the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.

BackgroundThe purpose of this study was to conduct a 20-week controlled trial of lovastatin (10 to 40 mg/day) in youth with fragile X syndrome (FXS) ages 10 to 17 years, combined with an open-label treatment of a parent-implemented language intervention (PILI), delivered via distance video teleconferencing to both treatment groups, lovastatin and placebo.MethodA randomized, double-blind trial was conducted at one site in the Sacramento, California, metropolitan area. Fourteen participants were assigned to the lovastatin group; two participants terminated early from the study. Sixteen participants were assigned to the placebo group. Lovastatin or placebo was administered orally in a capsule form, starting at 10 mg and increasing weekly or as tolerated by 10 mg increments, up to a maximum dose of 40 mg daily. A PILI was delivered to both groups for 12 weeks, with 4 activities per week, through video teleconferencing by an American Speech-Language Association-certified Speech-Language Pathologist, in collaboration with a Board-Certified Behavior Analyst. Parents were taught to use a set of language facilitation strategies while interacting with their children during a shared storytelling activity. The main outcome measures included absolute change from baseline to final visit in the means for youth total number of story-related utterances, youth number of different word roots, and parent total number of story-related utterances.ResultsSignificant increases in all primary outcome measures were observed in both treatment groups. Significant improvements were also observed in parent reports of the severity of spoken language and social impairments in both treatment groups. In all cases, the amount of change observed did not differ across the two treatment groups. Although gains in parental use of the PILI-targeted intervention strategies were observed in both treatment groups, parental use of the PILI strategies was correlated with youth gains in the placebo group and not in the lovastatin group.ConclusionParticipants in both groups demonstrated significant changes in the primary outcome measures. The magnitude of change observed across the two groups was comparable, providing additional support for the efficacy of the use of PILI in youth with FXS.Trial registrationUS National Institutes of Health (ClinicalTrials.gov), NCT02642653. Registered 12/30/2015.

To advance probiotic research, a comprehensive understanding of bacterial interactions with human physiology at the molecular and cellular levels is fundamental. Lacticaseibacillus rhamnosus LGG® is a bacterial strain that has long been recognized for its beneficial effects on human health. Probiotic effector molecules derived from LGG®, including secreted proteins, surface-anchored proteins, polysaccharides, and lipoteichoic acids, which interact with host physiological processes have been identified. In vitro and animal studies have revealed that specific LGG® effector molecules stimulate epithelial cell survival, preserve intestinal barrier integrity, reduce oxidative stress, mitigate excessive mucosal inflammation, enhance IgA secretion, and provide long-term protection through epigenetic imprinting. Pili on the cell surface of LGG® promote adhesion to the intestinal mucosa and ensure close contact to host cells. Extracellular vesicles produced by LGG® recapitulate many of these effects through their cargo of effector molecules. Collectively, the effector molecules of LGG® exert a significant influence on both the gut mucosa and immune system, which promotes intestinal homeostasis and immune tolerance.

The Gram-positive human pathogen Group A Streptococcus (GAS, Streptococcus pyogenes) employs an arsenal of virulence factors that contribute to its pathogenesis. The pilus is an important factor that enables the pathogen to adhere to and colonize host tissues. Emerging research in pilus function shows that pili’s involvement in establishing infection extends beyond host adhesion. The diversity of GAS pilus types reflect the varying characteristics identified in different pili. With the development of new experimental systems and animal models, a wider range of biological functions have been explored. This brief review summarizes recent reports of new functions in different GAS pilus types and the methodologies that contributed to the findings. The established importance of the pilus in GAS pathogenesis makes this surface structure a promising vaccine target. This article also reviews recent advancements in pilus-based vaccine strategies and discusses certain aspects that should be considered in vaccine development according to the newly defined properties of pili.

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra‐positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild‐type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus‐related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence‐related phenotypes of the serotype M49 strain of S. pyogenes. [ABSTRACT FROM AUTHOR]

Sortase‐mediated pili are flexible rod proteins composed of major and minor/tip pilins, playing important roles in the initial adhesion of bacterial cells to host tissues. The pilus shaft is formed by covalent polymerization of major pilins, and the minor/tip pilin is covalently attached to the tip of the shaft involved in adhesion to the host cell. The Gram‐positive bacterium Clostridium perfringens has a major pilin, and a minor/tip pilin (CppB) with the collagen‐binding motif. Here, we report X‐ray structures of CppB collagen‐binding domains, collagen‐binding assays and mutagenesis analysis, demonstrating that CppB collagen‐binding domains adopt an L‐shaped structure in open form, and that a small β‐sheet unique to CppB provides a scaffold for a favourable binding site for collagen peptide. [ABSTRACT FROM AUTHOR]

Objective: To characterise a collection of pili-carrying and none pili-carrying pneumococcal isolates of clinical origin for serotypes, antibiotic resistance and genotype. Methods: In total, 42 clinical isolates were collected between October 2017 and December 2019. Those isolates were analysed for antimicrobial susceptibility, serotype distribution, detection of pneumococcal virulence and pilus genes. Multilocus sequence typing was performed only for piliated isolates, followed by phylogenetic analysis. Results: The common isolation sites among the pneumococcal isolates were tracheal aspirate (28.6%), blood (26.2%), and sputum (23.8%). Fifty percent isolates were resistant to erythromycin, tetracycline (50.0%) and trimethoprim-sulfamethoxazole (43.0%). The most frequent were serotypes 19F (28.6%), 6A/B (23.8%) and 19A (14.3%). Piliated isolates were detected in a small proportion (33.3%); 64.3% were multidrug-resistant. ST320 was the prevalent sequence type among the piliated isolates and genetically related to the Pneumococcal Molecular Epidemiology Network clones Taiwan19F-14 (CC271). In the phylogenetic analysis, some piliated isolates showed a close association having similar ST320, carrying serotype 19A and both pilus genes indicating their clonal spread. Conclusions: Pneumococcal lineages of piliated isolates have been globally disseminated and pili could have played a role in the spread of antibiotic resistant clones.

Objective: To characterise a collection of pili-carrying and none pili-carrying pneumococcal isolates of clinical origin for serotypes, antibiotic resistance and genotype. Methods: In total, 42 clinical isolates were collected between October 2017 and December 2019. Those isolates were analysed for antimicrobial susceptibility, serotype distribution, detection of pneumococcal virulence and pilus genes. Multilocus sequence typing was performed only for piliated isolates, followed by phylogenetic analysis. Results: The common isolation sites among the pneumococcal isolates were tracheal aspirate (28.6%), blood (26.2%), and sputum (23.8%). Fifty percent isolates were resistant to erythromycin, tetracycline (50.0%) and trimethoprim-sulfamethoxazole (43.0%). The most frequent were serotypes 19F (28.6%), 6A/B (23.8%) and 19A (14.3%). Piliated isolates were detected in a small proportion (33.3%); 64.3% were multidrug-resistant. ST320 was the prevalent sequence type among the piliated isolates and genetically related to the Pneumococcal Molecular Epidemiology Network clones Taiwan19F-14 (CC271). In the phylogenetic analysis, some piliated isolates showed a close association having similar ST320, carrying serotype 19A and both pilus genes indicating their clonal spread. Conclusions: Pneumococcal lineages of piliated isolates have been globally disseminated and pili could have played a role in the spread of antibiotic resistant clones. [ABSTRACT FROM AUTHOR]

Abstract Streptococcus parasuis (S. parasuis) is a close relative of Streptococcus suis (S. suis), composed of former members of S. suis serotypes 20, 22 and 26. S. parasuis could infect pigs and cows, and recently, human infection cases have been reported, making S. parasuis a potential opportunistic zoonotic pathogen. In this study, we analysed the genomic characteristics of S. parasuis, using pan-genome analysis, and compare some phenotypic determinants such as capsular polysaccharide, integrative conjugative elements, CRISPR-Cas system and pili, and predicted the potential virulence genes by associated analysis of the clinical condition of isolated source animals and genotypes. Furthermore, to discuss the relationship with S. suis, we compared these characteristics of S. parasuis with those of S. suis. We found that the characteristics of S. parasuis are similar to those of S. suis, both of them have “open” pan-genome, their antimicrobial resistance gene profiles are similar and a srtF pilus cluster of S. suis was identified in S. parasuis genome. But S. parasuis still have its unique characteristics, two novel pilus clusters are and three different type CRISPR-Cas system were found. Therefore, this study provides novel insights into the interspecific and intraspecific genetic characteristics of S. parasuis, which can be useful for further study of this opportunistic pathogen, such as serotyping, diagnostics, vaccine development, and study of the pathogenesis mechanism.

The bacterial cell pole has long been recognized as a defined compartment for enzymatic activities that are important or even vital for the cell. Polarity of diguanylate cyclases and phosphodiesterases, enzymes that synthesize and degrade the second messenger c-di-GMP, has now been demonstrated for several bacterial systems. Here we review these polar regulatory systems and show how the asymmetry of c-di-GMP production and turnover in concert with different modes of activation and deactivation creates heterogeneity in cellular c-di-GMP levels. We highlight how this heterogeneity generates a diverse set of phenotypic identities or states and how this may benefit the cell population, and we discuss reasons why the polarity of c-di-GMP signaling is probably widespread among bacteria. [ABSTRACT FROM AUTHOR]

Biological redox reactions often use a set‐up in which final redox partners are localized in different compartments and electron transfer (ET) among them is mediated by redox‐active molecules. In enzymes, these ET processes occur over nm distances, whereas multi‐protein filaments bridge μm ranges. Electrons are transported over cm ranges in cable bacteria, which are formed by thousands of cells. In this review, we describe molecular mechanisms that explain how respiration in a compartmentalized set‐up ensures redox homeostasis. We highlight mechanistic studies on ET through metal‐free peptides and proteins demonstrating that long‐distance ET is possible because amino acids Tyr, Trp, Phe, and Met can act as relay stations. This cuts one long ET into several short reaction steps. The chances and challenges of long‐distance ET for cellular redox reactions are then discussed. [ABSTRACT FROM AUTHOR]

Enterococcus faecalis virulence requires cell wall‐associated proteins, including the sortase‐assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase‐assembled pilus biogenesis in Gram‐positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co‐localize with newly synthesized PG. Instead, surface‐exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis. [ABSTRACT FROM AUTHOR]

Members of the oral mitis group streptococci including Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii are the most abundant inhabitants of human oral cavity and dental plaque, and have been implicated in infectious complications such as bacteremia and infective endocarditis. Oral mitis group streptococci are genetically close to Streptococcus pneumoniae; however, they do not produce cytolysin (pneumolysin), which is a key virulence factor of S. pneumoniae. Similar to S. pneumoniae, oral mitis group streptococci possess several cell surface proteins that bind to the cell surface components of host mammalian cells. S. sanguinis expresses long filamentous pili that bind to the matrix proteins of host cells. The cell wall–anchored nuclease of S. sanguinis contributes to the evasion of the neutrophil extracellular trap by digesting its web‐like extracellular DNA. Oral mitis group streptococci produce glucosyltransferases, which synthesize glucan (glucose polymer) from sucrose of dietary origin. Neuraminidase (NA) is a virulent factor in oral mitis group streptococci. Influenza type A virus (IAV) relies on viral NA activity to release progeny viruses from infected cells and spread the infection, and NA‐producing oral streptococci elevate the risk of IAV infection. Moreover, oral mitis group streptococci produce hydrogen peroxide (H2O2) as a by‐product of sugar metabolism. Although the concentrations of streptococcal H2O2 are low (1–2 mM), they play important roles in bacterial competition in the oral cavity and evasion of phagocytosis by host macrophages and neutrophils. In this review, we intended to describe the diverse pathogenicity of oral mitis group streptococci. [ABSTRACT FROM AUTHOR]

This study explored the use of pili nutshells as aggregates and the economic value compared to standard aggregates. It involved observation, experimentation, analysis, and interpretation of data to determine the most appropriate design mixture of pili nutshells in concrete and the compressive strength of concrete using shells to ascertain its suitability as a replacement to aggregates in the production of the concrete mixture. Findings revealed that standard mixture and concrete with 25% pili nutshells replacement as fine aggregates has high workability. However, concrete with 25% pili nutshells replacement as coarse aggregates had medium workability. Furthermore, standard concrete is more economical compared to concrete with pili nutshells as coarse and fine aggregates. Researchers concluded that pili nutshells are more effective as fine aggregates than as coarse aggregates. Further studies on the properties/components of pili nutshells shall be made to determine the necessary treatment before they can be used as aggregates.

Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid. [Display omitted] • Cryo-EM of the R388 pilus shows a polymer with a subunit/PG 32 complex • The two acyl chains of PG 32 are splayed • The pilus is shown not to be able to retract • Pilus production is induced by the presence of recipient cells Vadakkepat et al. report the structure of the conjugative pilus produced by the plasmid R388. The structure is a polymer of subunits/phospholipid units, with the phospholipid acyl chain splayed consistent with findings by fluorescence microscopy that pilus production is induced by recipient cells and that the pilus is unable to retract. [ABSTRACT FROM AUTHOR]

Extracellular cellular adhesins facilitate microbial aggregation; however, most of the information about extracellular adhesins is based on pure culture studies. In this study, we characterized the hydrophobic characteristics and distribution of the extracellular adhesins in environmental biofilms and flocs. The hydrophobic characteristics of the extracellular adhesins were studied by sonicating the microbial aggregates to disperse the cells and by fractionating them using the microbial adhesion to the hydrocarbon method. Furthermore, we probed environmental biofilms and flocs using immunohistochemistry coupled with confocal laser scanning microscopy for reimaging the microbial aggregates based on extracellular adhesins. Small flocs have a relatively dispersed distribution of extracellular adhesins (flagella, fimbriae, pili, and amyloid adhesins). The stratified distribution of extracellular adhesins was observed in environmental biofilms. It was observed that the pili and amyloid adhesins were predominantly present in the core of biofilms, whereas flagella and fimbriae were present in the outer layer of the microbial aggregates. The dispersion of microbial aggregates is one of the limiting factors that challenge the sustainable application of wastewater treatment processes. Greater attention to the components of extracellular protein (such as the adhesins) is required to understand the aggregation of dispersible environmental microbial aggregates. [Display omitted] • Environmental microbial aggregates reimagined incorporating extracellular adhesins. • Adhesins are dispersed in floc, whereas adhesins in biofilms are stratified. • Flagella and fimbriae were relatively predominant in the outer layer of biofilms. • Amyloid adhesins and pili were relatively predominant in the core of biofilms. • Adhesins were differentially distributed in hydrophobic and hydrophilic regions. [ABSTRACT FROM AUTHOR]

Background: Pili producing genes in different life cycles of Mycobacterium tuberculosis (M. tuberculosis) were assessed. M. tuberculosis has two life cycles: dormant and active states. We aimed to assess the pili producing genes such as curli pili of M. tuberculosis (mtp) encoded by the mtp gene (Rv3312A) and fimbrial low-molecular-weight protein encoded by flp gene (Rv3656c) which were compared and analyzed. Methods: Two hundred M. tuberculosis isolates were investigated both at active and dormant states for production and expression of pili. The dormant M. tuberculosis was achieved by incubation in a sealed tube (modified Wayne method). The susceptibility of M. tuberculosis was evaluated on genes, rpob, inh, katg, and gyra by using multiplex polymerase chain reaction (PCR) and single-strand conformational polymorphism methods. The PCR–restriction fragment length polymorphism was used to express pili genes mtp and flp and then the PCR products was digested using restriction enzyme Fnu4HI, XmaI, and MspJI and AciI, TagII, and HaeII, respectively. The transmission electron microscopy was also used to detect pili in different isolates. The result was compared and analyzed using H37RV as a gold standard. Results: The mtp and flp PCR products were 263 and 122 bp in the studied strains irrespective of M. tuberculosis different life cycles, respectively. The PCR products were analyzed on 8% Polyacrylamide gel electrophoresis (PAGE), and in the 180/200 (20%), producing five fragments of 25,40,45,63,90 bp with the Fun4HI and two fragments of 126,138 bp with the XmaI and uncut with the MspJI for mtp gen were obtained at the dormant and active states of M. tuberculosis (P < 0.05). Similarly in flp gene producing three fragments of 22,35,65 bp with AciI and two fragments of 35.87 bp with TagII and two fragments of 38.84 bp with HaeII were obtained (P < 0.05). In contrast to genotyping analysis, the electron microscopy examination showed protruding of pili from M. tuberculosis, especially in dormant mycobacterium (15/100; 15%), that was multidrug resistance and extensive drug resistance isolates (P > 0.05). Conclusion: Pili were shown by electron microscopy, although at the gene expression, the insignificant difference was observed at the dormant strains in comparison to active states. Therefore, we may conclude that other genes might be involved in pili production of M. tuberculosis that needs further investigation. Although, the resistance phenomena might influence the pili producing gene expression that showed in our results.

ABSTRACT Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

The surface of Gram-positive and Gram-negative bacteria contains long hair-like proteinaceous protrusion known as pili or fimbriae. Historically, pilin proteins were considered to play a major role in the transfer of genetic material during bacterial conjugation. Recent findings however elucidate their importance in virulence, biofilm formation, phage transduction, and motility. Therefore, it is crucial to gain mechanistic insights on the subcellular assembly of pili and the localization patterns of their subunit proteins (major and minor pilins) that aid the macromolecular pilus assembly at the bacterial surface. In this article, we review the current knowledge of pilus assembly mechanisms in a wide range of Gram-positive and Gram-negative bacteria, including subcellular localization patterns of a few pilin subunit proteins and their role in virulence and pathogenesis.

Fecal microbiota transplantation (FMT) is an efficient treatment for recurrent Clostridioides difficile infection and currently investigated as a treatment for other intestinal and systemic diseases. Better understanding of the species potentially transferred in FMT is needed. We isolated from a healthy fecal donor a novel strain E10-96H of Pseudoruminococcus massiliensis, a recently described strictly anaerobic species currently represented only by the type strain. The whole genome sequence of E10-96H had over 98% similarity with the type strain. E10-96H carries 20 glycoside hydrolase encoding genes, degrades starch in vitro and thus may contribute to fiber degradation, cross-feeding of other species and butyrate production in the intestinal ecosystem. The strain carries pilus-like structures, harbors pilin genes in its genome and adheres to enterocytes in vitro but does not provoke a proinflammatory response. P. massiliensis seems to have commensal behavior with the host epithelium, and its role in intestinal ecology should be studied further.

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intraand extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy. [ABSTRACT FROM AUTHOR]

Actinobacterial species are mostly thought to be nonmotile. Recent studies have revealed the degenerate evolution of flagella in this phylum and different flagellar rod compositions from the classical model. Moreover, flagella-independent motility by various means has been reported in Streptomyces spp. and Mycobacterium spp., but the underlying mechanisms remain elusive., Competing Interests: Declaration of interests None are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.

Fimbrial adhesins promote bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified new fimbriae belonging to the π group, which we named PL (P-like) fimbriae since the genetic organization and sequence are similar to those of P and related fimbriae. Genes encoding PL fimbriae located on IncF plasmids are present in diverse E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit divergence in adhesin-encoding genes and could be divided into 5 classes based on sequence differences in the PlfG adhesin. plf genes from two predominant PlfG adhesin classes, PlfG class I (PlfGI) and PlfGII, were cloned. PL fimbriae were visualized by electron microscopy, associated with increased biofilm, demonstrated distinct hemagglutination profiles, and promoted adherence to human bladder and kidney epithelial cells. The genes encoding hybrid fimbriae were comprised of genes from plfQT598, wherein plfG was replaced by papG; the adhesin-encoding genes were also functional and mediated adherence to epithelial cells, demonstrating compatibility between the components of these two types of fimbriae. Deletion of plf genes did not reduce colonization of the mouse urinary tract in a single-strain infection model. In contrast, loss of plf genes significantly reduced competitive colonization in the mouse kidneys. Furthermore, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences from and similarities to P fimbriae, which mediated adherence to host cells and improved competitive colonization of the mouse kidney. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum (Rs), the causative agent of devastating wilt disease in several major and minor economic crops, is considered one of the most destructive bacterial plant pathogens. However, the mechanism(s) by which Rs counteracts host-associated environmental stress is still not clearly elucidated. To investigate possible stress management mechanisms, orthologs of stress-responsive genes in the Rs genome were searched using a reference set of known genes. The genome BLAST approach was used to find the distributions of these orthologs within different Rs strains. BLAST results were first confirmed from the KEGG Genome database and then reconfirmed at the protein level from the UniProt database. The distribution pattern of these stress-responsive factors was explored through multivariate analysis and STRING analysis. STRING analysis of stress-responsive genes in connection with different secretion systems of Rs was also performed. Initially, a total of 28 stress-responsive genes of Rs were confirmed in this study. STRING analysis revealed an additional 7 stress-responsive factors of Rs, leading to the discovery of a total of 35 stress-responsive genes. The segregation pattern of these 35 genes across 110 Rs genomes was found to be almost homogeneous. Increasing interactions of Rs stress factors were observed in six distinct clusters, suggesting six different types of stress responses: membrane stress response (MSR), osmotic stress response (OSR), oxidative stress response (OxSR), nitrosative stress response (NxSR), and DNA damage stress response (DdSR). Moreover, a strong network of these stress responses was observed with type 3 secretion system (T3SS), general secretory proteins (GSPs), and different types of pili (T4P, Tad, and Tat). To the best of our knowledge, this is the first report on overall stress response management by Rs and the potential connection with secretion systems. [ABSTRACT FROM AUTHOR]

Streptococcus parasuis (S. parasuis) is a close relative of Streptococcus suis (S. suis), composed of former members of S. suis serotypes 20, 22 and 26. S. parasuis could infect pigs and cows, and recently, human infection cases have been reported, making S. parasuis a potential opportunistic zoonotic pathogen. In this study, we analysed the genomic characteristics of S. parasuis, using pan-genome analysis, and compare some phenotypic determinants such as capsular polysaccharide, integrative conjugative elements, CRISPR-Cas system and pili, and predicted the potential virulence genes by associated analysis of the clinical condition of isolated source animals and genotypes. Furthermore, to discuss the relationship with S. suis, we compared these characteristics of S. parasuis with those of S. suis. We found that the characteristics of S. parasuis are similar to those of S. suis, both of them have "open" pan-genome, their antimicrobial resistance gene profiles are similar and a srtF pilus cluster of S. suis was identified in S. parasuis genome. But S. parasuis still have its unique characteristics, two novel pilus clusters are and three different type CRISPR-Cas system were found. Therefore, this study provides novel insights into the interspecific and intraspecific genetic characteristics of S. parasuis, which can be useful for further study of this opportunistic pathogen, such as serotyping, diagnostics, vaccine development, and study of the pathogenesis mechanism. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET) is an effective mechanism for microbial species to exchange electrons cooperatively during syntrophic metabolism. It is generally accepted that DIET is mainly mediated by electrically conductive pili and outer surface c -type cytochromes (c -Cyts). However, as an extracellular matrix is ubiquitous and abundant on the surface of microorganisms, the effect and mechanism of exopolysaccharides on DIET are still unclear. This study constructed a co-culture of exopolysaccharides-deficient Geobacter sulfurreducens with Geobacter metallireducens to explore the role of exopolysaccharides in DIET. Results revealed that the deficiency of exopolysaccharides extended the metabolic period of the co-culture by 44.4% and changed the proportions of each species in the co-culture. The exopolysaccharides-deficient co-culture failed to form large, tight spherical aggregates and the expression of c -Cyts and pili was decreased. The addition of magnetite and granular activated carbon (GAC), respectively, might compensate for the functions of c -Cyts and pili in the first generation of co-culture, but the stimulatory effect on the metabolic stable period co-culture was fairly limited. These findings demonstrate that non-conductive exopolysaccharides are an important component of DIET aggregates and an extracellular matrix for DIET-required c -Cyts. [ABSTRACT FROM AUTHOR]

ABSTRACT Protein glycosylation systems are widely recognized in bacteria, including members of the genus Neisseria. In most bacterial species, the molecular mechanisms and evolutionary contexts underpinning target protein selection and the glycan repertoire remain poorly understood. Broad-spectrum O-linked protein glycosylation occurs in all human-associated species groups within the genus Neisseria, but knowledge of their individual glycoprotein repertoires is limited. Interestingly, PilE, the pilin subunit of the type IV pilus (Tfp) colonization factor, is glycosylated in Neisseria gonorrhoeae and Neisseria meningitidis but not in the deeply branching species N. elongata subsp. glycolytica. To examine this in more detail, we assessed PilE glycosylation status across the genus and found that PilEs of commensal clade species are not modified by the gonococcal PglO oligosaccharyltransferase. Experiments using PglO oligosaccharyltransferases from across the genus expressed in N. gonorrhoeae showed that although all were capable of broad-spectrum protein glycosylation, those from a deep-branching group of commensals were unable to support resident PilE glycosylation. Further glycoproteomic analyses of these strains using immunoblotting and mass spectrometry revealed other proteins differentially targeted by otherwise remarkably similar oligosaccharyltransferases. Finally, we generated pglO allelic chimeras that begin to localize PglO protein domains associated with unique substrate targeting activities. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest in the neisserial protein substrates and oligosaccharyltransferases has significant potential to inform the structure-function relationships operating in these and related bacterial protein glycosylation systems. IMPORTANCE Although general protein glycosylation systems have been well recognized in prokaryotes, the processes governing their distribution, function, and evolution remain poorly understood. Here, we have begun to address these gaps in knowledge by comparative analyses of broad-spectrum O-linked protein glycosylation manifest in species within the genus Neisseria that strictly colonize humans. Using N. gonorrhoeae as a well-defined model organism in conjunction with comparative genomics, intraspecies gene complementation, and glycoprotein phenotyping, we discovered clear differences in both glycosylation susceptibilities and enzymatic targeting activities of otherwise largely conserved proteins. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest within Neisseria species has significant potential to elucidate the structure-function relationships operating in these and related systems and to inform novel approaches to applied glycoengineering strategies.

Direct interspecies electron transfer (DIET) is an effective mechanism for microbial species to exchange electrons cooperatively during syntrophic metabolism. It is generally accepted that DIET is mainly mediated by electrically conductive pili and outer surface c-type cytochromes (c-Cyts). However, as an extracellular matrix is ubiquitous and abundant on the surface of microorganisms, the effect and mechanism of exopolysaccharides on DIET are still unclear. This study constructed a co-culture of exopolysaccharides-deficient Geobacter sulfurreducens with Geobacter metallireducens to explore the role of exopolysaccharides in DIET. Results revealed that the deficiency of exopolysaccharides extended the metabolic period of the co-culture by 44.4% and changed the proportions of each species in the co-culture. The exopolysaccharides-deficient co-culture failed to form large, tight spherical aggregates and the expression of c-Cyts and pili was decreased. The addition of magnetite and granular activated carbon (GAC), respectively, might compensate for the functions of c-Cyts and pili in the first generation of co-culture, but the stimulatory effect on the metabolic stable period co-culture was fairly limited. These findings demonstrate that non-conductive exopolysaccharides are an important component of DIET aggregates and an extracellular matrix for DIET-required c-Cyts.

Silver (Ag) is a pivotal transition metal with applications in multiple industries, necessitating efficient recovery techniques. Despite various proposed methods for silver recovery from wastewaters, challenges persist especially for low concentrations. In this context, bioreduction by bacteria like Geobacter sulfurreducens , offers a promising approach by converting Ag(I) to Ag nanoparticles. To reveal the mechanisms driving microbial Ag(I) reduction, we conducted transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition. Integrated transcriptomic and protein-protein interaction network analyses identified significant transcriptional shifts, predominantly linked to c -type cytochromes, NADH, and pili. When compared to a pilus-deficient strain, the wild-type strain exhibited distinct cytochrome gene expressions, implying specialized functional roles. Additionally, despite a down-regulation in NADH dehydrogenase genes, we observed up-regulation of specific downstream cytochrome genes, highlighting NADH's potential role as an electron donor in the Ag(I) reduction process. Intriguingly, our findings also highlight the significant influence of pili on the morphology of the resulting Ag nanoparticles. The presence of pili led to the formation of smaller and more crystallized Ag nanoparticles. Overall, our findings underscore the intricate interplay of cytochromes, NADH, and pili in Ag(I) reduction. Such insights suggest potential strategies for further enhancing microbial Ag(I) reduction. [Display omitted] • Transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition was conducted. • Cytochromes, NADH, and pili were involved in Ag(I) reduction by G. sulfurreducens. • PCA and PCA-Δ pilA showed distinct cytochrome expression, implying specialized roles. • Pili contributed to the formation of small and well-crystallized Ag-NPs. [ABSTRACT FROM AUTHOR]

Background: Pili producing genes in different life cycles of Mycobacterium tuberculosis (M. tuberculosis) were assessed. M. tuberculosis has two life cycles: dormant and active states. We aimed to assess the pili producing genes such as curli pili of M. tuberculosis (mtp) encoded by the mtp gene (Rv3312A) and fimbrial low-molecular-weight protein encoded by flp gene (Rv3656c) which were compared and analyzed. Methods: Two hundred M. tuberculosis isolates were investigated both at active and dormant states for production and expression of pili. The dormant M. tuberculosis was achieved by incubation in a sealed tube (modified Wayne method). The susceptibility of M. tuberculosis was evaluated on genes, rpob, inh, katg, and gyra by using multiplex polymerase chain reaction (PCR) and single-strand conformational polymorphism methods. The PCR–restriction fragment length polymorphism was used to express pili genes mtp and flp and then the PCR products was digested using restriction enzyme Fnu4HI, XmaI, and MspJI and AciI, TagII, and HaeII, respectively. The transmission electron microscopy was also used to detect pili in different isolates. The result was compared and analyzed using H37RV as a gold standard. Results: The mtp and flp PCR products were 263 and 122 bp in the studied strains irrespective of M. tuberculosis different life cycles, respectively. The PCR products were analyzed on 8% Polyacrylamide gel electrophoresis (PAGE), and in the 180/200 (20%), producing five fragments of 25,40,45,63,90 bp with the Fun4HI and two fragments of 126,138 bp with the XmaI and uncut with the MspJI for mtp gen were obtained at the dormant and active states of M. tuberculosis (P < 0.05). Similarly in flp gene producing three fragments of 22,35,65 bp with AciI and two fragments of 35.87 bp with TagII and two fragments of 38.84 bp with HaeII were obtained (P < 0.05). In contrast to genotyping analysis, the electron microscopy examination showed protruding of pili from M. tuberculosis, especially in dormant mycobacterium (15/100; 15%), that was multidrug resistance and extensive drug resistance isolates (P > 0.05). Conclusion: Pili were shown by electron microscopy, although at the gene expression, the insignificant difference was observed at the dormant strains in comparison to active states. Therefore, we may conclude that other genes might be involved in pili production of M. tuberculosis that needs further investigation. Although, the resistance phenomena might influence the pili producing gene expression that showed in our results. [ABSTRACT FROM AUTHOR]