Flagellar Synthesis Research Articles

Transcriptomics analysis of the role of SdiA in desiccation tolerance of Cronobacter sakazakii in powdered infant formula

The quorum-sensing receptor SdiA is vital for regulating the desiccation tolerance of C. sakazakii, yet the specific mechanism remains elusive. Herein, transcriptomics and phenotypic analysis were employed to explore the response of C. sakazakii wild type (WT) and sdiA knockout strain (ΔsdiA) under drying conditions. Following 20 days of drying in powdered infant formula (PIF), WT exhibited 4 log CFU/g higher survival rates compared to ΔsdiA. Transcriptome revealed similar expression patterns between csrA and sdiA, their interaction was confirmed both by protein-protein interaction analysis and yeast two-hybrid assays. Notably, genes associated with flagellar assembly and chemotaxis (flg, fli, che, mot regulon) showed significantly higher expression levels in WT than in ΔsdiA, indicating a reduced capacity for flagellar synthesis in ΔsdiA, which was consistent with cellular morphology observations. Similarly, genes involved in trehalose biosynthesis (ostAB, treYZS) and uptake (thuEFGK) exhibited similar expression patterns to sdiA, with higher levels of trehalose accumulation observed in WT under desiccation conditions compared to ΔsdiA. Furthermore, WT demonstrated enhanced protein and DNA synthesis capabilities under desiccation stress. Higher expression levels of genes related to oxidative phosphorylation were also noted in WT, ensuring efficient cellular ATP synthesis. This study offers valuable insights into how SdiA influences the desiccation tolerance of C. sakazakii, paving the way for targeted strategies to inhibit and control this bacterium.

RpoS sigma factor mediates adaptation and virulence in Vibrio mimicus

The alternative sigma factor RpoS functions as a regulator of stress and virulence response in numerous bacterial species. Vibrio mimicus is a critical opportunistic pathogen causing huge losses to aquaculture. However, the exact role of RpoS in V. mimicus remains unclear. In this study, rpoS deletion mutant of V. mimicus was constructed through allelic exchange and the phenotypic and transcriptional changes were investigated to determine the function of RpoS. The abilities of growth, motility, biofilm production, hemolytic activity and pathogenicity were significantly impaired in ΔrpoS strain. Stationary-phase cells of ΔrpoS strain showed lower tolerance to H2O2, heat, ethanol, and starvation stress than the wild-type strain. Transcriptome analyses revealed the involvement of rpoS in various cellular processes, notably bacterial-type flagellum synthesis and assembly, membrane synthesis and assembly and response to various stimuli. Phenotypic and RNA-seq analysis revealed that RpoS is required for biofilm formation, stress resistance, and pathogenicity in V. mimicus. Furthermore, β-galactosidase activity showed that rpoS is essential for optimal transcription of the flgK, fliA, cheA, mcpH mRNA. These results offer significant insight into the function and regulatory network of rpoS/RpoS, thereby improving our understanding and facilitating selection of molecular targets for future prevention strategies against V. mimicus.

Systems Metabolic Engineering to Elucidate and Enhance Intestinal Metabolic Activities of Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is one of the most widely used probiotics to treat gastrointestinal diseases. Recently, many studies have engineered EcN to release therapeutic proteins to treat specific diseases. However, because EcN exhibits intestinal metabolic activities, it is difficult to predict outcomes after administration. In silico and fermentation profiles revealed mucin metabolism of EcN. Multiomics revealed that fucose metabolism contributes to the intestinal colonization of EcN by enhancing the synthesis of flagella and nutrient uptake. The multiomics results also revealed that excessive intracellular trehalose synthesis in EcN, which is responsible for galactose metabolism, acts as a metabolic bottleneck, adversely affecting growth. To improve the ability of EcN to metabolize galactose, otsAB genes for trehalose synthesis were deleted, resulting in the ΔotsAB strain; the ΔotsAB strain exhibited a 1.47-fold increase in the growth rate and a 1.37-fold increase in the substrate consumption rate relative to wild-type EcN.

BolA-like protein (IbaG) promotes biofilm formation and pathogenicity of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium widespread in temperate and tropical coastal waters; it is considered to be the most frequent cause of Vibrio-associated gastroenteritis in many countries. BolA-like proteins, which reportedly affect various growth and metabolic processes including flagellar synthesis in bacteria, are widely conserved from prokaryotes to eukaryotes. However, the effects exerted by BolA-like proteins on V. parahaemolyticus remain unclear, and thus require further investigation. In this study, our purpose was to investigate the role played by BolA-like protein (IbaG) in the pathogenicity of V. parahaemolyticus. We used homologous recombination to obtain the deletion strain ΔibaG and investigated the biological role of BolA family protein IbaG in V. parahaemolyticus. Our results showed that IbaG is a bacterial transcription factor that negatively modulates swimming capacity. Furthermore, overexpressing IbaG enhanced the capabilities of V. parahaemolyticus for swarming and biofilm formation. In addition, inactivation of ibaG in V. parahaemolyticus SH112 impaired its capacity for colonizing the heart, liver, spleen, and kidneys, and reduced visceral tissue damage, thereby leading to diminished virulence, compared with the wild-type strain. Finally, RNA-sequencing revealed 53 upregulated and 71 downregulated genes in the deletion strain ΔibaG. KEGG enrichment analysis showed that the two-component system, quorum sensing, bacterial secretion system, and numerous amino acid metabolism pathways had been altered due to the inactivation of ibaG. The results of this study indicated that IbaG exerts a considerable effect on gene regulation, motility, biofilm formation, and pathogenicity of V. parahaemolyticus. To the best of our knowledge, this is the first systematic study on the role played by IbaG in V. parahaemolyticus infections. Thus, our findings may lead to a better understanding of the metabolic processes involved in bacterial infections and provide a basis for the prevention and control of such infections.

Applications of the Microalgae Chlamydomonas and Its Bacterial Consortia in Detoxification and Bioproduction.

The wide metabolic diversity of microalgae, their fast growth rates, and low-cost production make these organisms highly promising resources for a variety of biotechnological applications, addressing critical needs in industry, agriculture, and medicine. The use of microalgae in consortia with bacteria is proving valuable in several areas of biotechnology, including the treatment of various types of wastewater, the production of biofertilizers, and the extraction of various products from their biomass. The monoculture of the microalga Chlamydomonas has been a prominent research model for many years and has been extensively used in the study of photosynthesis, sulphur and phosphorus metabolism, nitrogen metabolism, respiration, and flagellar synthesis, among others. Recent research has increasingly recognised the potential of Chlamydomonas-bacteria consortia as a biotechnological tool for various applications. The detoxification of wastewater using Chlamydomonas and its bacterial consortia offers significant potential for sustainable reduction of contaminants, while facilitating resource recovery and the valorisation of microalgal biomass. The use of Chlamydomonas and its bacterial consortia as biofertilizers can offer several benefits, such as increasing crop yields, protecting crops, maintaining soil fertility and stability, contributing to CO2 mitigation, and contributing to sustainable agricultural practises. Chlamydomonas-bacterial consortia play an important role in the production of high-value products, particularly in the production of biofuels and the enhancement of H2 production. This review aims to provide a comprehensive understanding of the potential of Chlamydomonas monoculture and its bacterial consortia to identify current applications and to propose new research and development directions to maximise their potential.

Functional metagenomic analysis reveals potential inflammatory triggers associated with genetic risk for autoimmune disease

To assess functional differences between the microbiomes of individuals with autoimmune risk-associated human leukocyte antigen (HLA) genetics and autoimmune protection-associated HLA, we performed a metagenomic analysis of stool samples from 72 infants in the All Babies in Southeast Sweden general-population cohort and assessed haplotype-peptide binding affinities. Infants with risk-associated HLA DR3-DQ2.5 and DR4-DQ8 had a higher abundance of known pathogen-associated molecular patterns and virulence related genes than infants with protection-associated HLA DR15-DQ6.2. However, there was limited overlap in the type of inflammatory trigger between risk groups. Supported by a high Firmicutes/Bacteroides ratio and differentially abundant flagellated species, genes related to the synthesis of flagella were prominent in those with HLA DR3-DQ2.5. However, this haplotype had a significantly lower likelihood of binding affinity to flagellin peptides. O-antigen biosynthesis genes were significantly correlated with the risk genotypes and absent from protective genotype association, supported by the differential abundance of gram-negative bacteria seen in the risk-associated groups. Genes related to vitamin B biosynthesis stood out in higher abundance in infants with HLA DR3-DQ2.5/DR4-DQ8 heterozygosity compared to those with autoimmune-protective genetics. Prevotella species and genus were significantly abundant in all infant groups with high risk for autoimmune disease. The potential inflammatory triggers associated with genetic risk for autoimmunity have significant implications. These results suggest that certain HLA haplotypes may be creating the opportunity for dysbiosis and subsequent inflammation early in life by clearing beneficial microbes or not clearing proinflammatory microbes. This HLA gatekeeping may prevent genetically at-risk individuals from benefiting from probiotic therapies by restricting the colonization of those beneficial bacteria.

The Microalgae Chlamydomonas for Bioremediation and Bioproduct Production.

The extensive metabolic diversity of microalgae, coupled with their rapid growth rates and cost-effective production, position these organisms as highly promising resources for a wide range of biotechnological applications. These characteristics allow microalgae to address crucial needs in the agricultural, medical, and industrial sectors. Microalgae are proving to be valuable in various fields, including the remediation of diverse wastewater types, the production of biofuels and biofertilizers, and the extraction of various products from their biomass. For decades, the microalga Chlamydomonas has been widely used as a fundamental research model organism in various areas such as photosynthesis, respiration, sulfur and phosphorus metabolism, nitrogen metabolism, and flagella synthesis, among others. However, in recent years, the potential of Chlamydomonas as a biotechnological tool for bioremediation, biofertilization, biomass, and bioproducts production has been increasingly recognized. Bioremediation of wastewater using Chlamydomonas presents significant potential for sustainable reduction in contaminants and facilitates resource recovery and valorization of microalgal biomass, offering important economic benefits. Chlamydomonas has also established itself as a platform for the production of a wide variety of biotechnologically interesting products, such as different types of biofuels, and high-value-added products. The aim of this review is to achieve a comprehensive understanding of the potential of Chlamydomonas in these aspects, and to explore their interrelationship, which would offer significant environmental and biotechnological advantages.

The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus.

Porins are crucial proteins located in the outer membrane that directly influence antimicrobial resistance mechanisms and virulence in bacteria. In this study, a porin gene (Vp-porin) was cloned in V. parahaemolyticus, and the function of Vp-Porin in biological characteristics and virulence was investigated. The results of sequence analysis showed that Vp-Porin is highly conserved in Vibrio spp., and the predicted 3D structure showed it could form a 20-strand transmembrane β-barrel domian. Membrane permeabilization provides evidence that the membrane integrity of ∆Vp-porin was damaged and the sensitivity to tetracycline, polymyxin B, rifampicin and cephalothin of ∆Vp-porin obviously increased. In addition, loss of Vp-porin damaged motility due to downregulated flagellar synthesis. In addition, ∆Vp-porin exhibited attenuated cytotoxicity to Tetrahymena. The relative survival rate of Tetrahymena infection with ∆Vp-porin was 86%, which is much higher than that with WT (49%). Taken together, the results of this study indicate that Vp-Porin in V. parahaemolyticus plays various roles in biological characteristics in membrane integrity, antimicrobial resistance and motility and contributes to virulence.

Effects of chlorogenic acid-grafted-chitosan on biofilms, oxidative stress, quorum sensing and c-di-GMP in Pseudomonas fluorescens

This study determined the inhibitory mechanism as well as anti-biofilm activity of chlorogenic acid-grafted-chitosan (CS-g-CA) against Pseudomonas fluorescens (P. fluorescens) in terms of biofilm content, oxidative stress, quorum sensing and cyclic diguanosine monophosphate (c-di-GMP) concentration, and detected the changes in the expression levels of related genes by quantitative real-time PCR (qRT-PCR). Results indicated that treatment with sub-concentrations of CS-g-CA for P. fluorescens led to reduce the biofilm size of large colonies, decrease the content of biofilm and extracellular polymers, weaken the motility and adhesion of P. fluorescens. Moreover, CS-g-CA resulted in higher ROS levels, diminished catalase activity (CAT), and increased superoxide dismutase (SOD) in P. fluorescens. CS-g-CA reduced the production of quorum-sensing signaling molecules (AHLs) and the concentration of c-di-GMP in bacteria. Genes for flagellar synthesis (flgA), the resistance to stress (rpoS and hfq), and pde (phosphodiesterases that degrade c-di-GMP) were significantly down-regulated as determined by RT-PCR. Overall, CS-g-CA leads to the accumulation of ROS in bacteria via P. fluorescens environmental resistance genes and decreases the activity of enzymes in the bacterial antioxidant system, and interferes with the production and reception of quorum-sensing signaling molecules and the synthesis of c-di-GMP in P. fluorescens, which regulates the generation of biofilms.

A trans-acting sRNA SaaS targeting hilD, cheA and csgA to inhibit biofilm formation of S. Enteritidis

IntroductionSalmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a critical role in microbial cross-contamination. Small non-coding RNAs (sRNAs) have been demonstrated to be responsible for regulating the formation of biofilm. The sRNA SaaS has been identified previously, that promotes pathogenicity by regulating invasion and virulence factors. However, whether the SaaS is implicated in regulating biofilm formation in abiotic surfaces remains unclear. ObjectivesThis study aimed to clarify the effect of SaaS in Salmonella Enteritidis and explore the modulatory mechanism on the biofilm formation. MethodsMotility characteristics and total biomass of biofilm of test strains were investigated by the phenotypes in three soft agar plates and crystal violet staining in polystyrene microplates. Studies of microscopic structure and extracellular polymeric substances (EPS) of biofilm on solid surfaces were carried out using confocal laser scanning microscope (CLSM) and Raman spectra. Transcriptomics and proteomics were applied to analyze the changes of gene expression and EPS component. The RNA-protein pull-down and promoter–reporter β-galactosidase activity assays were employed to analyze RNA binding proteins and identify target mRNAs, respectively. ResultsSaaS inhibits biofilm formation by repressing the adhesion potential and the secretion of EPS components. Integration of transcriptomics and proteomics analysis revealed that SaaS strengthened the expression of the flagellar synthesis system and downregulated the expression of curli amyloid fibers. Furthermore, RNA-protein pull-down interactome datasets indicated that SaaS binds to Hfq (an RNA molecular chaperone protein, known as a host factor for phage Qbeta RNA replication) uniquely among 193 candidate proteins, and promoter–reporter β-galactosidase activity assay confirmed target mRNAs including hilD, cheA, and csgA. ConclusionSaaS inhibits the properties of bacterial mobility, perturbs the secretion of EPS, and contributes to the inhibition of biofilm formation by interacting with target mRNA (hilD, cheA, and csgA) through the Hfq-mediated pathway.

Escherichia coli self-organizes developmental rosettes

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

Role of the sigma factor AlgU in regulating growth, virulence, motility, exopolysaccharide production, and environmental stress adaptation of Pseudomonas syringae pv. actinidiae QSY6

The extracytoplasmic function (ECF) sigma factor AlgU is involved in the regulation of various virulence-related pathways in Pseudomonas syringae, especially alginate biosynthesis and motility, and the role of AlgU differs among P. syringae pathovars. However, to date, the mechanism of its regulation in virulence of P. syringae pv. actinidiae (Psa) is still unclear. ECF sigma factors are a class of alternative sigma factors that typically function with anti-sigma factors as part of cell-surface signaling systems. Under non-inducing conditions, AlgU remains inhibited by anti-sigma factors such as MucA and MucB. To investigate the function of AlgU in Psa, mutant strains lacking algU or lacking algU with mucA and mucB genes, as well as complementary and overexpression strains of algU were generated, respectively. The results showed that AlgU was highly conserved among P. syringae pathovars and positively regulated growth rate, pathogenicity, and resistance to osmotic and oxidative stress of Psa QSY6. While AlgU did not affect the motility and exopolysaccharide production of Psa, its abundant expression enhanced the swimming ability of QSY6 and reduced its production of extracellular polysaccharides. Furthermore, AlgU regulates a number of virulence-related factors, including the Hrp system, the type VI secretion system, and flagellar synthesis. Specifically, AlgU induced the expression of hrpL and hrpRS in vivo, and repressed the transcription of hrpL and tssC in vitro, while promoting the expression of hrpS, fliC, and tssJ. This study contributes to a better understanding of the mechanisms of virulence regulation of AlgU in Psa.

Multiple pathways impact the swarming motility of Pseudomonas fluorescens Pf0-1.

Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the wild-type Pseudomonas fluorescens Pf0-1 is swarming deficient due to a point mutation in the gacA gene, which until recently was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by P. fluorescens Pf0-1. Here, we demonstrate that a ΔrsmA ΔrsmE ΔrsmI mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the ΔrsmA ΔrsmE ΔrsmI mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impacts swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that P. fluorescens Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility.IMPORTANCESwarming motility is a coordinated process that allows communities of bacteria to collectively move across a surface. For P. fluorescens Pf0-1, this phenotype is notably absent in the parental strain, and to date, little is known about the regulation of swarming in this strain. Here, we identify RsmA and RsmE as key repressors of swarming motility via modulating the levels of biosurfactant production/secretion. Using transposon mutagenesis and subsequent genetic analyses, we further identify potential regulatory mechanisms of swarming motility and link Gacamide A biosynthesis and transport machinery to swarming motility.

Differential mechanism between Listeria monocytogenes strains with different virulence contaminating ready-to-eat sausages during the simulated gastrointestinal tract

Listeria monocytogenes exhibits varying levels of pathogenicity when entering the host through contaminated food. However, little is known regarding the stress response and environmental tolerance mechanism of different virulence strains to host gastrointestinal (GI) stimuli. This study analyzed the differences in the survival and genes of stress responses among two strains of L. monocytogenes 10403S (serotype 1/2a, highly virulent strain) and M7 (serotype 4a, low-virulence strain) during simulated gastrointestinal digestion. The results indicated that L. monocytogenes 10403S showed greater acid and bile salt tolerance than L. monocytogenes M7, with higher survival rates and less cell deformation and cell membrane permeability during the in vitro digestion. KEGG analysis of the transcriptomes indicated that L. monocytogenes 10403S displayed significant activity in amino acid metabolism, such as glutamate and arginine, associated with acid tolerance. Additionally, L. monocytogenes 10403S demonstrated a higher efficacy in promoting activities that preserve bacterial cell membrane integrity and facilitate flagellar protein synthesis. These findings will contribute valuable practical insights into the tolerance distinctions among different virulence strains of L. monocytogenes in the GI environment.

Differences in Biofilm Formation of Listeria monocytogenes and Their Effects on Virulence and Drug Resistance of Different Strains.

Listeria monocytogenes is recognized as one of the primary pathogens responsible for foodborne illnesses. The ability of L. monocytogenes to form biofilms notably increases its resistance to antibiotics such as ampicillin and tetracycline, making it exceedingly difficult to eradicate. Residual bacteria within the processing environment can contaminate food products, thereby posing a significant risk to public health. In this study, we used crystal violet staining to assess the biofilm-forming capacity of seven L. monocytogenes strains and identified ATCC 19112 as the strain with the most potent biofilm-forming. Subsequent fluorescence microscopy observations revealed that the biofilm-forming capacity was markedly enhanced after two days of culture. Then, we investigated into the factors contributing to biofilm formation and demonstrated that strains with more robust extracellular polymer secretion and self-agglutination capabilities exhibited a more pronounced ability to form biofilms. No significant correlation was found between surface hydrophobicity and biofilm formation capability. In addition, we found that after biofilm formation, the adhesion and invasion of cells were enhanced and drug resistance increased. Therefore, we hypothesized that the formation of biofilm makes L. monocytogenes more virulent and more difficult to remove by antibiotics. Lastly, utilizing RT-PCR, we detected the expression levels of genes associated with biofilm formation, including those involved in quorum sensing (QS), flagellar synthesis, and extracellular polymer production. These genes were significantly upregulated after biofilm formation. These findings underscore the critical relationship between extracellular polymers, self-agglutination abilities, and biofilm formation. In conclusion, the establishment of biofilms not only enhances L. monocytogenes' capacity for cell invasion and adhesion but also significantly increases its resistance to drugs, presenting a substantial threat to food safety.

Comparative transcriptome analysis explored the molecular mechanisms of a luxR-type regulator regulating intracellular survival of Aeromonas hydrophila.

Aeromonas hydrophila is not a traditional intracellular bacterium. However, previous studies revealed that pathogenic A. hydrophila B11 could temporarily survive for at least 24 h in fish phagocytes, and the regulation of intracellular survival in bacteria was associated with regulators of the LuxR-type. The mechanisms of luxR08110 on the A. hydrophila's survival in macrophages were investigated using comprehensive transcriptome analysis and biological phenotype analysis in this study. The results showed that after luxR08110 was silenced, the intracellular survival ability of bacteria was significantly diminished. Comparative transcriptome analysis revealed that luxR08110 was a critical regulator of A. hydrophila, which regulated the expression of over 1200 genes, involving in bacterial flagellar assembly and chemotaxis, ribosome, sulphur metabolism, glycerolipid metabolism, and other mechanisms. Further studies confirmed that after the inhibition of expression of luxR08110, the motility, chemotaxis and adhesion of A. hydrophila significantly decreased. Moreover, compared with the wild-type strain, the survival rates of silencing strain were all considerably reduced under both H2O2 and low pH stress conditions. According to both transcriptome analysis and phenotypic tests, the luxR08110 of A. hydrophila could act as global regulator in bacteria intracellular survival. This regulator regulated intracellular survival of A. hydrophila mainly through two ways. One way is to regulate bacterial flagellar synthesis and further affects the motility, chemotaxis and adhesion of bacteria. The other way is to regulate sulphur and glycerolipid metabolisms, thus affecting bacterial energy production and the ability to resist environmental stress.

Clostridioides difficile Flagella.

Clostridioides difficile is an important pathogen for humans with a lead in nosocomial infection, but it is also more and more common in communities. Our knowledge of the pathology has historically been focused on the toxins produced by the bacteria that remain its major virulence factors. But the dysbiosis of the intestinal microbiota creating the conditions for the colonization appears to be fundamental for our understanding of the disease. Colonization implies several steps for the bacteria that do or do not use their capacity of motility with the synthesis of flagella. In this review, we focus on the current understanding of different topics on the C. difficile flagellum, ranging from its genetic organization to the vaccinal interest in it.

BolA promotes the generation of multicellular behavior in S. typhimurium by regulating the c-di-GMP pathway genes yeaJ and yhjH

The generation of multicellular behavior enhances the stress adaptability, antibiotic resistance, and pathogenic potential of Salmonella enterica serovar Typhimurium (S. typhimurium), which is challenging for its prevention and control. Therefore, determination of the mechanism of multicellular behavior development is urgently required. Accordingly, this study investigated BolA, a transcription factor that promotes bacterial survival under different stresses. We found that BolA promoted the generation of multicellular behavior. Furthermore, transcriptome analysis revealed that BolA affected the expression of numerous genes, including biofilm formation and motility-related genes. In terms of biofilm formation, compared with the wild-type strain, bolA overexpression (269BolA+) increased the extracellular matrix content (extracellular polysaccharide, extracellular protein, and extracellular DNA (eDNA) by upregulating gene expression, ultimately increasing the biofilm formation ability by 2.56 times. For motility, bolA overexpression inhibited the expression of flagella synthesis genes, resulting in a 91.15 % decrease in motility compared with the wild-type (6 h). Further mechanistic analysis demonstrated that BolA affected the expression of the C-di-GMP pathway genes yeaJ and yhjH, which influenced the generation of multicellular behavior. In terms of biofilms, the extracellular polysaccharide content of 269BolA + ∆Yeaj (bolA overexpression and yeaJ deletion) was reduced by 89.91 % compared with 269BolA+, resulting in a 71.1 % reduction in biofilm forming ability. The motility of the 269∆BolA∆Yhjh (bolA/yhjH double deletion) strain was significantly decreased compared with that of 269∆BolA. Finally, the LacZ gene reporting showed that BolA promoted and inhibited the expression of yeaJ and yhjH, respectively. In conclusion, BolA mainly improves the content of extracellular polysaccharide by promoting the expression of yeaJ, thus enhancing the formation of biofilms. BolA also restricts flagellar synthesis by inhibiting yhjH expression, therefore reducing motility, ultimately promoting multicellular behavior arises. These findings lay a theoretical foundation for the prevention and control of S. typhimurium.

Age, metabolisms, and potential origin of dominant anammox bacteria in the global oxygen-deficient zones.

Anammox bacteria inhabiting oxygen-deficient zones (ODZs) are a major functional group mediating fixed nitrogen loss in the global ocean. However, many basic questions regarding the diversity, broad metabolisms, origin, and adaptive mechanisms of ODZ anammox bacteria remain unaddressed. Here we report two novel metagenome-assembled genomes of anammox bacteria affiliated with the Scalindua genus, which represent most, if not all, of the anammox bacteria in the global ODZs. Metagenomic read-recruiting and comparison with historical data show that they are ubiquitously present in all three major ODZs. Beyond the core anammox metabolism, both organisms contain cyanase, and the more dominant one encodes a urease, indicating most ODZ anammox bacteria can utilize cyanate and urea in addition to ammonium. Molecular clock analysis suggests that the evolutionary radiation of these bacteria into ODZs occurred no earlier than 310 million years ago, ~1 billion years after the emergence of the earliest modern-type ODZs. Different strains of the ODZ Scalindua species are also found in benthic sediments, and the first ODZ Scalindua is likely derived from the benthos. Compared to benthic strains of the same clade, ODZ Scalindua uniquely encodes genes for urea utilization but has lost genes related to growth arrest, flagellum synthesis, and chemotaxis, presumably for adaptation to thrive in the global ODZ waters. Our findings expand the known metabolisms and evolutionary history of the bacteria controlling the global nitrogen budget.

Adaptive evolution of plasmid and chromosome contributes to the fitness of a blaNDM-bearing cointegrate plasmid in Escherichia coli.

Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts.