Flagellar Synthesis Research Articles

Phenotype, Virulence and Immunogenicity of Edwardsiella piscicida Cyclic AMP Receptor Protein (Crp) Mutants in Catfish Host.

Edwardsiella piscicida, a facultative aerobic pathogen belonging to the Enterobacteriaceae family, is the etiological agent of edwardsiellosis that causes significant economic loses in the aquaculture industry. cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. Here we characterize the crp gene and report the effect of a crp deletion in E. piscicida. The crp-deficient mutant lost the capacity to utilize maltose, and showed significantly reduced motility due to the lack of flagella synthesis. We further constructed a ΔPcrp mutant to support that the phenotype above was caused by the crp deletion. Evidence obtained in fish serum killing assay and competitive infection assay strongly indicated that the inactivation of crp impaired the ability of E. piscicida to evade host immune clearance. More importantly, the virulence of the crp mutant was attenuated in both zebrafish and channel catfish, with reductions in mortality rates. In the end, we found that crp mutant could confer immune protection against E. piscicida infection to zebrafish and channel catfish, indicating its potential as a live attenuated vaccine.

Investigating the Role of FlhF Identifies Novel Interactions With Genes Involved in Flagellar Synthesis in Campylobacter jejuni.

FlhF is a key protein required for complete flagellar synthesis, and its deletion results in the complete absence of a flagella and thus motility in Campylobacter jejuni. However, the specific mechanism still remains unknown. In this study, RNA-Seq, EMSAs, ChIP-qPCR and β-Galactosidase assays were performed to elucidate the novel interactions between FlhF and genes involved in flagellar synthesis. Results showed that FlhF has an overall influence on the transcription of flagellar genes with an flhF mutant displaying down-regulation of most flagellar related genes. FlhF can directly bind to the flgI promoter to regulate its expression, which has significant expression change in an flhF mutant. The possible binding site of FlhF to the flgI promoter was explored by continuously narrowing the flgI promoter region and performing further point mutations. Meanwhile, FlhF can directly bind to the promoters of rpoD, flgS, and fliA encoding early flagellin regulators, thereby directly or indirectly regulating the synthesis of class I, II, and III flagellar genes, respectively. Collectively, this study demonstrates that FlhF may directly regulate the transcription of flagellar genes by binding to their promoters as a transcriptional regulator, which will be helpful in understanding the mechanism of FlhF in flagellar biosynthetic and bacterial flagellation in general.

Deteriorated biofilm-forming capacity and electroactivity of Shewanella oneidnsis MR-1 induced by insertion sequence (IS) elements

Shewanella oneidensis MR-1, a model species of exoelectrogenic bacteria (EEB), has been widely applied in bioelectrochemical systems. Biofilms of EEB grown on electrodes are essential in governing the current output and power density of bioelectrochemical systems. The MR-1 genome is exceptionally dynamic due to the existence of a large number of insertion sequence (IS) elements. However, to date, the impacts of IS elements on the biofilm-forming capacity of EEB and performance of bioelectrochemical systems remain unrevealed. Herein, we isolated a non-motile mutant (NMM) with biofilm-deficient phenotype from MR-1. We found that the insertion of an ISSod2 element into the flrA (encoding the master regulator for flagella synthesis and assembly) of MR-1 resulted in the non-motile and biofilm-deficient phenotypes in NMM cells. Notably, such a variant was readily confused with the wild-type strain because there were no obvious differences in growth rates and colonial morphologies between the two strains. However, the reduced biofilm formation on the electrodes and the deteriorated performances of bioelectrochemical systems and Cr(VI) immobilization for the strain NMM were observed. Given the wide distribution of IS elements in EEB, appropriate cultivation and preservation conditions should be adopted to reduce the likelihood that IS elements-mediated mutation occurs in EEB. These findings reveal the negative impacts of IS elements on the biofilm-forming capacity of EEB and performance of bioelectrochemical systems and suggest that great attention should be given to the actual physiological states of EEB before their applications.

A Polar Flagellar Transcriptional Program Mediated by Diverse Two-Component Signal Transduction Systems and Basal Flagellar Proteins Is Broadly Conserved in Polar Flagellates.

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

Molecular Mechanisms Underpinning Aggregation in Acidiphilium sp. C61 Isolated from Iron-Rich Pelagic Aggregates.

Iron-rich pelagic aggregates (iron snow) are hot spots for microbial interactions. Using iron snow isolates, we previously demonstrated that the iron-oxidizer Acidithrix sp. C25 triggers Acidiphilium sp. C61 aggregation by producing the infochemical 2-phenethylamine (PEA). Here, we showed slightly enhanced aggregate formation in the presence of PEA on different Acidiphilium spp. but not other iron-snow microorganisms, including Acidocella sp. C78 and Ferrovum sp. PN-J47. Next, we sequenced the Acidiphilium sp. C61 genome to reconstruct its metabolic potential. Pangenome analyses of Acidiphilium spp. genomes revealed the core genome contained 65 gene clusters associated with aggregation, including autoaggregation, motility, and biofilm formation. Screening the Acidiphilium sp. C61 genome revealed the presence of autotransporter, flagellar, and extracellular polymeric substances (EPS) production genes. RNA-seq analyses of Acidiphilium sp. C61 incubations (+/− 10 µM PEA) indicated genes involved in energy production, respiration, and genetic processing were the most upregulated differentially expressed genes in the presence of PEA. Additionally, genes involved in flagellar basal body synthesis were highly upregulated, whereas the expression pattern of biofilm formation-related genes was inconclusive. Our data shows aggregation is a common trait among Acidiphilium spp. and PEA stimulates the central cellular metabolism, potentially advantageous in aggregates rapidly falling through the water column.

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction.IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system's fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

Rariglobus hedericola gen. nov., sp. nov., belonging to the Verrucomicrobia, isolated from a temperate freshwater habitat.

The bacterial strain 53C-WASEF was isolated from a small freshwater ditch located in Eugendorf, Austria. Phylogenetic reconstructions with 16S rRNA gene sequences and genome based, with amino acid sequences obtained from 105 single copy genes, suggested that the strain represents a new genus and a new species within the family Opitutaceae, which belongs to the class Opitutae of the phylum Verrucomicrobia. Comparisons of the 16S rRNA gene sequence of strain 53C-WASEF with those of related type strains revealed a highest sequence similarity of 93.5 % to Nibricoccus aquaticus and of 92.9 % to Geminisphaera colitermitum. Interestingly, phylogentic trees indicated the latter as being the closest known relative of the new strain. Phenotypic, chemotaxonomic and genomic traits were investigated. Cells were observed to be small, spherical, motile and unpigmented, and grew chemoorganotrophically and aerobically. The respiratory quinone was MK-7, the predominant fatty acids were anteiso-C15 : 0, C16 : 1ω5c and C16 : 0. The identified polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Genome sequencing revealed genes putatively encoding for flagella synthesis and cellulose degradation. The genome size was 4.1 Mbp and the G+C content 60.6 mol%. For the new genus and the new species, we propose the name Rariglobus hedericola gen. nov., sp. nov. (=CIP 111665T=DSM 109123T).

The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune-stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen-associated molecular pattern recognized by extracellular and intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum-specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest antitumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains.

Impact of the fliF Gene on Biotite Weathering of Rhizobium pusense S41

Rhizobia have been reported to weather silicate mineral, however, molecular mechanisms underlying mineral weathering of Rhizobium strain is unclear. In this study, biotite weathering behaviors were compared among the mineral-weathering Rhizobium pusense S41, its mutants created by deleting the fliF gene involved in flagellar protein synthesis, and its complemented strain. Flagellum was observed in strain S41 but not in its mutant S41ΔfliF. Mobilized Fe and Al concentrations decreased in the presence of S41ΔfliF during mineral weathering process compared to strain S41. No significant differences in the gluconic acid concentration and pH values were found between strains S41 and S41ΔfliF during the mineral weathering process except for day 2. A similar bacterial growth in the culture medium was observed between strains S41 and S41ΔfliF, however, significantly lower cell numbers on the mineral surface were observed for S41ΔfliF compared to strain S41. Furthermore, biofilm production was depressed for S41ΔfliF compared to strain S41. The results showed the important role of a flagellum of strain S41 in biotite weathering and that the fliF gene of strain S41 exhibited important impacts on mineral weathering activity and biofilm production.

C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Integrated Proteomics and Lipidomics Reveal That the Swarming Motility of Paenibacillus polymyxa Is Characterized by Phospholipid Modification, Surfactant Deployment, and Flagellar Specialization Relative to Swimming Motility.

Paenibacillus polymyxa is a Gram-positive bacterium commonly found associated with plant roots. P. polymyxa can exhibit two forms of flagellar motility: swimming in liquid culture and swarming on a surface. Here, swimming cells were compared to swarming cells using an integrated proteomic and lipidomic approach, yielding information about how lipid modifications and protein/enzyme pathways are tailored for these specific phenotypes. Observed differences in both phospholipid composition and metabolism between the two conditions suggest membrane remodeling in response to the surrounding environment. Key enzymes involved in glycerophospholipid metabolism were abundant in swimming bacteria, while enzymes associated with glycerol-3-phosphate metabolism were more abundant in swarming bacteria. Several glycoside hydrolases were either unique to or more abundant during swarming. This likely reflects the degradation of their own exopolysaccharides to both enhance swarming and supply the necessary chemical energy to compensate for increased flagellar synthesis. The observed upregulation of biosynthetic gene clusters (polyketides, lantibiotics, and surfactin) in swarming bacteria suggest the importance of signaling, antimicrobial activity, and surfactin production during this mode of motility – the latter of which is confirmed via RT-PCR.

Exposure to Broad-Spectrum Visible Light Causes Major Transcriptomic Changes in Listeria monocytogenes EGDe.

Listeria monocytogenes, the causative agent of the serious foodborne disease listeriosis, can rapidly adapt to a wide range of environmental stresses, including visible light. This study shows that exposure of the L. monocytogenes EGDe strain to low-intensity, broad-spectrum visible light inhibited bacterial growth and caused altered multicellular behavior during growth on semisolid agar compared to when the bacteria were grown in complete darkness. These light-dependent changes were observed regardless of the presence of the blue light receptor (Lmo0799) and the stressosome regulator sigma B (SigB), which have been suggested to be important for the ability of L. monocytogenes to respond to blue light. A genome-wide transcriptional analysis revealed that exposure of L. monocytogenes EGDe to broad-spectrum visible light caused altered expression of 2,409 genes belonging to 18 metabolic pathways compared to bacteria grown in darkness. The light-dependent differentially expressed genes are involved in functions such as glycan metabolism, cell wall synthesis, chemotaxis, flagellar synthesis, and resistance to oxidative stress. Exposure to light conferred reduced bacterial motility in semisolid agar, which correlates well with the light-dependent reduction in transcript levels of flagellar and chemotaxis genes. Similar light-induced reduction in growth and motility was also observed in two different L. monocytogenes food isolates, suggesting that these responses are typical for L. monocytogenes Together, the results show that even relatively small doses of broad-spectrum visible light cause genome-wide transcriptional changes, reduced growth, and motility in L. monocytogenesIMPORTANCE Despite major efforts to control L. monocytogenes, this pathogen remains a major problem for the food industry, where it poses a continuous risk of food contamination. The ability of L. monocytogenes to sense and adapt to different stressors in the environment enables it to persist in many different niches, including food production facilities and in food products. The present study shows that exposure of L. monocytogenes to low-intensity broad-spectrum visible light reduces its growth and motility and alters its multicellular behavior. Light exposure also caused genome-wide changes in transcript levels, affecting multiple metabolic pathways, which are likely to influence the bacterial physiology and lifestyle. In practical terms, the data presented in this study suggest that broad-spectrum visible light is an important environmental variable to consider as a strategy to improve food safety by reducing L. monocytogenes contamination in food production environments.

221. Newly-Named Klebsiella aerogenes Is Associated with Poor Clinical Outcomes Relative to Enterobacter cloacae Complex in Patients with Bloodstream Infection

BackgroundWhole-genome-based comparative bacterial phylogenetics led to Enterobacter aerogenes being renamed Klebsiella aerogenes. It is unclear how infections with K. aerogenes differ from Enterobacter cloacae complex (Ecc) regarding clinical characteristics, antibiotic resistance, patient outcomes, and bacterial virulence genes.MethodsAdult inpatients with K. aerogenes or Ecc bloodstream infection (BSI) were prospectively enrolled at Duke from 2002–2015. Whole-genome bacteria sequencing was performed. Chi-squared, Fisher’s exact, Mann–Whitney U, t-tests, and multivariable logistic regression models identified risk factors for infection and clinical outcomes. PanOCT algorithm identified flexible genomic islands (fgi). Multidrug resistance (MDR) was defined as resistance to ≥3 drug classes and poor clinical outcome as death before discharge and/or BSI complication (septic shock, acute kidney injury, acute lung injury/acute respiratory distress syndrome, disseminated intravascular coagulation).ResultsWe identified 104 (69%) patients with Ecc BSI and 46 (31%) with K. aerogenes BSI (N = 150). Patients with Ecc BSI more often required hemodialysis (23% vs. 9%, P = 0.04). MDR was similar between Ecc and K. aerogenes (30% vs. 33%; P = 0.85). Total (21% vs. 28%; P = 0.3) and attributable in-hospital mortality (12% vs. 20%; P = 0.3) did not differ between the two genera. Poor clinical outcome was more common with K. aerogenes BSI (70% vs. 40%, P = 0.001) and remained significant after adjusting for age, source of BSI, site of acquisition (e.g., hospital), days to appropriate antibiotic, and chronic APACHE-II score (odds ratio 2.8, 95% CI 1.2–6.4, P = 0.001). Ecc and K. aerogenes isolates formed 14 and 3 phylogenetic clades, respectively (Fig 1A). K. aerogenes contained 983 core genes, grouped within 324 fgi, that were not present in Ecc (Fig 1B). These included homologs to virulence genes involved in iron acquisition, flagella synthesis, and fimbriae production.ConclusionPatients with BSI due to K. aerogenes had poor clinical outcomes relative to Ecc. Multiple unique K. aerogenes genes homologous to virulence factors may contribute to this difference. Disclosures All authors: No reported disclosures.

Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Abstract Background Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac. How to cite: Laia ML, Moreira LM, Goncalves F, et al. Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.003 .

Metabolic perturbations in mutants of glucose transporters and their applications in metabolite production in Escherichia coli

BackgroundMost microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate.ResultsAs glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13C metabolic flux analysis (13C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related to glycolysis, chemotaxis, and flagella synthesis was downregulated, and that of gluconeogenesis, Krebs cycle, alternative transporters, quorum sensing, and stress induced proteins was upregulated in the sugar transporter mutants. The specific production yields of value-added compounds (enhanced green fluorescent protein, γ-aminobutyrate, lycopene) were improved significantly in the sugar transporter mutants.ConclusionsThe elimination of sugar transporter resulted in alteration of global gene expression and redirection of carbon flux distribution, which was purposed to increase energy yield and recycle carbon sources. When the pathways for several valuable compounds were introduced to mutant strains, specific yield of them were highly improved. These results showed that controlling the sugar uptake rate is a good strategy for ameliorating metabolite production.

Antimicrobial activity and virulence attenuation of citral against the fish pathogen Vibrio alginolyticus

Vibrio alginolyticus is an important fish pathogen causing pandemic diseases in marine animals and brings about severe economic losses in aquaculture all over the world. The emergence of multi-drug resistant V. alginolyticus and the prohibition of antibiotics both require the development of new anti-virulence strategies and therapeutic agents with alternative modes of action. In this study, the effect of citral on V. alginolyticus growth and virulence was investigated to determine the basis of its use in disease prevention and treatment in aquaculture. The minimum inhibitory concentration (MIC) of citral against V. alginolyticus was determined to be 0.125 mg/mL. Citral caused membrane invaginations and damages as shown by field emission scanning electron microscopy (FESEM) observations. Besides, membrane damage was further verified through the increase of conductivity and extracellular malondialdehyde content. After treatment with sub-inhibitory concentrations (SICs) of citral, the virulence factors of V. alginolyticus, including motility, biofilm formation, extracellular polysaccharide, and alkaline serine protease production, were all significantly inhibited. Transcription analysis showed that citral repressed genes involved in flagella synthesis (fliS, flaK, lafA, lafK), biofilm regulation gene (sypG), and quorum sensing genes. Moreover, citral reduced the virulence of V. alginolyticus to zebrafish. These findings suggest that citral has the potential to be developed as an alternative or supplemental agent to mitigate the infections caused by V. alginolyticus in aquaculture.

Crosstalk between bacterial conjugation and motility is mediated by plasmid-borne regulators.

Plasmid conjugation is a major horizontal gene transfer mechanism. The acquisition of a plasmid may cause a perturbation of the cell functions in addition to provide advantageous properties for the recipient cell, such as the gaining of antibiotic resistances. The interplay between plasmid and chromosomal functions has been studied using the IncHI1 plasmid R27. Plasmids of the incompatibility group HI1, isolated from several Gram-negative pathogens, are associated with the spread of multidrug resistance. Their conjugation is tightly regulated by temperature, being repressed at temperatures within the host (37°C). In this report, we described that at permissive temperature, when conjugation of plasmid R27 is prompted, a reduction in the motility of the cells is observed. This reduction is mediated by the plasmid-encoded regulators TrhR/TrhY, which together with HtdA form a plasmid-borne regulatory circuit controlling R27 conjugation. TrhR/TrhY, required to induce R27 conjugation, is responsible for the downregulation of the flagella synthesis and the consequent decrease in motility. TrhR/TrhY repress, direct or indirectly, the expression of the specific flagellar sigma subunit FliA and, consequently, the expression of all genes located bellow in the flagellar expression cascade.

Orf20 in prophage phiv142-3 contributes to the adhesion and colonization ability of avian pathogenic Escherichia coli strain DE142 by affecting the formation of flagella and I fimbriae

We have previously demonstrated that prophage phiv142-3 enhances the colonization ability of avian pathogenic Escherichia coli (APEC) strain DE142. However, the mechanism of this action remains unknown. In this study, we demonstrate that deletion of phiv142-3 orf20 leads to a decrease in the survival ability in chicken serum, adhesion, and ability to invade DF-1 cells of mutant strain DE142Δorf20 compared with that of wild type (WT). Avian infection assays showed that bacterial loads in lungs and hearts of chickens challenged with the mutant are decreased to 7% and 8.3% compared with those challenged with the WT. The number of flagella and I fimbriae of the mutant are decreased and the mutant exhibits filamentation. However, protein ORF20 shows no adhesion ability to DF-1 cells in adherence inhibition experiments, indicating that it does not directly participate in adhesion. qRT-PCR revealed that the deletion of orf20 leads to reduction in the expression of nine genes related to the exportation of flagellar protein and two I-fimbriae-related genes (fimA and fimH), but does not affect genes related to the synthesis of flagella and other adhesins. Compared with the WT, the transcription level of the cell-division-associated genes minC and minD was increased 1.4-fold and 2.5-fold in mutant DE142Δorf20, respectively, indicating that orf20 affects the morphology of DE142 by regulating expression of minC and minD. Thus, our study revealed that orf20 in prophage phiv142-3 played a role in flagellar exportation, cell morphology, and I fimbriae synthesis.

A Dual Role of Amino Acids from Sesbania rostrata Seed Exudates in the Chemotaxis Response of Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans ORS571 can induce nodule formation on the roots and the stems of its host legume, Sesbania rostrata. Plant exudates are essential in the dialogue between microbes and their host plant and, in particular, amino acids can play an important role in the chemotactic response of bacteria. Histidine, arginine, and aspartate, which are the three most abundant amino acids present in S. rostrata seed exudates, behave as chemoattractants toward A. caulinodans. A position-specific-iterated BLAST analysis of the methyl-accepting chemotaxis proteins (MCPs) (chemoreceptors) in the genome of A. caulinodans was performed. Among the 43 MCP homologs, two MCPs harboring a dCache domain were selected as possible cognate amino acid MCPs. After analysis of relative gene expression levels and construction of a gene-deleted mutant strain, one of them, AZC_0821 designed as TlpH, was confirmed to be responsible for the chemotactic response to the three amino acids. In addition, it was found that these three amino acids can also influence chemotaxis of A. caulinodans independently of the chemosensory receptors, by being involved in the increase of the expression level of several che and fla genes involved in the chemotaxis pathway and flagella synthesis. Thus, the contribution of amino acids present in seed exudates is directly related to the role as chemoattractants and indirectly related to the role in the regulation of expression of key genes involved in chemotaxis and motility. This "dual role" is likely to influence the formation of biofilms by A. caulinodans and the host root colonization properties of this bacterium.

Exploring Molecular Signs of Sex in the Marine Diatom Skeletonema marinoi.

Sexual reproduction plays a fundamental role in diatom life cycles. It contributes to increasing genetic diversity through meiotic recombination and also represents the phase where large-sized cells are produced to counteract the cell size reduction process that characterizes these microalgae. With the aim to identify genes linked to the sexual phase of the centric planktonic diatom Skeletonema marinoi, we carried out an RNA-seq experiment comparing the expression level of transcripts in sexualized cells with that of large cells not competent for sex. A set of genes involved in meiosis were found upregulated. Despite the fact that flagellate gametes were observed in the sample, we did not detect the expression of genes involved in the synthesis of flagella that were upregulated during sexual reproduction in another centric diatom. A comparison with the set of genes changing during the first phases of sexual reproduction of the pennate diatom Pseudo-nitzschia multistriata revealed the existence of commonalities, including the strong upregulation of genes with an unknown function that we named Sex Induced Genes (SIG). Our results further broadened the panel of genes that can be used as a marker for sexual reproduction of diatoms, crucial for the interpretation of metatranscriptomic datasets.