Pilus Gene Research Articles

Inhibition of biofilm formation and exopolysaccharide synthesis of Enterococcus faecalis by phenyllactic acid

This study aimed to evaluate the inhibitory activity of phenyllactic acid (PLA) against the biofilm formation of Enterococcus faecalis and to explore its potential molecular mechanism. The MIC value of PLA that inhibited the growth of E. faecalis R612-Z1 in BHI broth was 5 mg/mL. PLAs at subinhibitory concentrations of 1.25 and 2.50 mg/mL were found to inhibit biofilm formation by a crystal violet staining assay. The cell swimming and swarming motilities of E. faecalis were reduced in the presence of PLA. An apparent decrease in the thickness of PLA-treated biofilms was observed through confocal laser scanning microscopy analysis. The exopolysaccharide production in E. faecalis biofilms was inhibited by EPS quantification assay and scanning electron microscopy (SEM). qRT-PCR analyses showed that PLA down-regulated the transcription of Ebp pili genes (ebpABC) and Epa polysaccharide genes (epaABE). PLA inhibited the biofilm formation by interfering with cell mobility and EPS production of E. faecalis. In addition, PLA at concentrations of 10.0 mg/mL can effectively control the bacterial cells in a three-day-old mature biofilm of E. faecalis grown on 24-well flat-bottom polystyrene plates and stainless-steel surfaces. Thus, PLA is potentially an effective agent to control E. faecalis biofilms.

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av.

Whole-genome sequence analysis of Vibrio cholerae from three outbreaks in Uganda, 2014 - 2016

Background: Cholera remains a serious public health problem in Uganda and Africa. The aim of this study was to provide the complete array of antimicrobial resistance genes, integrative and conjugative elements, virulence genes, pathogenicity islands, plasmids, and insertion sequences in the strains. In addition, this study also aimed to provide a single nucleotide polymorphism (SNP) based phylogenetic analysis of the strains. Methods: In the analysis, both Linux and web-based bioinformatics approaches were used to analyze the study sequences. Databases used included; FastQC, MultiQC, Snippy, PANTHER, PATRIC, Unicycler, ISFinder, Center for Genomic Epidemiology pipelines (i.e. MLST, PlasmidFinder, MyDbFinder, and ResFinder), MashTree and IcyTree. Results: The 10 sequenced strains of Vibrio cholerae were found to carry virulence-associated genes including MakA, ctxA, ctxB, carA, carB, trpB, clpB, ace, toxR, zot, rtxA, ompW, ompR, gmhA, fur, hlyA, and rstR. Also identified were: genes of the Type VI secretion system including vasA-L, vgrG-2, vgrG-3, vipA/mglA, and vipB/mglB; alsD (VC1589), involved in the synthesis of 2,3-butanediol; alsR, involved in the acetate-responsive LysR-type regulation; makA, the flagella-mediated cytotoxin gene; Type VI pilus genes including tcpA-F, tcpH-J, tcpN, tcpP-T, and icmF/vasK; adherence genes acfA-D and IlpA; and quorum sensing system genes luxS and cqsA. Pathogenicity islands identified comprised of VSP-1 and VSP-2, as well as VPI-1 and VPI-2. In addition, strA and B, APH(3'')-I, APH(3'')-Ib, APH(6)-Id, APH(6)-Ic, murA, pare, dfrA1, floR, catB, and catB9 were among the antimicrobial resistance genes found in the sequences. Analysis for SNPs shared among the sequences showed that the sequenced strains shared 218 SNPs and of these, 98 SNPs were missense. Gene enrichment analysis of these SNPs showed enrichment in genes that mediate transmembrane-signaling receptor activity, peptidyl-prolyl cis-trans isomerase activity, and phosphor-relay response regulator activity. Conclusions: This study applied bioinformatics approaches to provide comprehensive genomic analysis of V. cholerae genomes obtained from Uganda.

CRISPR-Cas9 knockout of qseB induced asynchrony between motility and biofilm formation in Escherichia coli.

Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.

Genomic Analysis of Pseudomonas sp. Strain SCT, an Iodate-Reducing Bacterium Isolated from Marine Sediment, Reveals a Possible Use for Bioremediation.

Strain SCT is an iodate-reducing bacterium isolated from marine sediment in Kanagawa Prefecture, Japan. In this study, we determined the draft genome sequence of strain SCT and compared it to complete genome sequences of other closely related bacteria, including Pseudomonas stutzeri. A phylogeny inferred from concatenation of core genes revealed that strain SCT was closely related to marine isolates of P. stutzeri. Genes present in the SCT genome but absent from the other analyzed P. stutzeri genomes comprised clusters corresponding to putative prophage regions and possible operons. They included pil genes, which encode type IV pili for natural transformation; the mer operon, which encodes resistance systems for mercury; and the pst operon, which encodes a Pi-specific transport system for phosphate uptake. We found that strain SCT had more prophage-like genes than the other P. stutzeri strains and that the majority (70%) of them were SCT strain-specific. These genes, encoded on distinct prophage regions, may have been acquired after branching from a common ancestor following independent phage transfer events. Thus, the genome sequence of Pseudomonas sp. strain SCT can provide detailed insights into its metabolic potential and the evolution of genetic elements associated with its unique phenotype.

Genomic analysis of a novel nontoxigenic Corynebacterium diphtheriae strain isolated from a cancer patient.

The genome of a novel nontoxigenic Corynebacterium diphtheriae, strain 5015, isolated from a patient with adenoid cystic carcinoma was sequenced and compared with 117 publically available genomes. This strain is phylogenetically distinct and lacks virulence genes encoding the toxin, BigA and Sdr-like adhesins. Strain 5015 possesses spaD-type and spaH-type pilus gene clusters with a loss of some gene functions, and 31 unique genes that need molecular characterization to understand their potential role in virulence characteristics.

A ‘pathogenic needle’ in a ‘commensal haystack’: Genetic virulence signatures of Corynebacterium glucuronolyticum that may drive its infectious propensity for the male urogenital system

The predominance of the genus Corynebacterium in the healthy male urogenital system contributes to the resident microbiome of not only the distal urethra, but potentially the proximal urethra and urinary bladder as well. However, for certain species in this genus, pathogenic potential was described, and the salient representative is Corynebacterium glucuronolyticum (C. glucuronolyticum) implicated in cases of urethritis and prostatitis in men. Nonetheless, some still question whether C. glucuronolyticum can actually be considered pathogenic or rather just a commensal species fortuitously isolated in patients with urogenital symptoms and/or syndromes. Although pathogen/commensal dichotomy is not always clear-cut, we hypothesize that specific genetic markers may expose C. glucuronolyticum as a convincingly pathogenic Corynebacterium. More specifically, characteristic pathogenic gene constellation inherent to this species (most notably the presence of specific sortase/SpaA-type pili gene clusters, but also the augmentative role of type VII secretion system) may significantly facilitate host tissue adhesion, with subsequent suppression/evasion of the immune response and acquisition of vitally important nutrients. Consequently, these genetic markers differentiate C. glucuronolyticum from its commensal counterparts, and give this species a pathogenic facet, which can be even further influenced by the Allee effect. In this paper we also propose a specific methodological approach on how to analyze C. glucuronolyticum epithelial colonization capacity and explore inceptive host cell-pathogen interactions that manipulate host environment and immune responses. This entails moving from approaches based primarily on overall homology of primary sequences towards specific structure-function studies to precisely evaluate all stakeholders involved in pili assemblage, cell adhesion and the expression of other virulence traits. In the era of high precision medicine, the hypothesized roles of C. glucuronolyticum adhesion systems in both virulence and nutrient acquisition may also reveal promising targets for future drug developments.

Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release.

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (ldhA) mutant had an increased level of biofilm formation. Deletion of ldhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-deficient mutant, suggesting a critical role for eDNA. Compared to wild-type, the ldhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the ldhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of ldhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.

Phase variation of Opa proteins in hypervirulent serogroup W meningococcal isolates

Serotype W, sequence type 11 Neisseria meningitidis are an increasing cause of morbidity and mortality. The mechanisms underpinning the success of this clone remain unclear. Meningococci express up to four Opa proteins, which mediate adhesion to/invasion of host cells. Opa expression undergoes phase variation – a high frequency ON/OFF switch in gene expression due to insertion/deletions of pentameric repeats (5′-CTCTT-3′) in the coding region. NadA functions as an adhesion, and is phase-variable through tetrameric repeats in a regulatory. WGS and PCR-based fragment size analysis of 121 invasive and 51 carriage MenW:ST11 strains was conducted to determine gene expression patterns between invasive and carriage. Inactivating mutations were constructed in opa, pilE and nadA genes of two representative MenW isolates. These mutants were tested in in vitro infection assays for adhesion and invasion of A549 cells. We observe that four opa loci are present in all MenW:cc11 isolates and that OpaB and OpaD share 100 % sequence similarity. Repeat number variability was detected between the ‘original UK’ strain and the novel ‘2013-strain’ of the hypervirulent MenW:cc11 South American sublineage. No significant differences in the patterns of Opa expression were observed between invasive and carriage isolates. In vitro assays with pilE and nadA deletion suggest that NadA has an effect on invasion, while the double mutant shows a reduction in both adhesion and invasion. Our findings indicate that the Opa proteins do not contribute to the invasiveness of MenW:cc11 strains, but may give a niche specific advantage by enhancing phase variation-mediated immune evasion.

Molecular Characterization of Diarrhoegenic Escherichia coli Isolated from Neonatal Goat-Kids

Faecal samples (n=300) from diarrhoeic neonatal goat-kids of different livestock sheds of ICAR-CIRG, Makhdoom, and field goat-kids of Mathura, UP were aseptically collected, and used for E. coli isolation. On the basis of cultural, morphological, biochemical and molecular characteristics, a total of 193 E. coli isolates were identified from 300 fecal samples. Out of 140 E. coli isolates, only 90 isolates could be serotyped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, and the most common serogroups responsible for neonatal diarrhoea were found as O88 (n=11), O22 (n=10), O11 (n=8) and O83 (n=7). Congo red dye agar test was done to determine invasiveness of the isolates, and 77.20% (149/193)E. coli isolates showed Congo red binding activity. Identification of shiga toxin producing E. coli (STEC) was done by PCR amplification of its stx-1 gene, and 5.69% (11/193) isolates were identified as STEC. Pathotype specific primers were used to amplify bundle forming pilus (bfpA) gene of enteropathogenic E. coli (EPEC), and 35.23% (68/193) isolates were identified as EPEC. A multiplex PCR was performed to detect labile toxin producing enterotoxigenic E. coli (ETEC-lt), stable toxin producing enterotoxigenic E. coli (ETEC-st) and enteroinvasive E. coli (EIEC), and 24.35% (47/193), 2.59% (5/193) and 2.07% (4/193) isolates were determined as ETEC-st, ETEC-lt and EIEC, respectively. EPEC and ETEC-st were found as the most prevalent pathotypes associated with neonatal diarrhoea in goat-kids whereas; O88 and O22 were observed as the most common serogroups in causing diarrhoea in the neonatal goat-kids.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.

Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.

Development of a multiplex PCR assay based on the pilA gene sequences to detect different types of Acidovorax citrulli

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli, is a major threat to commercial watermelon and melon production worldwide. At present, there are at least two genetically distinct sub-populations (group I and II) of A. citrulli that differ in host preference among cucurbit species and copper sensitivity. In this study, we analyzed the pilA gene sequences of 103 A. citrulli strains from China and other countries. Based on these data, we classified all tested A. citrulli strains into three types. The pilA-based type 1 strains in this study coincided with the previously established group I strains; while the type 2 strains coincided with group II strains. Ten strains that did not cluster with group I or II strains were classified into a new type, designated type 3. Based on differences in pilA sequences, we designed a multiplex PCR assay to distinguish the three A. citrulli pilus types. This multiplex PCR assay has proven to be viable for strain typing of 139 A. citrulli strains and for the detection of this pathogen in artificially inoculated seeds and leaves and naturally infected leaves and fruits. This assay proved to be rapid, accurate, reliable and applicable for early distinction of A. citrulli types associated with BFB epidemics. It may also inform the judicious and environmentally sound use of bactericides, especially copper-based compounds.

The structure of PilA from Acinetobacter baumannii AB5075 suggests a mechanism for functional specialization in Acinetobacter type IV pili

Type IV pili (T4P) are bacterial appendages composed of protein subunits, called pilins, noncovalently assembled into helical fibers. T4P are essential, in many bacterial species, for processes as diverse as twitching motility, natural competence, biofilm or microcolony formation, and host cell adhesion. The genes encoding type IV pili are found universally in the Gram-negative, aerobic, nonflagellated, and pathogenic coccobacillus Acinetobacter baumannii, but there is considerable variation in PilA, the major protein subunit, both in amino acid sequence and in glycosylation patterns. Here we report the X-ray crystal structure of PilA from AB5075, a recently characterized, highly virulent isolate, at 1.9 Å resolution and compare it to homologues from A. baumannii strains ACICU and BIDMC57, which are C-terminally glycosylated. These structural comparisons revealed that PilAAB5075 exhibits a distinctly electronegative surface chemistry. To understand the functional consequences of this change in surface electrostatics, we complemented a ΔpilA knockout strain with divergent pilA genes from ACICU, BIDMC57, and AB5075. The resulting transgenic strains showed differential twitching motility and biofilm formation while maintaining the ability to adhere to epithelial cells. PilAAB5075 and PilAACICU, although structurally similar, promote different characteristics, favoring twitching motility and biofilm formation, respectively. These results support a model in which differences in pilus electrostatics affect the equilibrium of microcolony formation, which in turn alters the balance between motility and biofilm formation in Acinetobacter.

Population Structure, Antibiotic Resistance, and Uropathogenicity of Klebsiella variicola.

Klebsiella variicola is a member of the Klebsiella genus and often misidentified as Klebsiella pneumoniae or Klebsiella quasipneumoniae The importance of K. pneumoniae human infections has been known; however, a dearth of relative knowledge exists for K. variicola Despite its growing clinical importance, comprehensive analyses of K. variicola population structure and mechanistic investigations of virulence factors and antibiotic resistance genes have not yet been performed. To address this, we utilized in silico, in vitro, and in vivo methods to study a cohort of K. variicola isolates and genomes. We found that the K. variicola population structure has two distant lineages composed of two and 143 genomes, respectively. Ten of 145 K. variicola genomes harbored carbapenem resistance genes, and 6/145 contained complete virulence operons. While the β-lactam blaLEN and quinolone oqxAB antibiotic resistance genes were generally conserved within our institutional cohort, unexpectedly 11 isolates were nonresistant to the β-lactam ampicillin and only one isolate was nonsusceptible to the quinolone ciprofloxacin. K. variicola isolates have variation in ability to cause urinary tract infections in a newly developed murine model, but importantly a strain had statistically significant higher bladder CFU than the model uropathogenic K. pneumoniae strain TOP52. Type 1 pilus and genomic identification of altered fim operon structure were associated with differences in bladder CFU for the tested strains. Nine newly reported types of pilus genes were discovered in the K. variicola pan-genome, including the first identified P-pilus in Klebsiella spp.IMPORTANCE Infections caused by antibiotic-resistant bacterial pathogens are a growing public health threat. Understanding of pathogen relatedness and biology is imperative for tracking outbreaks and developing therapeutics. Here, we detail the phylogenetic structure of 145 K. variicola genomes from different continents. Our results have important clinical ramifications as high-risk antibiotic resistance genes are present in K. variicola genomes from a variety of geographic locations and as we demonstrate that K. variicola clinical isolates can establish higher bladder titers than K. pneumoniae Differential presence of these pilus genes inK. variicola isolates may indicate adaption for specific environmental niches. Therefore, due to the potential of multidrug resistance and pathogenic efficacy, identification of K. variicola and K. pneumoniae to a species level should be performed to optimally improve patient outcomes during infection. This work provides a foundation for our improved understanding of K. variicola biology and pathogenesis.

Molecular genetic basis of biofilm formation as a component of Vibrio Cholerae persistence in water reservoirs of Russian Federation

Background. The problem of cholera remains acute for world health service and risks of importation of Vibrio cholerae strains from endemic countries to Russia do exist. Toxigenic strains (carrying cholera toxin genes ctxAB) can cause epidemic outbreaks of cholera and non-toxigenic (ctxAB-) single or multiple cases of cholera-like diarrhea. Investigation of their ability to survive in water reservoirs in climatic conditions of middle latitudes by means of forming biofilms is essential for potential threat evaluation.&#x0D; Materials and methods. Biofilm formation by 15 V. cholerae strains on abiotic surfaces was studied in microcosms with tap water and cover glasses. Identification of responsible genetic determinants in whole genome sequences and bioinformatics analysis were performed using BioEdit 7.2.5, BLASTN 2.2.29, Blastp and Vector NTI Advance 11 software.&#x0D; Results. The strains investigated differed in terms of biofilm formation which correlated with structural features of genes for MSHA pili (msh), matrix polysaccharides (vps) and proteins (rbm) as well as for certain regulatory factors. Strains with none or few genetic deviations from prototypes formed mature biofilms in 5-7 days while those containing truncated genes mshL, mshN, rbmC only in 13 days. One strain with truncated gene for positive regulator vpsR formed an immature biofilm. Acceleration of the process in some strains up to 2-3 days correlated with either truncated gene hapR (negative regulator) or altered structure of both msh and vps-rbm gene clusters.&#x0D; Conclusion. Analysis of genetic determinants responsible for biofilm formation may be used for prediction of V. cholerae ability to survive in environmental objects of Russia and thus the potential danger of the latters as sources of infection.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus.

Two-component signaling systems (TCS) regulate bacterial responses to environmental signals through the process of protein phosphorylation. Specifically, sensor histidine kinases (SK) recognize signals and propagate the response via phosphorylation of a cognate response regulator (RR) that functions to initiate transcription of specific genes. Signaling within a single TCS is remarkably specific and cross-talk between TCS is limited. However, regulation of the flow of information through complex signaling networks that include closely related TCS remains largely unknown. Additionally, many bacteria utilize multi-component signaling networks which provide additional genetic and biochemical interactions that must be regulated for signaling fidelity, input and output specificity, and phosphorylation kinetics. Here we describe the characterization of an NtrC-like RR that participates in regulation of Type-IV pilus-dependent motility of Myxococcus xanthus and is thus named NmpR, NtrC Modulator of Pili Regulator. A complex multi-component signaling system including NmpR was revealed by suppressor mutations that restored motility to cells lacking PilR, an evolutionarily conserved RR required for expression of pilA encoding the major Type-IV pilus monomer found in many bacterial species. The system contains at least four signaling proteins: a SK with a protoglobin sensor domain (NmpU), a hybrid SK (NmpS), a phospho-sink protein (NmpT), and an NtrC-like RR (NmpR). We demonstrate that ΔpilR bypass suppressor mutations affect regulation of the NmpRSTU multi-component system, such that NmpR activation is capable of restoring expression of pilA in the absence of PilR. Our findings indicate that pilus gene expression in M. xanthus is regulated by an extended network of TCS which interact to refine control of pilus function.

A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location.

Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

Complete and assembled genome sequence of an NDM-5- and CTX-M-15-producing Escherichia coli sequence type 617 isolated from wastewater in Switzerland.

Carbapenem-resistant Escherichia coli have emerged worldwide and represent a major challenge to effective healthcare management. Here we report the genome sequence of an NDM-5- and CTX-M-15-producing E. coli belonging to sequence type 617 isolated from wastewater treatment plant effluent in Switzerland. Whole-genome sequencing of E. coli 657SK2 was performed using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology RS2 reads (C4/P6 chemistry). De novo assembly was carried out using Canu 1.6, and sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome of E. coli 657SK2 consists of a 4.9-Mbp chromosome containing blaCTX-M-15, genes associated with virulence [fyuA, hlyE, the pyelonephritis-associated pili (pap) gene cluster and the yad gene cluster], the copper resistance gene pco, and genes associated with resistance to quaternary ammonium compound (QAC) disinfectants (emrA, mdfA and sugE). A 173.9-kb multidrug resistance IncFII-FIA-FIB plasmid was detected harbouring aadA2, aadA5, blaNDM-5, blaOXA-1, cat, drfA, drfA17, the mph(A)-mrx-mphR cluster, the tetA-tetC-tetR cluster, and the virulence genes iutA and ylpA. The genome sequence of E. coli 657SK2 provides information on resistance mechanisms and virulence characteristics of pathogenic E. coli harbouring blaNDM-5 and blaCTX-M-15 that are spreading into the environment via urban wastewater.

Biological role of Actinobacillus pleuropneumoniae type IV pilus proteins encoded by the apf and pil operons

Actinobacillus pleuropneumoniae is a species of Gram-negative bacteria that colonizes the respiratory tract, causing porcine pleuropneumoniae. Given the high agricultural impact of this disease, a detailed understanding of the pathogenesis of A. pleuropneumoniae is crucial. Previous work found that only the apfABCD (apf) operon encodes type IV pili (Tfp), which promotes bacterial adherence to host cells, in A. pleuropneumoniae. However, the detailed biogenesis and function of Tfp in A. pleuropneumoniae remained unclear. Here, we found that the apfABCD and pilMNOPQ (pil) gene clusters are operons, and the products of each gene within the apf and pil loci are required for Tfp biogenesis and function. Sequencing analysis revealed that the apf and pil operons are highly conserved among different serotypes of A. pleuropneumoniae. Our data demonstrate that the transcription of the pil operon was greatly upregulated upon bacterial contact with host cells. Mutants with single deletions of each gene in the pil and apf operons had defective adherence when they were in contact with host cells and, additionally, these mutants were also deficient in biofilm formation in vitro. In conclusion, these results indicate that the products of each gene within the apf and pil operons are necessary for the normal biogenesis and function of Tfp in A. pleuropneumoniae.