A mutagenic screen reveals NspS residues important for regulation of Vibrio cholerae biofilm formation

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

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Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽

10.1099/mic.0.001098 ◽

2021 ◽

Vol 167 (10) ◽

Author(s):

Mengting Shi ◽

Yue Zheng ◽

Xianghong Wang ◽

Zhengjia Wang ◽

Menghua Yang

Keyword(s):

Mouse Model ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Type Species ◽

Wild Type ◽

Expression Library ◽

Content Type ◽

Genomic Expression ◽

Infant Mouse ◽

Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

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PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽

10.1099/mic.0.001031 ◽

2021 ◽

Vol 167 (3) ◽

Author(s):

Sathi Mallick ◽

Shanti Kiran ◽

Tapas Kumar Maiti ◽

Anindya S. Ghosh

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Type Species ◽

Pentose Phosphate ◽

Bacterial Cells ◽

Surface Adhesion ◽

Content Type ◽

Penicillin Binding Proteins ◽

E Coli ◽

Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

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Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

Journal of Medical Microbiology ◽

10.1099/jmm.0.001467 ◽

2022 ◽

Vol 71 (1) ◽

Author(s):

Bailey F. Keefe ◽

Luiz E. Bermudez

Keyword(s):

Cystic Fibrosis ◽

Host Cell ◽

Biofilm Formation ◽

Type Species ◽

Divalent Cations ◽

Mycobacterium Abscessus ◽

Content Type ◽

Link Type ◽

Populations At Risk

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

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Molecular characterization of Vibrio cholerae O1 isolates obtained from outbreaks in the Philippines, 2015–2016

Journal of Medical Microbiology ◽

10.1099/jmm.0.001443 ◽

2021 ◽

Vol 70 (11) ◽

Author(s):

Mark Philip Bugayong ◽

Hidemasa Izumiya ◽

Josie M. Bilar ◽

Masatomo Morita ◽

Eiji Arakawa ◽

...

Keyword(s):

Vibrio Cholerae ◽

Type Species ◽

Variable Number Tandem Repeat ◽

The Philippines ◽

Large Chromosome ◽

Content Type ◽

Link Type ◽

Mlva Type ◽

El Tor

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon. Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies. Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines. Methodology. A total of 139 V . cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping. Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant (n=67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries. Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

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The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00139-17 ◽

2017 ◽

Vol 199 (18) ◽

Cited By ~ 15

Author(s):

Jennifer K. Teschler ◽

Andrew T. Cheng ◽

Fitnat H. Yildiz

Keyword(s):

Signal Transduction ◽

Vibrio Cholerae ◽

Histidine Kinase ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Response Regulator ◽

Systematic Analysis ◽

Content Type ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. cholerae. vxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels. IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.

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Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

Access Microbiology ◽

10.1099/acmi.0.000211 ◽

2021 ◽

Vol 3 (3) ◽

Author(s):

Aya Ahmad Elnegery ◽

Wafaa Kamel Mowafy ◽

Tarek Ahmed Zahra ◽

Noha Tharwat Abou El-Khier

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Proteolytic Activity ◽

Virulence Factors ◽

Biofilm Formation ◽

Type Species ◽

Assay Method ◽

Content Type ◽

Burn Wounds ◽

Link Type

Background. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection. Purpose. To estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes. Methods. A cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR. Results. In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain. Conclusions. QS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

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gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

Microbial Genomics ◽

10.1099/mgen.0.000594 ◽

2021 ◽

Vol 7 (6) ◽

Author(s):

Thea G. Fennell ◽

Grace A. Blackwell ◽

Nicholas R. Thomson ◽

Matthew J. Dorman

Keyword(s):

Vibrio Cholerae ◽

Metabolic Pathway ◽

Type Species ◽

Gene Encoding ◽

Natural Competence ◽

Content Type ◽

Metabolic Substrate ◽

Link Type ◽

Reference Strains ◽

Present Experimental Evidence

Members of the bacterial genus Vibrio utilize chitin both as a metabolic substrate and a signal to activate natural competence. Vibrio cholerae is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in V. cholerae has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse V. cholerae . We show that the gene encoding GbpA, known to be an important colonization and virulence factor in pandemic isolates, is not ubiquitous amongst V. cholerae . We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within V. cholerae is more complex than previously thought, and emphasize the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains.

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Transcriptome profiling of Variovorax paradoxus EPS under different growth conditions reveals regulatory and structural novelty in biofilm formation

Access Microbiology ◽

10.1099/acmi.0.000121 ◽

2020 ◽

Vol 2 (6) ◽

Author(s):

Richard J. Fredendall ◽

Jenny L. Stone ◽

Michael J. Pehl ◽

Paul M. Orwin

Keyword(s):

Gene Expression ◽

Stationary Phase ◽

Exponential Growth ◽

Biofilm Formation ◽

Type Species ◽

Extracellular Dna ◽

Differentially Expressed ◽

Content Type ◽

Variovorax Paradoxus ◽

Link Type

We used transcriptome analysis by paired-end strand-specific RNA-seq to evaluate the specific changes in gene expression associated with the transition to static biofilm growth in the rhizosphere plant growth-promoting bacterium Variovorax paradoxus EPS. Triplicate biological samples of exponential growth, stationary phase and static biofilm samples were examined. DESeq2 and Rockhopper were used to identify robust and widespread shifts in gene expression specific to each growth phase. We identified 1711 protein-coding genes (28%) using DESeq2 that had altered expression greater than twofold specifically in biofilms compared to exponential growth. Fewer genes were specifically differentially expressed in stationary-phase culture (757, 12%). A small set of genes (103/6020) were differentially expressed in opposing fashions in biofilm and stationary phase, indicating potentially substantial shifts in phenotype. Gene-ontology analysis showed that the only class of genes specifically upregulated in biofilms was associated with nutrient transport, highlighting the importance of nutrient uptake in the biofilm. The biofilm-specific genes did not overlap substantially with the loci identified by mutagenesis studies, although some were present in both sets. The most highly upregulated biofilm-specific gene is predicted to be a part of the RNA degradosome, which indicates that RNA stability is used to regulate the biofilm phenotype. Two small putative proteins, Varpa_0407 and Varpa_3832, are highly expressed specifically in biofilms and are predicted to be secreted DNA-binding proteins, which may stabilize extracellular DNA as a component of the biofilm matrix. An flp/tad type-IV pilus locus (Varpa_5148–60) is strongly downregulated specifically in biofilms, in contrast with results from other systems for these pili. Mutagenesis confirms that this locus is important in surface motility rather than biofilm formation. These experimental results suggest that V. paradoxus EPS biofilms have substantial regulatory and structural novelty.

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Phylogenetic and antimicrobial drug resistance analysis of Vibrio cholerae O1 isolates from Ghana

Microbial Genomics ◽

10.1099/mgen.0.000668 ◽

2021 ◽

Vol 7 (10) ◽

Author(s):

Japheth A. Opintan ◽

Robert C. Will ◽

George K. Kuma ◽

Mary Osei ◽

Amos Akumwena ◽

...

Keyword(s):

Vibrio Cholerae ◽

Type Species ◽

Lag Phase ◽

Environmental Isolates ◽

Content Type ◽

Antimicrobial Drug Resistance ◽

Link Type ◽

Pathogen Persistence ◽

Vibrio Cholerae O1 ◽

Vaccination Campaigns

We investigated the evolution, phylogeny and antimicrobial resistance of Vibrio cholerae O1 isolates (VCO1) from Ghana. Outbreak and environmental sources of VCO1 were characterized, whole-genome sequenced and compared to globally available seventh pandemic (7P) strains of V. cholerae at SNP resolution. Final analyses included 636 isolates. Novel Ghanaian isolates clustered into three distinct clades (clades 1, 2 and 3) in wave 3 of the 7P lineage. The closest relatives of our novel Ghanaian isolates were from Benin, Cameroon, Togo, Niger and Nigeria. All novel Ghanaian isolates were multi-drug resistant. Environmental isolates clustered into clade 2, despite being isolated years later, showing the possibility of persistence and re-emergence of older clades. A lag phase of several years from estimated introduction to reported cases suggests pathogen persistence in the absence of reported cholera cases. These results highlight the importance of deeper surveillance for understanding transmission routes between bordering countries and planning tailored vaccination campaigns in an effort to eradicate cholera.

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Characterizing species interactions that contribute to biofilm formation in a multispecies model of a potable water bacterial community

Microbiology ◽

10.1099/mic.0.000849 ◽

2020 ◽

Vol 166 (1) ◽

pp. 34-43 ◽

Cited By ~ 2

Author(s):

Alex F. Thompson ◽

Erika L. English ◽

Adam M. Nock ◽

Graham G. Willsey ◽

Korin Eckstrom ◽

...

Keyword(s):

Drinking Water ◽

Bacterial Community ◽

Species Interactions ◽

Biofilm Formation ◽

Type Species ◽

Potable Water ◽

Space Station ◽

Water Systems ◽

Content Type ◽

Link Type

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans , Chryseobacterium gleum , Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis , but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum / S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans . These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

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