Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

Rhizosphere colonization by bacteria involves molecular and cellular mechanisms, such as motility and chemotaxis, biofilm formation, metabolic versatility, or biosynthesis of secondary metabolites, among others. Nonetheless, there is limited knowledge concerning the main regulatory factors that drive the rhizosphere colonization process. Here we show the importance of the AmrZ and FleQ transcription factors for adaption in the plant growth-promoting rhizobacterium (PGPR) and rhizosphere colonization model Pseudomonas ogarae F113. RNA-Seq analyses of P. ogarae F113 grown in liquid cultures either in exponential and stationary growth phase, and rhizosphere conditions, revealed that rhizosphere is a key driver of global changes in gene expression in this bacterium. Regarding the genetic background, this work has revealed that a mutation in fleQ causes considerably more alterations in the gene expression profile of this bacterium than a mutation in amrZ under rhizosphere conditions. The functional analysis has revealed that in P. ogarae F113, the transcription factors AmrZ and FleQ regulate genes involved in diverse bacterial functions. Notably, in the rhizosphere, these transcription factors antagonistically regulate genes related to motility, biofilm formation, nitrogen, sulfur, and amino acid metabolism, transport, signalling, and secretion, especially the type VI secretion systems. These results define the regulon of two important bifunctional transcriptional regulators in pseudomonads during the process of rhizosphere colonization.

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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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Investigation of LuxS-mediated quorum sensing in Klebsiella pneumoniae

Journal of Medical Microbiology ◽

10.1099/jmm.0.001148 ◽

2020 ◽

Vol 69 (3) ◽

pp. 402-413 ◽

Cited By ~ 3

Author(s):

Lijiang Chen ◽

Jonathan J. Wilksch ◽

Haiyang Liu ◽

Xiaoxiao Zhang ◽

Von V. L. Torres ◽

...

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Type Species ◽

Microtiter Plate ◽

Resistant Isolate ◽

Wild Type ◽

Content Type ◽

Link Type

Introduction. Autoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires luxS activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation. Aim. To investigate the role of luxS in biofilm formation and gene expression in the nosocomial pathogen Klebsiella pneumoniae . Methodology. A ΔluxS gene deletion was made in K. pneumoniae KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and ΔluxS strains grown in media supplemented with different carbohydrates. Potential roles of luxS in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide (wzm and wbbM), polysaccharide (pgaA), and type 3 fimbriae (mrkA) synthesis genes in wild-type and ΔluxS mutant biofilm extracts. Results. AI-2 production was dependent on the presence of luxS. AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the ΔluxS mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and ΔluxS mutant using a microtiter plate assay were observed. In ΔluxS mutant biofilm extracts, the expression of wzm was down-regulated, and the expression of pgaA, which encodes a porin for poly-β−1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated. Conclusion. Relationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in K. pneumoniae . These inter-connected processes could be important for bacterial group behaviour and persistence.

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Eucalyptol inhibits biofilm formation of Streptococcus pyogenes and its mediated virulence factors

Journal of Medical Microbiology ◽

10.1099/jmm.0.001253 ◽

2020 ◽

Vol 69 (11) ◽

pp. 1308-1318

Author(s):

Karuppiah Vijayakumar ◽

Vajravelu Manigandan ◽

Danaraj Jeyapragash ◽

Veeraiyan Bharathidasan ◽

Balaiyan Anandharaj ◽

...

Keyword(s):

Gene Expression ◽

Streptococcus Pyogenes ◽

Biofilm Formation ◽

Type Species ◽

Inhibitory Concentration ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Content Type ◽

Link Type

Introduction. Streptococcus pyogenes is a diverse virulent synthesis pathogen responsible for invasive systemic infections. Establishment of antibiotic resistance in the pathogen has produced a need for new antibiofilm agents to control the biofilm formation and reduce biofilm-associated resistance development. Aim. The present study investigates the in vitro antibiofilm activity of eucalyptol against S. pyogenes . Methodology. The antibiofilm potential of eucalyptol was assessed using a microdilution method and their biofilm inhibition efficacy was visualized by microscopic analysis. The biochemical assays were performed to assess the influence of eucalyptol on virulence productions. Real-time PCR analysis was performed to evaluate the expression profile of the virulence genes. Results. Eucalyptol showed significant antibiofilm potential in a dose-dependent manner without affecting bacterial growth. Eucalyptol at 300 µg ml−1 (biofilm inhibitory concentration) significantly inhibited the initial stage of biofilm formation in S. pyogenes . However, eucalyptol failed to diminish the mature biofilms of S. pyogenes at biofilm inhibitory concentration and it effectively reduced the biofilm formation on stainless steel, titanium, and silicone surfaces. The biochemical assay results revealed that eucalyptol greatly affects the cell-surface hydrophobicity, auto-aggregation, extracellular protease, haemolysis and hyaluronic acid synthesis. Further, the gene-expression analysis results showed significant downregulation of virulence gene expression upon eucalyptol treatment. Conclusion. The present study suggests that eucalyptol applies its antibiofilm assets by intruding the initial biofilm formation of S. pyogenes . Supplementary studies are needed to understand the mode of action involved in biofilm inhibition.

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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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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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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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Chinese herbal medicines and nutraceuticals inhibit Pseudomonas aeruginosa biofilm formation

Access Microbiology ◽

10.1099/acmi.0.000254 ◽

2021 ◽

Vol 3 (8) ◽

Author(s):

Minami Hayashi ◽

Hiroshi Kaneko ◽

Tetsuya Yamada ◽

Hideaki Ikoshi ◽

Norihisa Noguchi ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Type Species ◽

Antimicrobial Agents ◽

Herbal Medicines ◽

Bacterial Strains ◽

Chinese Herbal Medicines ◽

Content Type ◽

Chinese Herbal ◽

Link Type

Pseudomonas aeruginosa is a major biofilm-forming, opportunistic pathogen. Tolerance to antimicrobial agents due to biofilm formation may lead to the emergence of antimicrobial-resistant bacterial strains. Thus, adjunctive agents that can inhibit biofilm formation are necessary to enhance the therapeutic efficacy of antimicrobial agents. In this study, we evaluated the anti-biofilm formation activity of selected Chinese herbal medicines and nutraceuticals, which are commercially available in Japan. Among the eight agents evaluated for their potential to inhibit biofilm formation, Eiekikaryu S, Iribakuga and Hyakujunro significantly reduced P. aeruginosa biofilm formation (P <0.05) without inhibiting bacterial growth. Additionally, the expression of biofilm-associated genes (rhlR, rhlA and lasB) in P. aeruginosa was significantly suppressed by Eiekikaryu S, Iribakuga and Hyakujunro (P <0.001). Our findings indicate that some Chinese herbal medicines and nutraceuticals can be potential adjunctive agents for antimicrobial therapy against P. aeruginosa .

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Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing

Microbiology ◽

10.1099/mic.0.001027 ◽

2021 ◽

Author(s):

Ian Vandenbussche ◽

Andrea Sass ◽

Filip Van Nieuwerburgh ◽

Marta Pinto-Carbó ◽

Olga Mannweiler ◽

...

Keyword(s):

Gene Expression ◽

Single Molecule ◽

Type Species ◽

Cytosine Methylation ◽

Regulation Of Gene Expression ◽

Burkholderia Cenocepacia ◽

Smrt Sequencing ◽

Content Type ◽

Link Type ◽

Genome Bisulfite Sequencing

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia , an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia .

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