Involvement of RpoN in Regulating Motility, Biofilm, Resistance, and Spoilage Potential of Pseudomonas fluorescens

Pseudomonas fluorescens is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen assimilation and virulence in many pathogens. However, its exact role in regulating the spoilage caused by P. fluorescens is unknown. Here, an in-frame deletion mutation of rpoN was constructed to investigate its global regulatory function through phenotypic and RNA-seq analysis. The results of phenotypic assays showed that the rpoN mutant was deficient in swimming motility, biofilm formation, and resistance to heat and nine antibiotics, while the mutant increased the resistance to H2O2. Moreover, the rpoN mutant markedly reduced extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized fish juice at 4°C; meanwhile, the juice with the rpoN mutant showed significantly higher sensory scores than that with the wild-type strain. To identify RpoN-controlled genes, RNA-seq-dependent transcriptomics analysis of the wild-type strain and the rpoN mutant was performed. A total of 1224 genes were significantly downregulated, and 474 genes were significantly upregulated by at least two folds at the RNA level in the rpoN mutant compared with the wild-type strain, revealing the involvement of RpoN in several cellular processes, mainly flagellar mobility, adhesion, polysaccharide metabolism, resistance, and amino acid transport and metabolism; this may contribute to the swimming motility, biofilm formation, stress and antibiotic resistance, and spoilage activities of P. fluorescens. Our results provide insights into the regulatory role of RpoN of P. fluorescens in food spoilage, which can be valuable to ensure food quality and safety.

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RstA, a two-component response regulator, plays important roles in multiple virulence-associated processes in enterohemorrhagic Escherichia coli O157:H7

Gut Pathogens ◽

10.1186/s13099-019-0335-4 ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Yutao Liu ◽

Shujie Li ◽

Wendi Li ◽

Peisheng Wang ◽

Peng Ding ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Acid Tolerance ◽

Enterohemorrhagic Escherichia Coli ◽

Regulatory Function ◽

Wild Type ◽

Two Component

Abstract Background Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157. Results In the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP. Conclusions In summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections.

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Rhizosphere Selection of Highly Motile Phenotypic Variants of Pseudomonas fluorescens with Enhanced Competitive Colonization Ability

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3429-3434.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3429-3434 ◽

Cited By ~ 62

Author(s):

Francisco Martï¿½nez-Granero ◽

Rafael Rivilla ◽

Marta Martï¿½n

Keyword(s):

Pseudomonas Fluorescens ◽

Type Strain ◽

Wild Type Strain ◽

Root Tip ◽

Wild Type ◽

Swimming Motility ◽

Rhizosphere Colonization ◽

Prolonged Cultivation ◽

Genes Encoding ◽

Different Populations

ABSTRACT Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks.

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Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

mBio ◽

10.1128/mbio.01464-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 6

Author(s):

Jan Kampf ◽

Jan Gerwig ◽

Kerstin Kruse ◽

Robert Cleverley ◽

Miriam Dormeyer ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Selective Pressure ◽

Wild Type ◽

Master Regulator ◽

Form Complex ◽

Content Type

ABSTRACT Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains. IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

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vpaH, a Gene Encoding a Novel Histone-Like Nucleoid Structure-Like Protein That Was Possibly Horizontally Acquired, Regulates the Biogenesis of Lateral Flagella in trh-Positive Vibrio parahaemolyticus TH3996

Infection and Immunity ◽

10.1128/iai.73.9.5754-5761.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5754-5761 ◽

Cited By ~ 19

Author(s):

Kwon-Sam Park ◽

Michiko Arita ◽

Tetsuya Iida ◽

Takeshi Honda

Keyword(s):

Biofilm Formation ◽

Vibrio Parahaemolyticus ◽

Type Strain ◽

Wild Type Strain ◽

Enteric Bacteria ◽

Wild Type ◽

Gene Encoding ◽

Lateral Flagellum ◽

Global Gene Regulation ◽

Nucleoid Structure

ABSTRACT A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.

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Quorum-Sensing Systems in the Plant Growth-Promoting Bacterium Paraburkholderia phytofirmans PsJN Exhibit Cross-Regulation and Are Involved in Biofilm Formation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0008-r ◽

2017 ◽

Vol 30 (7) ◽

pp. 557-565 ◽

Cited By ~ 16

Author(s):

Ana Zúñiga ◽

Raúl A. Donoso ◽

Daniela Ruiz ◽

Gonzalo A. Ruz ◽

Bernardo González

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Homoserine Lactone ◽

Wild Type ◽

Transcript Levels ◽

Sensing Systems ◽

Plant Growth Promoting ◽

Growth Promoting

Quorum-sensing systems play important roles in host colonization and host establishment of Burkholderiales species. Beneficial Paraburkholderia species share a conserved quorum-sensing (QS) system, designated BraI/R, that controls different phenotypes. In this context, the plant growth-promoting bacterium Paraburkholderia phytofirmans PsJN possesses two different homoserine lactone QS systems BpI.1/R.1 and BpI.2/R.2 (BraI/R-like QS system). The BpI.1/R.1 QS system was previously reported to be important to colonize and produce beneficial effects in Arabidopsis thaliana plants. Here, we analyzed the temporal variations of the QS gene transcript levels in the wild-type strain colonizing plant roots. The gene expression patterns showed relevant differences in both QS systems compared with the wild-type strain in the unplanted control treatment. The gene expression data were used to reconstruct a regulatory network model of QS systems in P. phytofirmans PsJN, using a Boolean network model. Also, we examined the phenotypic traits and transcript levels of genes involved in QS systems, using P. phytofirmans mutants in homoserine lactone synthases genes. We observed that the BpI.1/R.1 QS system regulates biofilm formation production in strain PsJN and this phenotype was associated with the lower expression of a specific extracytoplasmic function sigma factor ecf26.1 gene (implicated in biofilm formation) in the bpI.1 mutant strain.

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Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

10.1101/536201 ◽

2019 ◽

Cited By ~ 3

Author(s):

Philippe Vogeleer ◽

Antony T. Vincent ◽

Samuel M. Chekabab ◽

Steve J. Charette ◽

Alexey Novikov ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Inorganic Phosphate ◽

Type Strain ◽

Wild Type Strain ◽

Pho Regulon ◽

Wild Type ◽

E Coli ◽

Pi Starvation ◽

Pst System

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. InE. coliO157:H7, the Δpstmutant, biofilm, and autoagglutination were increased. In the double-deletion mutant ΔpstΔphoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from Δpstmutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation andpstmutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the Δpstmutant. Interestingly,waaH, encoding a glycosyltransferase associated with LPS modifications inE. coliK-12, was highly expressed in the Δpstmutant ofE. coliO157:H7. Deletion ofwaaHfrom the Δpstmutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the Δpstmutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how theE. coliO157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

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Porphyromonas gingivalis Induces Insulin Resistance by Increasing BCAA Levels in Mice

Journal of Dental Research ◽

10.1177/0022034520911037 ◽

2020 ◽

Vol 99 (7) ◽

pp. 839-846 ◽

Cited By ~ 2

Author(s):

J. Tian ◽

C. Liu ◽

X. Zheng ◽

X. Jia ◽

X. Peng ◽

...

Keyword(s):

Insulin Resistance ◽

Insulin Sensitivity ◽

Plasma Level ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Alveolar Bone ◽

Alveolar Bone Loss ◽

Wild Type

Insulin resistance is one of the critical pathogeneses of type 2 diabetes mellitus (T2DM). Elevated levels of plasma branched-chain amino acids (BCAAs) are associated with insulin resistance. Recent studies have demonstrated the role of Porphyromonas gingivalis in the development of insulin resistance. However, the mechanisms by which P. gingivalis induces insulin resistance are still unclear. The purpose of this study was to investigate whether P. gingivalis induces insulin resistance through BCAA biosynthesis. We established a murine model of periodontitis by infecting mice with P. gingivalis. Alveolar bone loss, insulin sensitivity, and the plasma level of BCAAs were measured. A P. gingivalis BCAA aminotransferase-deficient strain ( ∆bcat) was constructed, and its kinetic growth, biofilm formation, and in vivo colonization were compared with its wild-type strain. Alveolar bone loss, insulin sensitivity, and the plasma level of BCAAs of the mice infected with either wild-type strain or ∆bcat strain were further measured. We found that periodontal infection with P. gingivalis significantly upregulated the plasma level of BCAAs and aggravated the high-fat diet (HFD)–induced insulin resistance. Bcat deletion did not alter the growth, biofilm formation, and in vivo colonization of P. gingivalis. More important, the ∆bcat strain was unable to upregulate the plasma level of BCAAs and induce insulin resistance in HFD-fed mice. These findings suggest that the BCAA biosynthesis of P. gingivalis plays a critical role in the development of insulin resistance in the HFD-fed mice. The BCAA biosynthesis pathways may provide a potential target for the disruption of linkage between periodontitis and T2DM.

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The Edwardsiella piscicida Type III Translocon Protein EseC Inhibits Biofilm Formation by Sequestering EseE

Applied and Environmental Microbiology ◽

10.1128/aem.02133-18 ◽

2019 ◽

Vol 85 (8) ◽

Cited By ~ 3

Author(s):

Ying Li Liu ◽

Tian Tian He ◽

Lu Yi Liu ◽

Jia Yi ◽

Pin Nie ◽

...

Keyword(s):

Type Iii Secretion ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Type Iii Secretion System ◽

Secretion System ◽

Wild Type ◽

Content Type ◽

Type Iii ◽

Edwardsiella Piscicida

ABSTRACT The type III secretion system (T3SS) is one of the most important virulence factors of the fish pathogen Edwardsiella piscicida. It contains three translocon proteins, EseB, EseC, and EseD, required for translocation of effector proteins into host cells. We have previously shown that EseB forms filamentous appendages on the surface of E. piscicida, and these filamentous structures mediate bacterial cell-cell interactions promoting autoaggregation and biofilm formation. In the present study, we show that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida. At 18 h postsubculture, a ΔeseC strain developed strong autoaggregation and mature biofilm formation, accompanied by enhanced formation of EseB filamentous appendages. This is in contrast to the weak autoaggregation and immature biofilm formation seen in the E. piscicida wild-type strain. EseE, a protein that directly binds to EseC and also positively regulates the transcription of the escC-eseE operon, was liberated and showed increased levels in the absence of EseC. This led to augmented transcription of the escC-eseE operon, thereby increasing the steady-state protein levels of intracellular EseB, EseD, and EseE, as well as biofilm formation. Notably, the levels of intracellular EseB and EseD produced by the ΔeseE and ΔeseC ΔeseE strains were similar but remarkably lower than those produced by the wild-type strain at 18 h postsubculture. Taken together, we have shown that the translocon protein EseC inhibits biofilm formation through sequestering EseE, a positive regulator of the escC-eseE operon. IMPORTANCE Edwardsiella piscicida, previously known as Edwardsiella tarda, is a Gram-negative intracellular pathogen that mainly infects fish. The type III secretion system (T3SS) plays a pivotal role in its pathogenesis. The T3SS translocon protein EseB is required for the assembly of filamentous appendages on the surface of E. piscicida. The interactions between the appendages facilitate autoaggregation and biofilm formation. In this study, we explored the role of the other two translocon proteins, EseC and EseD, in biofilm formation. We have demonstrated that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida, providing new insights into the regulatory mechanism involved in E. piscicida biofilm formation.

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Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence

Microbiology ◽

10.1099/mic.0.046185-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1798-1805 ◽

Cited By ~ 20

Author(s):

Hongyan Dong ◽

Daxin Peng ◽

Xinan Jiao ◽

Xiaorong Zhang ◽

Shizhong Geng ◽

...

Keyword(s):

Biofilm Formation ◽

Type Strain ◽

Salmonella Enteritidis ◽

Wild Type Strain ◽

Host Cells ◽

Wild Type ◽

Important Food ◽

Food Borne ◽

Gene Mutant ◽

Intracellular Proliferation

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

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Crp-Like Protein Plays Both Positive and Negative Roles in Regulating the Pathogenicity of Bacterial Pustule Pathogen Xanthomonas axonopodis pv. glycines

Phytopathology ◽

10.1094/phyto-07-18-0225-r ◽

2019 ◽

Vol 109 (7) ◽

pp. 1171-1183 ◽

Cited By ~ 3

Author(s):

Wei Guo ◽

Jie Gao ◽

Qingshan Chen ◽

Bojun Ma ◽

Yuan Fang ◽

...

Keyword(s):

Cell Motility ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Extracellular Polysaccharide ◽

Polymerase Chain Reaction Analysis ◽

Single Copy ◽

Wild Type ◽

Cell Wall Degrading Enzymes ◽

In The Wild

The global regulator Crp-like protein (Clp) is positively involved in the production of virulence factors in some of the Xanthomonas spp. However, the functional importance of Clp in X. axonopodis pv. glycines has not been investigated previously. Here, we showed that deletion of clp led to significant reduction in the virulence of X. axonopodis pv. glycines in soybean, which was highly correlated with the drastic reductions in carbohydrates utilization, extracellular polysaccharide (EPS) production, biofilm formation, cell motility, and synthesis of cell wall degrading enzymes (CWDEs). These significantly impaired properties in the clp mutant were completely rescued by a single-copy integration of the wild-type clp into the mutant chromosome via homologous recombination. Interestingly, overexpression of clp in the wild-type strain resulted in significant increases in cell motility and synthesis of the CWDEs. To our surprise, significant reductions in carbohydrates utilization, EPS production, biofilm formation, and the protease activity were observed in the wild-type strain overexpressing clp, suggesting that Clp also plays a negative role in these properties. Furthermore, quantitative reverse transcription polymerase chain reaction analysis suggested that clp was positively regulated by the diffusible signal factor-mediated quorum-sensing system and the HrpG/HrpX cascade. Taken together, our results reveal that Clp functions as both activator and repressor in multiple biological processes in X. axonopodis pv. glycines that are essential for its full virulence.

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