A Pleiotropic Regulator, Frp, Affects Exopolysaccharide Synthesis, Biofilm Formation, and Competence Development in Streptococcus mutans

ABSTRACT Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development.

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Role of Unsaturated Fatty Acid Biosynthesis in Virulence of Streptococcus mutans

Infection and Immunity ◽

10.1128/iai.01938-06 ◽

2007 ◽

Vol 75 (3) ◽

pp. 1537-1539 ◽

Cited By ~ 45

Author(s):

Elizabeth M. Fozo ◽

Kathy Scott-Anne ◽

Hyun Koo ◽

Robert G. Quivey

Keyword(s):

Fatty Acids ◽

Streptococcus Mutans ◽

Type Strain ◽

Wild Type Strain ◽

Fatty Acid Biosynthesis ◽

Wild Type ◽

Acid Biosynthesis ◽

Carious Lesions ◽

In The Wild

ABSTRACT An insertionally inactivated fabM strain of Streptococcus mutans does not produce unsaturated membrane fatty acids and is acid sensitive (E. M. Fozo and R. G. Quivey, Jr., J. Bacteriol. 186:4152-4158, 2004). In this study, the strain was shown to be poorly transmissible from host to host. Animals directly infected with the fabM strain exhibited fewer and less severe carious lesions than those observed in the wild-type strain.

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The Role of a Host-Induced Arginase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice

Phytopathology ◽

10.1094/phyto-02-19-0058-r ◽

2019 ◽

Vol 109 (11) ◽

pp. 1869-1877

Author(s):

Yuqiang Zhang ◽

Guichun Wu ◽

Ian Palmer ◽

Bo Wang ◽

Guoliang Qian ◽

...

Keyword(s):

Biofilm Formation ◽

Bacterial Blight ◽

Wild Type Strain ◽

Bacterial Pathogen ◽

Extracellular Polysaccharide ◽

Rice Cultivar ◽

Xanthomonas Oryzae ◽

Wild Type ◽

Bacterial Blight Of Rice

The plant bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial blight of rice, which is one of the most destructive rice diseases prevalent in Asia and parts of Africa. Despite many years of research, how X. oryzae pv. oryzae causes bacterial blight of rice is still not completely understood. Here, we show that the loss of the rocF gene caused a significant decrease in the virulence of X. oryzae pv. oryzae in the susceptible rice cultivar IR24. Bioinformatics analysis demonstrated that rocF encodes arginase. Quantitative real-time PCR and Western blot assays revealed that rocF expression was significantly induced by rice and arginine. The rocF deletion mutant strain showed elevated sensitivity to hydrogen peroxide, reduced extracellular polysaccharide (EPS) production, and reduced biofilm formation, all of which are important determinants for the full virulence of X. oryzae pv. oryzae, compared with the wild-type strain. Taken together, the results of this study revealed a mechanism by which a bacterial arginase is required for the full virulence of X. oryzae pv. oryzae on rice because of its contribution to tolerance to reactive oxygen species, EPS production, and biofilm formation.

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Influence of thePhotorhabdus luminescensPhosphomannose Isomerase Gene,manA, on Mannose Utilization, Exopolysaccharide Structure, and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.02326-10 ◽

2010 ◽

Vol 77 (3) ◽

pp. 776-785 ◽

Cited By ~ 8

Author(s):

Matthew R. Amos ◽

Maria Sanchez-Contreras ◽

Robert W. Jackson ◽

Xavier Muñoz-Berbel ◽

Todd A. Ciche ◽

...

Keyword(s):

Biofilm Formation ◽

Wild Type Strain ◽

Compositional Analysis ◽

Extracellular Polysaccharide ◽

Major Effect ◽

Photorhabdus Luminescens ◽

Wild Type ◽

Abiotic Surfaces ◽

Structural Matrix

ABSTRACTExtracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation inPhotorhabdus luminescensand demonstrate the key role of the phosphomannose isomerase gene,manA, in the structure of functional EPS. Phenotypic analyses of amanA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that themanAmutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis ofP. luminescensEPS reveals how themanAmutation has a major effect on the formation of a complete, branched EPS.

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In the absence of Lgt, lipoproteins are shed from Streptococcus uberis independently of Lsp

Microbiology ◽

10.1099/mic.0.022061-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 134-141 ◽

Cited By ~ 9

Author(s):

E. L. Denham ◽

P. N. Ward ◽

J. A. Leigh

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Bovine Mastitis ◽

Signal Peptidase ◽

Wild Type ◽

Streptococcus Uberis ◽

Degradation Analysis ◽

In The Wild ◽

Full Length Gene

The role of lipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) responsible for processing lipoproteins was investigated in Streptococcus uberis, a common cause of bovine mastitis. In the absence of Lgt, three lipoproteins [MtuA (SUB0473), Hap (SUB1625) and an extracellular solute-binding protein (SUB0365)] were detected in extracellular locations. All were shown by Edman degradation analysis to be cleaved on the carboxy side of the LXXC lipobox. Detection of MtuA, a lipoprotein shown previously to be essential for infectivity and virulence, was used as a surrogate lipoprotein marker to locate and assess processing of lipoproteins. The absence of Lgt did not prevent location of MtuA to the cell membrane, its location in the wild-type strain but, in contrast to the situation with wild-type, did result in a widespread location of this protein. In the absence of both Lgt and Lsp, MtuA was similarly released from the bacterial cell. In such strains, however, the cell-associated MtuA represented the full-length gene product, indicating that Lsp was able to cleave non-lipidated (lipo)proteins but was not responsible for their release from this bacterium.

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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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Role of glutathione in the growth of Bradyrhizobium sp. (peanut microsymbiont) under different environmental stresses and in symbiosis with the host plant

Canadian Journal of Microbiology ◽

10.1139/w06-007 ◽

2006 ◽

Vol 52 (7) ◽

pp. 609-616 ◽

Cited By ~ 15

Author(s):

Luciano Sobrevals ◽

Peter Müller ◽

Adriana Fabra ◽

Stella Castro

Keyword(s):

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Environmental Stresses ◽

Wild Type ◽

Glutamylcysteine Synthetase ◽

Bradyrhizobium Sp ◽

In The Wild ◽

Saline Medium

Glutathione (GSH) plays an important role in the defence of microorganisms and plants against different environmental stresses. To determine the role of GSH under different stresses, such as acid pH, saline shock, and oxidative shock, a GSH-deficient mutant (Bradyrhizobium sp. 6144-S7Z) was obtained by disruption of the gshA gene, which encodes the enzyme γ-glutamylcysteine synthetase. Growth of the mutant strain was significantly reduced in liquid minimal saline medium, and the GSH content was very low, about 4% of the wild-type level. The defect, caused by disruption of the gshA gene in the growth of mutant strain, cannot be reversed by the addition of GSH (up to 100 µmol/L) to the liquid minimal saline medium, and the endogenous GSH level was approximately the same as that observed without the addition of GSH. In contrast, the wild-type strain increased the GSH content under these conditions. However, the growth of the mutant strain in a rich medium (yeast extract – mannitol) increased, suggesting that at least some but not all of the functions of GSH could be provided by peptides and (or) amino acids. The symbiotic properties of the mutant were similar to those found in the wild-type strain, indicating that the mutation does not affect the ability of the mutant to form effective nodules.Key words: glutathione, γ-glutamylcysteine synthetase, Bradyrhizobium sp., peanut.

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Role of vimA in cell surface biogenesis in Porphyromonas gingivalis

Microbiology ◽

10.1099/mic.0.038331-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2180-2193 ◽

Cited By ~ 13

Author(s):

Devon O. Osbourne ◽

Wilson Aruni ◽

Francis Roy ◽

Christopher Perry ◽

Lawrence Sandberg ◽

...

Keyword(s):

Cell Surface ◽

Outer Membrane ◽

Porphyromonas Gingivalis ◽

Type Strain ◽

Wild Type Strain ◽

Phenotypic Expression ◽

Surface Proteins ◽

Wild Type ◽

In The Wild

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

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Role of the luxS Gene in Initial Biofilm Formation by Streptococcus mutans

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000371816 ◽

2015 ◽

Vol 25 (1) ◽

pp. 60-68 ◽

Cited By ~ 24

Author(s):

Zhiyan He ◽

Jingping Liang ◽

Zisheng Tang ◽

Rui Ma ◽

Huasong Peng ◽

...

Keyword(s):

Streptococcus Mutans ◽

Mutant Strain ◽

Biofilm Formation ◽

Type Strain ◽

Laser Scanning ◽

Wild Type Strain ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Wild Type ◽

Initial Biofilm

Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS luxS gene on initial biofilm formation by Streptococcus mutans. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The luxS mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the luxS mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the luxS mutant strain, except the gtfB gene. QS luxS gene can affect the initial biofilm formation by S. mutans.

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Role of the Type VI Secretion System in the Pathogenicity of Pseudomonas syringae pv. actinidiae, the Causative Agent of Kiwifruit Bacterial Canker

Frontiers in Microbiology ◽

10.3389/fmicb.2021.627785 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nana Wang ◽

Ning Han ◽

Runze Tian ◽

Jiliang Chen ◽

Xiaoning Gao ◽

...

Keyword(s):

Gene Cluster ◽

Pseudomonas Syringae ◽

Biofilm Formation ◽

Wild Type Strain ◽

Deletion Mutants ◽

Wild Type ◽

Type Vi Secretion System ◽

Bacterial Competition ◽

Type Vi Secretion

The type VI secretion system (T6SS), a macromolecular machine, plays an important role in the pathogenicity of many Gram-negative bacteria. However, the role of T6SS in the pathogenicity of Pseudomonas syringae pv. actinidiae (Psa), the pathogen of kiwifruit bacterial canker, is yet to be studied. Here, we found a T6SS gene cluster consisting of 13 core genes (A-J) in the genome of Psa M228 based on a genome-wide analysis. To determine whether the T6SS gene cluster affects the pathogenicity of Psa M228, T6SS and its 13 core gene deletion mutants were constructed and their pathogenicity was determined. The deletion mutants showed different degrees of reduction in pathogenicity compared with the wild-type strain M228; in tssM and tssJ mutants, pathogenicity was significantly reduced by 78.7 and 71.3%, respectively. The pathogenicity results were also confirmed by electron microscopy. To further confirm that the reduction in pathogenicity is related to the function of T6SS, we selected the T6SS gene cluster, comprising tssM and tssJ, for further analyses. Western blot results revealed that tssM and tssJ were necessary for hemolytic co-regulatory protein secretion, indicating that they encode a functional T6SS. Further, we explored the mechanism by which T6SS affects the pathogenicity of Psa M228. The ability of bacterial competition, biofilm formation, hydrogen peroxide tolerance, and proteolytic activity were all weakened in the deletion mutants M228ΔT6SS, M228ΔtssM, and M228ΔtssJ. All these properties of the two gene complementation mutants were restored to the same levels as those of the wild-type strain, M228. Quantitative real-time results showed that during the interaction between the deletion mutant M228ΔT6SS and the host, expression levels of T3SS transcriptional regulatory gene hrpR, structural genes hrpZ, hrcC, hopP1, and effector genes hopH1 and hopM1 were down-regulated at different levels. Taken together, our data provide evidence for the first time that the T6SS plays an important role in the pathogenicity of Psa, probably via effects on bacterial competition, biofilm formation, and environmental adaptability. Moreover, a complicated relationship exists between T6SS and T3SS.

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Effects of RelA on Key Virulence Properties of Planktonic and Biofilm Populations of Streptococcus mutans

Infection and Immunity ◽

10.1128/iai.72.3.1431-1440.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1431-1440 ◽

Cited By ~ 106

Author(s):

José A. C. Lemos ◽

Thomas A. Brown ◽

Robert A. Burne

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Stringent Response ◽

Strain Differences ◽

Acid Tolerance ◽

Normal Growth ◽

Wild Type ◽

Carbohydrate Source

ABSTRACT Streptococcus mutans is a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity. Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism. Here, we investigated the role of the S. mutans relA gene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase/hydrolase, in biofilm formation and acid tolerance. Two mutants in which relA was insertionally inactivated or replaced by an antibiotic resistance determinant were constructed. Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar. The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks. There was no difference in the sensitivities to acid killing of the parent and relA strains grown in planktonic cultures. However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain. Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants. Expression of the luxS gene was increased as much as fivefold in the relA mutants, suggesting a link between AI-2 quorum sensing and the stringent response.

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