scholarly journals The Exopolysaccharide of Xylella fastidiosa Is Essential for Biofilm Formation, Plant Virulence, and Vector Transmission

2013 ◽  
Vol 26 (9) ◽  
pp. 1044-1053 ◽  
Author(s):  
N. Killiny ◽  
R. Hernandez Martinez ◽  
C. Korsi Dumenyo ◽  
D. A. Cooksey ◽  
R. P. P. Almeida
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Xylella Fastidiosa ◽  
Growth Characteristics ◽  
Gas Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Mutant Strains ◽  
Plant Movement

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography–mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

scholarly journals Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

2020 ◽  
Vol 8 (1) ◽  
pp. 70 ◽  
Author(s):  
Bhumika Shokeen ◽  
Jane Park ◽  
Emily Duong ◽  
Sonam Rambhia ◽  
Manash Paul ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Fusobacterium Nucleatum ◽  
Interspecies Interaction ◽  
Wild Type ◽  
Oral Epithelial Cells ◽  
Small Proteins ◽  
Mutant Strains ◽  
Oral Epithelial ◽  
Wild Type Parent

RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

scholarly journals Glucomannan-Mediated Attachment of Rhizobium leguminosarum to Pea Root Hairs Is Required for Competitive Nodule Infection

2008 ◽  
Vol 190 (13) ◽  
pp. 4706-4715 ◽  
Author(s):  
Alan Williams ◽  
Adam Wilkinson ◽  
Martin Krehenbrink ◽  
Daniela M. Russo ◽  
Angeles Zorreguieta ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Rhizobium Leguminosarum ◽  
Root Hairs ◽  
The Other ◽  
Plant Interactions ◽  
Wild Type ◽  
Polysaccharide Synthesis ◽  
Surface Polysaccharides

ABSTRACT The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterized. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one of these loci (gmsA) determines glucomannan synthesis and one (gelA) determines a gel-forming polysaccharide, but the role of the other locus (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both of these characteristics, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but it did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but it was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria but are absent from closely related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterium-plant interactions.

A role of Candida albicans CDC4 in the negative regulation of biofilm formation

2015 ◽  
Vol 61 (4) ◽  
pp. 247-255 ◽  
Author(s):  
Tzu-Ling Tseng ◽  
Wei-Chung Lai ◽  
Tai-Lin Lee ◽  
Wan Hua Hsu ◽  
Yu Wen Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Negative Regulation ◽  
Null Mutant ◽  
Auxotrophic Strain ◽  
Reduction Assay ◽  
Mutant Strains

The CDC4 gene is nonessential in Candida albicans and plays a role in suppressing filamentous growth, in contrast to its homologues, which are involved in the G1–S transition of the cell cycle. While characterizing the function of C. albicans CDC4 (CaCDC4), we found that the loss of CaCDC4 resulted in a reduction in cell flocculation, indicating a possible role for CaCDC4 in biofilm formation. To elucidate the role of CaCDC4 in biofilm formation, Cacdc4 null mutant strains were constructed by using the mini-Ura-blaster method. To create a CaCDC4 rescued strain, the plasmid p6HF-ACT1p-CaCDC4 capable of constitutively expressing CaCDC4 was introduced into the Cacdc4 homozygous null mutant. To determine the biofilm formation ability, an in vitro XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-5-carboxanilide) reduction assay was used. Compared with the parental auxotrophic strain BWP17, the Cacdc4 homozygous null mutant was able to enhance biofilm formation significantly. This enhancement of biofilm formation in the Cacdc4 homozygous null mutant could be reversed by constitutively expressing CaCDC4. We conclude that CaCDC4 has a role in suppressing biofilm formation in C. albicans.

scholarly journals Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni

2021 ◽  
Author(s):  
Martin Tereň ◽  
Ekaterina Shagieva ◽  
Lucie Vondrakova ◽  
Jitka Viktorova ◽  
Viviana Svarcova ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Early Stage ◽  
Wild Type ◽  
Genetic Determinants ◽  
Autoinducer 2 ◽  
Naturally Occurring ◽  
Mutant Strains ◽  
The Impact

Abstract Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81–176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated (⸬luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

scholarly journals Salmonella typhimurium LT2 Catabolizes Propionate via the 2-Methylcitric Acid Cycle

1999 ◽  
Vol 181 (18) ◽  
pp. 5615-5623 ◽  
Author(s):  
Alexander R. Horswill ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Salmonella Typhimurium ◽  
Negative Ion ◽  
Resonance Spectroscopy ◽  
Ionization Mass ◽  
Wild Type ◽  
Acid Cycle ◽  
Mutant Strains ◽  
Methylcitrate Synthase

ABSTRACT We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism inSalmonella typhimurium. Results from13C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro.1H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

scholarly journals Role of Cyclic di-GMP in Xylella fastidiosa Biofilm Formation, Plant Virulence, and Insect Transmission

2010 ◽  
Vol 23 (10) ◽  
pp. 1356-1363 ◽  
Author(s):  
Subhadeep Chatterjee ◽  
Nabil Killiny ◽  
Rodrigo P. P. Almeida ◽  
Steven E. Lindow
Keyword(s):  
Cell Density ◽  
Intracellular Signaling ◽  
Biofilm Formation ◽  
Extracellular Enzymes ◽  
Xylella Fastidiosa ◽  
Amorphous Layer ◽  
Extracellular Polysaccharides ◽  
Wild Type ◽  
A Cell

Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

scholarly journals Chromosome-Based Genetic Complementation System for Xylella fastidiosa

2009 ◽  
Vol 75 (6) ◽  
pp. 1679-1687 ◽  
Author(s):  
Ayumi Matsumoto ◽  
Glenn M. Young ◽  
Michele M. Igo
Keyword(s):  
Dna Sequences ◽  
Xylella Fastidiosa ◽  
Cell Physiology ◽  
Multiple Cloning Site ◽  
Wild Type ◽  
In Planta ◽  
Catalase Peroxidase ◽  
The Stability

ABSTRACT Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

scholarly journals Edwardsiella piscicida YefM-YoeB: A Type II Toxin-Antitoxin System That Is Related to Antibiotic Resistance, Biofilm Formation, Serum Survival, and Host Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Dongmei Ma ◽  
Hanjie Gu ◽  
Yanjie Shi ◽  
Huiqin Huang ◽  
Dongmei Sun ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Wild Strain ◽  
Host Cells ◽  
Resistant Bacteria ◽  
Serum Resistance ◽  
Type Ii ◽  
Edwardsiella Piscicida

The emergence of drug resistant bacteria is a tricky and confronted problem in modern medicine, and one of important reasons is the widespread of toxin-antitoxin (TA) systems in pathogenic bacteria. Edwardsiella piscicida (also known as E. tarda) is the leading pathogen threatening worldwide fresh and seawater aquaculture industries and has been considered as a model organism for studying intracellular and systemic infections. However, the role of type II TA systems are completely unknown in aquatic pathogenic bacteria. In this study, we identified and characterized a type II TA system, YefM-YoeB, of E. piscicida, where YefM is the antitoxin and YoeB is the toxin. yefM and yoeB are co-expressed in a bicistronic operon. When expressed in E. coli, YoeB cause bacterial growth arrest, which was restored by the addition of YefM. To investigate the biological role of the TA system, two markerless yoeB and yefM-yoeB in-frame mutant strains, TX01ΔyoeB and TX01ΔyefM-yoeB, were constructed, respectively. Compared to the wild strain TX01, TX01ΔyefM-yoeB exhibited markedly reduced resistance against oxidative stress and antibiotic, and markedly reduced ability to form persistent bacteria. The deletion of yefM-yoeB enhanced the bacterial ability of high temperature tolerance, biofilm formation, and host serum resistance, which is the first study about the relationship between type II TA system and serum resistance. In vitro infection experiment showed that the inactivation of yefM-yoeB greatly enhanced bacterial capability of adhesion in host cells. Consistently, in vivo experiment suggested that the yefM-yoeB mutation had an obvious positive effect on bacteria dissemination of fish tissues and general virulence. Introduction of a trans-expressed yefM-yoeB restored the virulence of TX01ΔyefM-yoeB. These findings suggest that YefM-YoeB is involved in responding adverse circumstance and pathogenicity of E. piscicida. In addition, we found that YefM-YoeB negatively autoregulated the expression of yefM-yoeB and YefM could directly bind with own promoter. This study provides first insights into the biological activity of type II TA system YefM-YoeB in aquatic pathogenic bacteria and contributes to understand the pathogenesis of E. piscicida.

scholarly journals TatD DNases Contribute to Biofilm Formation and Virulence in Trueperella pyogenes

2021 ◽  
Vol 12 ◽  
Author(s):  
Zehui Zhang ◽  
Yinfeng Liang ◽  
Lihui Yu ◽  
Menghan Chen ◽  
Yuru Guo ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Wild Type Strain ◽  
Divalent Cations ◽  
Bacterial Load ◽  
Economic Losses ◽  
Wild Type ◽  
Trueperella Pyogenes ◽  
Mutant Strains ◽  
Sequence Characteristics

TatD DNases are conserved proteins in a variety of organisms and are considered potential virulence factors in Plasmodium falciparum and Streptococcus pneumoniae. However, the function of TatD DNases has not yet been determined in Trueperella pyogenes, which causes various infections in animals and leads to economic losses. In this study, we describe the roles of TatD DNases in T. pyogenes (TpTatDs). A bioinformatics analysis was performed to investigate the sequence characteristics of TpTatDs, and then the ability of recombinant TatD proteins to hydrolyze DNA was determined in the presence of divalent cations. Moreover, we constructed tatD-deficient mutants. The biofilms formed by the wild-type and mutant strains were observed under a microscope. The mortality and bacterial load in the spleen of mice infected with the wild-type strain and tatD-deficient mutants were determined to obtain insights into the role of TatDs in the virulence of T. pyogenes. Two TatD DNases were identified in T. pyogenes. They were Mg2+-dependent DNases and exhibited DNA endonuclease activity. Compared with those formed by the parental strain, biofilms formed by mutants showed a significantly reduced thickness and biomass. Moreover, mutants produced a lower bacterial load in the spleen of mice and compromised virulence. Our data indicated that TatD DNases in T. pyogenes are involved in biofilm formation and required for virulence during infections.

scholarly journals Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression

2021 ◽  
Author(s):  
Wei Wei ◽  
Lindsey Price Burbank ◽  
Teresa Sawyer
Keyword(s):  
Gene Expression ◽  
Nucleic Acid ◽  
Biofilm Formation ◽  
Cold Shock ◽  
Xylella Fastidiosa ◽  
Cold Shock Protein ◽  
Nucleic Acid Binding ◽  
Wild Type ◽  
Surface Attachment

Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant (Δcsp1). We observed decreases in cellular aggregation and surface attachment with the Δcsp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that Δcsp1 had reduced pili compared to the wild type and complemented strains. The Δcsp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and Δcsp1 revealed changes in expression of several genes important for attachment and biofilm formation in Δcsp1. One gene of intertest,pilA1, encodes a type IV pili subunit protein and was up regulated in Δcsp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines.X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation.

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