A Chemotaxis-Like Pathway of Azorhizobium caulinodans Controls Flagella-Driven Motility, Which Regulates Biofilm Formation, Exopolysaccharide Biosynthesis, and Competitive Nodulation

The genome of the Azorhizobium caulinodans ORS571 contains a unique chemotaxis gene cluster (che) including five chemotaxis genes: cheA, cheW, cheY1, cheB, and cheR. Analysis of the role of the chemotaxis cluster of A. caulinodans using deletion mutant strains revealed that CheA or the Che signaling pathway controls chemotaxis behavior and flagella-driven motility and plays important roles in formation of biofilms and production of extracellular polysaccharides (EPS). Furthermore, the deletion mutants (ΔcheA and ΔcheA-R) were defective in competitive adsorption and colonization on the root surface of host plants. In addition, a functional CheA or Che pathway promoted competitive nodulation on roots and stems. Interestingly, a nonflagellated mutant, ΔfliM, displayed a phenotype highly similar to that of the ΔcheA or ΔcheA-R mutant strains. These findings suggest that through controlling flagella-driven motility behavior, the chemotaxis signaling pathway in A. caulinodans coordinates biofilm formation, EPS, and competitive colonization and nodulation.

Download Full-text

RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri

Antibiotics ◽

10.3390/antibiotics10070823 ◽

2021 ◽

Vol 10 (7) ◽

pp. 823

Author(s):

Cristiana Mateus ◽

Ana Rita Nunes ◽

Mónica Oleastro ◽

Fernanda Domingues ◽

Susana Ferreira

Keyword(s):

Oxidative Stress ◽

Human Serum ◽

Ethidium Bromide ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Efflux Pumps ◽

Bacterial Virulence ◽

Relative Fitness ◽

Mutant Strains

Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence.

Download Full-text

The Role of Exopolysaccharides in Oral Biofilms

Journal of Dental Research ◽

10.1177/0022034519845001 ◽

2019 ◽

Vol 98 (7) ◽

pp. 739-745 ◽

Cited By ~ 7

Author(s):

C. Cugini ◽

M. Shanmugam ◽

N. Landge ◽

N. Ramasubbu

Keyword(s):

Biofilm Formation ◽

Bacterial Colonization ◽

Super Resolution ◽

Extracellular Proteins ◽

Extracellular Polysaccharides ◽

Oral Biofilms ◽

The Matrix ◽

Oral Microbes ◽

Biofilm Development

The oral cavity contains a rich consortium of exopolysaccharide-producing microbes. These extracellular polysaccharides comprise a major component of the oral biofilm. Together with extracellular proteins, DNA, and lipids, they form the biofilm matrix, which contributes to bacterial colonization, biofilm formation and maintenance, and pathogenesis. While a number of oral microbes have been studied in detail with regard to biofilm formation and pathogenesis, the exopolysaccharides have been well characterized for only select organisms, namely Streptococcus mutans and Aggregatibacter actinomycetemcomitans. Studies on the exopolysaccharides of other oral organisms, however, are in their infancy. In this review, we present the current research on exopolysaccharides of oral microbes regarding their biosynthesis, regulation, contributions to biofilm formation and stability of the matrix, and immune evasion. In addition, insight into the role of exopolysaccharides in biofilms is highlighted through the evaluation of emerging techniques such as pH probing of biofilm colonies, solid-state nuclear magnetic resonance for macromolecular interactions within biofilms, and super-resolution microscopy analysis of biofilm development. Finally, exopolysaccharide as a potential nutrient source for species within a biofilm is discussed.

Download Full-text

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

Microorganisms ◽

10.3390/microorganisms8010070 ◽

2020 ◽

Vol 8 (1) ◽

pp. 70 ◽

Cited By ~ 1

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.

Download Full-text

LuxR-Type Regulator AclR1 of Azorhizobium caulinodans Regulates Cyclic di-GMP and Numerous Phenotypes in Free-Living and Symbiotic States

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-19-0306-r ◽

2020 ◽

Vol 33 (3) ◽

pp. 528-538

Author(s):

Wei Liu ◽

Yan Li ◽

Xue Bai ◽

Haiguang Wu ◽

Lanxing Bian ◽

...

Keyword(s):

Biofilm Formation ◽

Host Plants ◽

Biological Processes ◽

Azorhizobium Caulinodans ◽

Free Living ◽

A Genome ◽

Living State ◽

And Control ◽

Functional Mechanisms ◽

Azorhizobium Caulinodans Ors571

LuxR-type regulators play important roles in transcriptional regulation in bacteria and control various biological processes. A genome sequence analysis showed the existence of seven LuxR-type regulators in Azorhizobium caulinodans ORS571, an important nitrogen-fixing bacterium in both its free-living state and in symbiosis with its host, Sesbania rostrata. However, the functional mechanisms of these regulators remain unclear. In this study, we identified a LuxR-type regulator that contains a cheY-homologous receiver (REC) domain in its N terminus and designated it AclR1. Interestingly, phylogenetic analysis revealed that AclR1 exhibited relatively close evolutionary relationships with MalT/GerE/FixJ/NarL family proteins. Functional analysis of an aclR1 deletion mutant (ΔaclR1) in the free-living state showed that AclR1 positively regulated cell motility and flocculation but negatively regulated exopolysaccharide production, biofilm formation, and second messenger cyclic diguanylate (c-di-GMP)-related gene expression. In the symbiotic state, the ΔaclR1 mutant was defective in competitive colonization and nodulation on host plants. These results suggested that AclR1 could provide bacteria with the ability to compete effectively for symbiotic nodulation. Overall, our results show that the REC-LuxR-type regulator AclR1 regulates numerous phenotypes both in the free-living state and during host plant symbiosis.

Download Full-text

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

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0526 ◽

2015 ◽

Vol 61 (4) ◽

pp. 247-255 ◽

Cited By ~ 1

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.

Download Full-text

A Dual Role of Amino Acids from Sesbania rostrata Seed Exudates in the Chemotaxis Response of Azorhizobium caulinodans ORS571

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-19-0059-r ◽

2019 ◽

Vol 32 (9) ◽

pp. 1134-1147 ◽

Cited By ~ 2

Author(s):

Xiaolin Liu ◽

Zhihong Xie ◽

Yixuan Wang ◽

Yu Sun ◽

Xiaoxiao Dang ◽

...

Keyword(s):

Amino Acids ◽

Dual Role ◽

Chemotactic Response ◽

Nodule Formation ◽

Sesbania Rostrata ◽

Azorhizobium Caulinodans ◽

Regulation Of Expression ◽

Chemotaxis Proteins ◽

Azorhizobium Caulinodans Ors571

Azorhizobium caulinodans ORS571 can induce nodule formation on the roots and the stems of its host legume, Sesbania rostrata. Plant exudates are essential in the dialogue between microbes and their host plant and, in particular, amino acids can play an important role in the chemotactic response of bacteria. Histidine, arginine, and aspartate, which are the three most abundant amino acids present in S. rostrata seed exudates, behave as chemoattractants toward A. caulinodans. A position-specific-iterated BLAST analysis of the methyl-accepting chemotaxis proteins (MCPs) (chemoreceptors) in the genome of A. caulinodans was performed. Among the 43 MCP homologs, two MCPs harboring a dCache domain were selected as possible cognate amino acid MCPs. After analysis of relative gene expression levels and construction of a gene-deleted mutant strain, one of them, AZC_0821 designed as TlpH, was confirmed to be responsible for the chemotactic response to the three amino acids. In addition, it was found that these three amino acids can also influence chemotaxis of A. caulinodans independently of the chemosensory receptors, by being involved in the increase of the expression level of several che and fla genes involved in the chemotaxis pathway and flagella synthesis. Thus, the contribution of amino acids present in seed exudates is directly related to the role as chemoattractants and indirectly related to the role in the regulation of expression of key genes involved in chemotaxis and motility. This “dual role” is likely to influence the formation of biofilms by A. caulinodans and the host root colonization properties of this bacterium.

Download Full-text

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

10.21203/rs.3.rs-204944/v1 ◽

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.

Download Full-text

Molecular Regulatory Network Involved in Biofilm Structure Development by Acidithiobacillus thiooxidans Includes Pel Exopolysaccharide Machinery

Solid State Phenomena ◽

10.4028/www.scientific.net/ssp.262.330 ◽

2017 ◽

Vol 262 ◽

pp. 330-333 ◽

Cited By ~ 3

Author(s):

Mauricio Diaz ◽

Nicolas Guiliani

Keyword(s):

Gene Cluster ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Industrial Process ◽

Genomic Analysis ◽

Mutant Strains ◽

Relevant Role ◽

Biofilm Structure

The Acidithiobacillus genus plays a relevant role in bioleaching. The molecular understanding of biofilm formation has been pointed out to design biological strategies to improve the efficiency of this industrial process and to prevent environmental damages caused by acid mine/rock drainages. In Acidithiobacillus spp., the molecular mechanisms involved in biofilm formation are currently emerging. The second messenger cyclic diguanylate (c-di-GMP) appears as a key player for biofilm formation by Acidithiobacillus sp. Here, results obtained from genomic analysis to characterize c-di-GMP pathway in At. thiooxidans are reported. Intracellular levels of c-di-GMP have been previously measured and data indicated that they are higher in adhered cells than planktonic ones. During the course of characterization of c-di-GMP effectors, a complete pel-like gene cluster has been identified in At. thiooxidans. By using total RNA obtained from planktonic and adhered sulfur-grown cells, transcriptomic analysis revealed that pelA belonging to the pel-like gene cluster is overexpressed in adhered cells. Moreover, genetic experiments were performed to compare wild type and null-mutant strains of At. thiooxidans for assessing the role of Pel exopolysaccharide. All together, the results obtained suggest a specific role for Pel machinery in the attachment to solid energy substrates by At. thiooxidans.

Download Full-text

TatD DNases Contribute to Biofilm Formation and Virulence in Trueperella pyogenes

Frontiers in Microbiology ◽

10.3389/fmicb.2021.758465 ◽

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.

Download Full-text

Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

Microorganisms ◽

10.3390/microorganisms8121975 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1975

Author(s):

Muzaffar Hussain ◽

Christian Kohler ◽

Karsten Becker

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Biofilm Formation ◽

Surface Proteins ◽

Transcription Level ◽

Coagulase Negative Staphylococci ◽

Staphylococcus Lugdunensis ◽

Cell Surface Proteins ◽

Mutant Strains

Among coagulase-negative staphylococci (CoNS), Staphylococcus lugdunensis has a special position as causative agent of aggressive courses of infectious endocarditis (IE) more reminiscent of IEs caused by Staphylococcus aureus than those by CoNS. To initiate colonization and invasion, bacterial cell surface proteins are required; however, only little is known about adhesion of S. lugdunensis to biotic surfaces. Cell surface proteins containing the LPXTG anchor motif are covalently attached to the cell wall by sortases. Here, we report the functionality of Staphylococcus lugdunensis sortase A (SrtA) to link LPXTG substrates to the cell wall. To determine the role of SrtA dependent surface proteins in biofilm formation and binding eukaryotic cells, we generated SrtA-deficient mutants (ΔsrtA). These mutants formed a smaller amount of biofilm and bound less to immobilized fibronectin, fibrinogen, and vitronectin. Furthermore, SrtA absence affected the gene expression of two different adhesins on transcription level. Surprisingly, we found no decreased adherence and invasion in human cell lines, probably caused by the upregulation of further adhesins in ΔsrtA mutant strains. In conclusion, the functionality of S. lugdunensis SrtA in anchoring LPXTG substrates to the cell wall let us define it as the pathogen’s housekeeping sortase.

Download Full-text