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Biofilm dispersal in Xanthomonas campestris is controlled by cell–cell signaling role for the DSF/rpf regulatory system in biofilm formation and/or dispersal. Biofilm formation and dispersal in the black rot pathogen Xanthomonas campestris pathovar campestris (Xcc) is influenced by a number of. Among them, Xanthomonas campestris is the most dominant species with at least .. Identification of other genes encoding for biofilm formation/dispersal and.

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It is now clear that cell—cell communication, often formaton to as quorum sensing QSis the norm in the prokaryotic kingdom and this community-wide genetic regulatory mechanism has been adopted for regulation of many important biological functions.

Since the s, several types of QS signals have been identified, which are associated commonly with different types of QS mechanisms. Among them, the diffusible signal factor DSF -dependent QS system, originally discovered from bacterial pathogen Xanthomonas campestris pv. The rapid research progress over the last few years has identified the chemical structure of xanthomons QS signal DSF, established the DSF regulon, and unveiled the general signaling pathways and mechanisms. Particular noteworthy are that DSF biosynthesis is modulated by a novel posttranslational autoinduction mechanism involving protein—protein interaction between the DSF synthase RpfF dampestris the sensor RpfC, and that QS signal sensing is coupled to intracellular regulatory networks through a second messenger cyclic-di-GMP and a global regulator Clp.

Genomic and genetic xanthompnas show that the DSF QS-signaling pathway regulates xanthomomas biological blofilm including virulence, biofilm dispersal, and ecological competence. Moreover, evidence is emerging that the DSF QS system is conserved in a range of plant and human bacterial pathogens. The Xanthomonas genus is one of the most ubiquitous groups of plant-associated bacterial pathogens.

Members of this genus have been shown to infect at least monocotyledonous and dicotyledonous plant species Leyns et al.

Among them, Xanthomonas campestris is the most dominant species with at least pathovars, which infect a wide range of plants including many of agricultural interest, e. Xcc is a small, rod-shaped, aerobic gram-negative, nonspore-forming bacterium Onsando, The bacterium has a single polar flagellum, is positive in catalase activity and hydrogen sulfide reaction, oxidase negative, and does not produce nitrate or indole Onsando, According to the Commonwealth Mycological Institute CMI distribution map, the disease has been recorded in bjofilm 90 countries representing all the five continents Anon, The most important hosts of Xcc are the members of Brassica oleraceaincluding cabbage, cauliflower, broccoli, Brussel sprouts, and kale.

Another study shows that Xcc normally gains entry into plants via hydathodes Hugouvieux et al. Once inside the plant, Xcc colonizes the vascular system where it produces an extracellular polysaccharide EPS called xanthan, which can obstruct the xylem vessels, causing tissue necrosis and severe leaf wilting Williams, ; Onsando, Evidence is accumulating that xanthan may also play other roles in infection.

Treatment of plants with xanthan suppresses the accumulation of callose and enhances plant susceptibility to Xcc Yun et al. Xanthan is also associated with the formation of biofilms, which was proposed to play a protective role for the bacterial cells inside the biofilm structure against plant defense responses Torres et al. Furthermore, Xcc produces a yellow pigment xanthomonadin, which appears to contribute to the bacterial epiphytic survival Poplawsky et al.

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In addition to its roles in Xcc virulence, xanthan has had valuable industry and biotechnology applications. It was known long time ago that Xcc is capable of producing substantial quantities of xanthan during the fermentation of carbohydrate substrates Lilly et al. Xanthan has distinctive rheological properties in camlestris solutions Morris et al. Owing to its unique properties, xanthan has utility in a wide range of applications. Because of its properties in thickening aqueous solutions, as a dispersing agent, and stabilizer of emulsions and suspensions, xanthan is used in pharmaceutical formulations, cosmetics, and agricultural products.

It is used in textile printing pastes, glazes, slurry explosive formulations, and rust removers. The widespread utility of xanthan has made it an important industrial biopolymer and its global for,ation continues to increase each year Sutherland, Research progress in the last few years shows that Xcc has evolved a unique quorum-sensing QS system, which plays a key role in the regulation of xanthan EPS biosynthesis and bacterial virulence.

The QS system of Xcc differs from other known QS systems in various aspects, including the signal chemistry, autoregulation of signal production, and ways to couple to the bacterial central regulatory networks. This review will focus on these fascinating features, as well as the latest findings on QS regulon and signaling networks.

The potential implications of these findings on other bacterial pathogens will also be discussed. QS is one of the sophisticated mechanisms of cell—cell communications, by which bacteria count their own number by producing, detecting, and responding to the accumulation of signaling molecules that they export into their environment for reviews, forkation Whitehead et al.

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Booming research on QS since the s has led to identification of several types of bacterial QS signals Fig. The chemical structure of the first AHL signal from marine bacterium Vibrio fischeri was unveiled more than two decades ago Eberhard et al.

Biofilm formation and dispersal in Xanthomonas campestris.

Up till now, AHL-type QS signals, which share a conserved homoserine lactone ring linked to a fatty acid side chain with variations in chain length and substitutions Fig. At present, two chemically similar AI-2 signals have been identified in Vibrio harveyi and Salmonella typhimuriumxanthoomonas. Chemical structures of representative microbial QS signals. The information was summarized from the following references: Similar to many other bacterial pathogens, Xcc has also evolved QS systems for genetic regulation at the community level.

An early transposon mutagenesis analysis unveiled that a cluster of Xcc genes, designated rpfABCDEFG for r egulation of p athogenicity f actors formatlon, are involved in the production of Dislersal and extracellular enzymes Tang et al. Subsequently, it was found that the protease production in the mutant of the rpfF gene, which encodes a putative enoyl CoA hydratase, was restored by cocultivation in proximity to Xcc wild-type strains, suggesting that Xcc wild-type strains could produce a diffusible signal factor DSF Barber et disperrsal.

Comparison of the biological activities of a range of DSF derivatives has led to identification of a few key structural features that determine the signaling activity.

Biofilm formation and dispersal in Xanthomonas campestris.

Interestingly, two groups of Gram-negative bacterial QS signals, i. The chemical structures of another two groups of QS signals produced by Gram-negative bacteria, i. Similarly, application of the DF extracts on cauliflower plants restores the virulence of the DF-deficient mutant to a level comparable to its wild-type Xcc parental strain Chun et al. A subsequent study has shown that pigB and DF are needed for epiphytic survival because xanthoonas xanthomonadin production formatlon critical for protection of the bacterial pathogen against UV light Poplawsky et al.

MS analysis of DF predicted a butyrolactone structure Chun et al. Further analysis of the pigB DNA sequence reveals the xanthoomnas of two genes and only one of them Xccwhich encodes a pteridine-dependent deoxygenase-like protein, is needed for production of the DF signal Poplawsky et al. DF and DSF are clearly distinct signals because they differ in biological activity, chromatographic mobility, chemical xanrhomonas, and the genes encoding biosynthesis Fromation et al.

Utilization of two QS signal molecules to regulate different sets of virulence genes may provide Xcc the plasticity in response to different environments. Further characterization of the chemical structure of DF, and its signaling mechanisms in regulation of xanthomonadin and EPS production will be essential for a clear understanding of the roles of the two sophisticated QS systems DSF and DF in modulation of Xcc physiology and virulence.

Null mutation of rpfF in both strains leads to reduced production of EPS and extracellular enzymes, including proteases and cellulases Tang et al. DSF activity could be extracted with ethyl acetate from supernatants of overnight cultures of Xccand addition of the extracted DSF could restore extracellular enzyme production of the DSF-deficient mutants Barber et al. Another important DSF-dependent biological function in Xcc is biofilm formation. Mutation of the rpfF gene in both Xcc strains and XC1 causes the formation of bacterial cell aggregates, but whose size varies depending on the strains Dow et al.

Addition of DSF to the bacterial cultures stops formation of cell aggregates by the rpfF deletion mutants, suggesting a critical role of DSF signal in modulation of Xcc switching between planktonic and biofilm growth forms. The availability of the Xcc genome sequence and advancement of microarray technology have greatly facilitated the determination of the scope of genes and biological functions regulated by the DSF signal.

Genome annotation of Xcc strains ATCC and leads to prediction of and genes, respectively da Silva et al. Measurement of gene expression levels has been performed using an oligonucleotide microarray based on the genome sequence of Xcc strain ATCC He et al.

Interestingly, the DSF-dependent genes are in general randomly distributed throughout the bacterial genome He et al. The pattern is similar to the P. Some of these newly identified DSF-mediated phenotypes, including resistance to toxin acriflavin and hydrogen peroxide and bacterial survival at different temperatures, have been verified by genetic and phenotype analysis He et al. Taken together, the available genetic and genomic data suggest that the DSF-signaling system is not only essential for coordinating the expression of virulence genes at the community level but also appears to be of critical importance for maintenance of Xcc ecological competence.

Intriguingly, in contrast to the positive-regulation pattern of the DSF signal on most virulence genes, microarray analysis reveals that in YEB medium mutation of rpfF encoding DSF biosynthesis significantly increases the expression of the genes encoding the type III secretion system T3SS, also known as Hrp He et al.

However, the other report shows that when grown in MME medium, mutation of rpfG encoding DSF signal transduction has no effect on the expression of hrpX and hrpGwhich encode the master regulators of the hrp operon Ryan et al. Given that T3SS is one set of key bacterial virulence determinants in many bacterial pathogens Hueck, ; Ghosh,thorough investigation of the regulatory mechanisms of T3SS by QS and other signaling mechanisms would be of key importance for designing and developing effective new treatments against bacterial virulence and infections.

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How is the DSF signal detected, transduced, and connected to the intracellular regulatory networks? This intriguing puzzle has been resolved recently by the collective works of several laboratories.

Although many details remain to be added on, the general outline of the DSF-signaling network has been established, which could be a useful platform for further in-depth investigations. In the Xcc genome, rpfGrpfHand rpfC are transcribed as the same operon although rpfC has its own weak promoter, suggesting that the three genes could be functionally related Slater et al.

RpfG protein contains two major domains: Schematic representation of a model of the DSF signal autoinduction. The dashed arrow indicates basal signal generation or no signal flow.

At a high cell density, accumulated extracellular DSF signal interacts with RpfC and induces a conformational change in the sensor, which undergoes autophosphorylation and facilitates release of RpfF and phosphorelay from the sensor to its response regulator RpfG. The solid arrow indicates signal flow or signal generation. The question mark indicates unknown precursor s. This speculation was later confirmed by site-directed mutagenesis and domain deletion analysis.

Peptide alignment analysis with other well-characterized two-component sensors and regulators reveals four conserved residues, i. Substitution of these residues with unrelated amino acids or deletion of any one of the domains containing these conserved residues results in decreased virulence factor production He, ; He et al. Different from the orthodox response regulator that typically contains a receiver domain and a DNA-binding domain for a review, see Stock et al.

In trans expression of the HD-GYP domain of RpfG alone in the null mutants of RpfG restores the production of EPS and extracellular enzymes to a level close to the wild-type strain, whereas expression of the receiver domain alone has no effect He, ; Ryan et al.

Firstly, the purified HD-GYP domain protein is able to degrade the model substrate bis p -nitrophenyl phosphate but has no activity against p -nitrophenyl phosphate, indicating that it is a phosphodiesterase but not phosphomonoesterase. Fourthly, substitution of the H and D residues of the HD-GYP domain abrogates both the enzymatic activity against cyclic-di-GMP and the regulatory activity on virulence factor production.

Data mining of the genes influenced by DSF shows that the clp gene, which encodes a transcriptional regulator with a nucleotide-binding domain and a DNA-binding domain, is likely to be a potential candidate He et al.

The clp C AP- l ike p rotein gene was named due to the observation that its product shares a high similarity to the catabolite activation factor CAP, also widely known as Crp derived from its function as a c AMP r eceptor p rotein of Escherichia coli de Crecy-Lagard et al. However, the null mutation of clp in Xcc strain NRRLB does not affect the utilization of various carbon sources but instead decreases the biosynthesis of EPS, extracellular cellulase, and polygalacturonate lyase de Crecy-Lagard et al.

In addition, the transcriptional expression of several DSF-regulated genes He et al. Moreover, microarray analysis shows that the DSF regulon and the Clp regulon are largely overlapping with similar expression patterns He et al.

Even though it is not clear how Clp detects and responds to the signal input from RpfG, the available lines of evidence have unequivocally established the important status of Clp in the DSF-signaling network Fig. Schematic representation of the QS-signaling network in Xcc. The solid arrow indicates the demonstrated or predicted directed protein—protein interaction or directed signal modulation.

The dashed arrows suggest a potential signal regulation pathway. Microarray analysis reveals that null mutation of Clp affects genes at the transcriptional level He et al.

The available data suggest that Clp modulates the transcriptional expression of this large set of camppestris by either direct activation or indirect regulation via other transcriptional regulators. Within this family, the Crp of E. Most proteins or enzymes encoded by these 86 genes belong to the following functional groups: These findings cakpestris that Clp may directly regulate the transcriptional expression of these genes Fig.

Consistent with these findings, electrophoretic mobility shift assays and point mutation analysis confirm that Clp binds to the consensus Clp-binding sites at the promoter of engXCA gene, which encodes an extracellular cellulase Hsiao et al.

The expression of both zur and fhrR is regulated by DSF and Clp and these two regulatory factors regulated xantuomonas sets of genes within the Clp regulon He et al.