All relevant data are within the paper and its Supporting Information files. Abstract Xanthomonas citri subsp. Xanthomonas spp. Herein, the role of extracellular DNA eDNA was evaluated in the formation and stabilization of the biofilm matrix at different stages of biofilm development. Fluorescence and light microscopy, as well as DNAse treatments, were used to determine the presence of eDNA in biofilms and bacterial cultures.
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Published online Sep 5. E-mail: ei. Edited by Steven E. Abstract The rpf gene cluster of Xanthomonas campestris pathovar campestris Xcc is required for the pathogenesis of this bacterium to plants. Several rpf genes are involved in the coordinate positive regulation of the production of virulence factors mediated by the small diffusible molecule DSF for diffusible signal factor.
In L medium, rpfF, rpfG, rpfC, and rpfGHC mutants grew as matrix-enclosed aggregates, whereas the wild type grew in a dispersed planktonic fashion. Synthesis of the extracellular polysaccharide xanthan was required for aggregate formation. Addition of DSF triggered dispersion of the aggregates formed by the rpfF strain, but not those of rpf strains defective in DSF signal transduction.
This enzyme had no detectable activity against soluble xanthan. It is evident that many bacterial species live predominantly in biofilms in both natural and artificial environments 1 — 4. Biofilms are dynamic structures in which transitions between the planktonic and biofilm modes of growth occur presumably in response to different environmental signals.
Although the amount of data on the regulation of initiation and maturation of biofilms is increasing, very little is known about the mechanisms of biofilm dispersal or the signals that trigger it 2. Despite the recognition of the ubiquity of biofilms, only relatively few species of bacteria have been studied in any detail.
Although most attention has been focused on animal and human pathogens, the ability of plant pathogenic bacteria to form and detach from biofilms may equally have considerable implications for the completion of their disease cycle. Xanthomonas campestris pathovar campestris Xcc is the causal agent of black rot disease of cruciferous plants. The ability of Xcc to incite disease depends on several factors, including the synthesis of extracellular plant cell wall-degrading enzymes and the extracellular polysaccharide EPS xanthan 5 , 6.
The rpf genes act to positively regulate the synthesis of extracellular enzymes, EPS, and pathogenicity 5 — 8. RpfH and the products of other genes within the rpf gene cluster rpfADEI have no apparent involvement in the DSF regulatory system and have minor regulatory roles.
The work in this paper was prompted by the observation that in certain liquid media, strains with mutations in rpfF, rpfG, and rpfC grow as large multicellular aggregates rather than the dispersed planktonic form seen with the wild-type or other rpf mutants.
Examination of the aggregates by scanning electron microscopy revealed that bacteria are held together in a matrix of extracellular material. With this model system, we have identified an enzyme that allows bacteria to escape from an aggregated state in response to the DSF signal and have established a role for this enzyme in the virulence of Xcc to plants.
Bacterial strains and plasmids and culture conditions and NYGB medium have been described 7 — 9. Degradation of xanthan was estimated both viscometrically and by measurement of the release of reducing sugars. The ethyl acetate extracts were evaporated to dryness and resuspended in H2O before use. Scanning Electron Microscopy. A drop of bacterial suspension was placed on the surface of a piece of moistened cellulose acetate membrane, which had been stuck to the aluminum scanning electron microscope stub by using OCT compound BDH.
Excess liquid was wicked away by using a piece of filter paper but without causing drying. The sample was viewed at 3 kV, and digital images were saved. Extracellular Enzymes and Aggregate Dispersal. The concentration factor was fold. The percentage of the bacteria in the culture that were present in aggregates was estimated as follows.
After 5 min without shaking, when the aggregated bacteria had settled, a 1-ml sample was removed from the top of the culture and the number of planktonic bacteria was estimated by measurement of OD at nm.
To estimate the total bacteria in the culture, a preparation of the dispersing enzyme was added. The enzyme activity capable of dispersing aggregates was partially purified from culture supernatants of the engXCA strain This choice was to allow easier analysis of less abundant proteins. The eluate which contained all of the activity was dialyzed overnight against distilled water, and 1 M sodium acetate buffer, pH 5, was added to give a final concentration of 20 mM.
The column was equilibrated with 20 mM sodium acetate buffer, pH 5, and all the activity bound to the column at this pH. One-milliliter fractions were collected and assayed for the ability to disperse aggregates. Peptide Mass Fingerprinting of Proteins. Bands were cut from the SDS-polyacrylamide gel stained with Coomassie blue, and the proteins were reduced, derivatized with iodoacetamide, and subjected to enzymatic hydrolysis with trypsin.
The sizes of the peptide fragments generated were determined by mass spectrometry on a Bruker Reflex III matrix-assisted laser desorption ionization—time-of-flight mass spectrometer Bruker Daltonics, Coventry, U.
The data were used to interrogate the National Center for Biotechnology Information nonredundant protein databases by using the mascot program available at www. For band 1, the only significant hit probability-based mowse score of 94 was a pectate lyase from Xcc GenBank accession no.
The identity of the cloned fragment was confirmed by sequencing. The fragment was excised from this construct by using EcoRI, which cuts at sites flanking the insert. Virulence Testing. The virulence of Xcc to Chinese radish Raphanus sativus variety niger was estimated after bacteria were introduced into the leaves by infiltration, leaf clipping, or spraying.
For leaf clipping the last completely expanded leaf was cut with scissors dipped in bacterial suspensions of OD 0. Lesion length was measured 14 days after inoculation on 15 leaves for each strain tested. For spraying, bacterial suspensions of OD 0.
Lesions appeared at the leaf margin, and the number of plants with lesions was measured 14 days after inoculation. For infiltration inoculation bacterial suspensions of OD of 0. Strains of Xcc carrying mutations in genes within the rpf gene cluster grow in a dispersed fashion in NYGB medium with growth characteristics similar to the wild type.
However in L medium, rpfF, rpfG, and rpfC mutants and the triple deletion mutant rpfGHC grew in an aggregated state, forming readily observable clumps Fig. The wild-type and other rpf mutants rpfA, rpfB, rpfE, rpfH, and rpfI did not form such aggregates in L medium and grew in a dispersed fashion.
Examination by scanning electron microscopy indicated that bacteria within aggregates were held together in a matrix of extracellular material to form a three-dimensional reticulated structure Fig. In contrast, bacteria growing in a dispersed fashion showed no aggregation by light microscopy and were not embedded in an extracellular matrix Fig. Supplementation of L medium with DSF before bacterial inoculation allowed for dispersed growth of the rpfF mutant Fig.
Addition of DSF to h cultures of the rpfF mutant in L medium triggered the dispersal of the aggregates, so that within3hofDSF addition, almost all the bacteria within the culture were in the planktonic state.
BIOFILM FORMATION AND DISPERSAL IN XANTHOMONAS CAMPESTRIS PDF
Nishicage Multiple studies have reported that QS-deficient mutants formed thinner and more disorganized biofilms compared to the wild-types Tomlin et al. Kaplan Journal of dental research Mar Ecol Prog Ser Appl Microbiol Biotechnol Given that there are limited effective antibacterial compounds for controlling Xoo, search to develop cost effective and novel strategies that have minimal environmental impact is the need of hour. Source for Bio-fertilizers, Bio-medicines and Agent Research. Several other virulence related phenotypes were assayed by assessing the extracellular enzyme production of XooAS29 both in the presence and absence of THY oil. One such new wrinkle is the use of anti-virulence or anti-quorum sensing QS agents that do not kill the bacteria but restrains the production of disease triggering virulence factors Tay and Yew, The molecular mechanism of QS inhibition by Gram-positive bacteria and eukaryotic e. Thus, inn observed for quorum sensing been reported that inhibition of the agr system increases inhibitors, it appears that promising targets for formatoon biofilm formation Vuong et al. Click here to sign up.
BIOFILM FORMATION DISPERSAL XANTHOMONAS CAMPESTRIS PDF
Kazisho The contribution of each of these proteins to virulence, extracellular enzyme synthesis, and biofilm formation has been investigated using a panel of mutants Ryan et al. Dispersao global regulators in transcriptional regulatory networks in bacteria. Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily. 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 formatlon Xcc wild-type strains, suggesting that Xcc wild-type strains could produce a diffusible signal factor DSF Barber 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. The solid arrow indicates the demonstrated or predicted directed protein—protein interaction or directed signal modulation. Biological role of xanthomonadin pigments fomation Xanthomonas campestris pv.