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.

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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.

DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development, as well as disrupted preformed biofilm. By comparison, no significant effect of the DNAse was detected for biofilm formation by Xc. DNAse effects on biofilm formation or disruption varied among Xcc strains and Xanthomonas species which suggest different roles for eDNA. Variation in the structure of fibers containing eDNA in biofilms, bacterial cultures, and in twitching motility was also visualized by microscopy.

The proposed roles for eDNA are as an adhesin in the early stages of biofilm formation, as an structural component of mature bacterial aggregates, and twitching motility structures. Introduction Biofilms adhere bacteria to surfaces within a tridimensional structure that protects the bacterium against antibiotics and abiotic stresses [ 1 — 4 ].

The extracellular matrix provides resistance to temperature, pH and other deleterious conditions and also confers mechanical stability. Biofilms are composed of water, proteins, exopolysaccharide, lipopolysaccharide, lipids, surfactants and extracellular DNA eDNA [ 5 , 6 ].

The presence of eDNA in the extracellular matrix was described in [ 14 ] and its importance in biofilm formation was first demonstrated for Pseudomonas aeruginosa in the early stages of bacterial adhesion [ 10 ]. Meanwhile, in clinical isolates of the same species, eDNA was found to be important for stability of the biofilm [ 11 ].

Besides biofilm formation, eDNA contributes to bacterial resistance to antibiotics, e. DNAse in P. At high concentrations, eDNA produces antimicrobial activity by chelating cations and destabilizing the bacterial outer membrane through lipopolysaccharide modification [ 19 ]. These findings identify eDNA as an important component of the extracellular bacterial matrix that plays a role in several processes related to bacterial colonization and virulence.

Xanthomonas citri subsp. Xcc produces necrotic lesions on leaves, twigs and fruits that reduces fruit quality and marketability and restricts commercialization of plants and fruits in markets free of CBC [ 20 ].

Xcc A strain type is by far the most severe and widespread CBC pathogen and affects the widest range of Citrus species. Black rot is a systemic vascular disease that causes symptoms including marginal leaf chlorosis, necrosis, darkening of leaf veins and vascular tissue within the stem [ 28 , 29 ]. Xcc and Xc produce biofilm to facilitate the infection process [ 3 , 30 , 31 ].

Several mechanisms in Xcc contribute to biofilm formation during the host-pathogen interaction: adhesins, the type III Secretion System, lipopolysaccharide, exopolysaccharide, type IV pili associated to twitching motility and chemotaxis [ 31 — 41 ]. Furthermore, biofilm formation in planta is related to the different host range of Xcc strains [ 42 ]. The roles that eDNA play in attachment and biofilm formation are not widely characterized for plant-bacterial interactions.

Indeed, presence of eDNA in the extracellular matrix and in the different phases of biofilm formation have not been studied for Xanthomonas spp. Herein, we report on the contribution of eDNA to biofilm formation and the related twitching motility for several Xcc strains with different host range and ability to form biofilms, and compare these features with those of Xc, a systemic pathogen of cabbage, and Xac, a citrus pathogen able to infect and produce biofilm on citrus leaves [ 20 , 22 , 42 ].

In these studies, presence of eDNA was corroborated after DNase treatments and visualized by fluorescence staining of bacterial cultures, biofilms and bacteria undergoing twitching motility associated with bacterial aggregation.

Furthermore, eDNA was observed in bacterial cultures at the exponential growth phase Fig 3 , after plate growth S1 Fig and in twitching motility assays Fig 4 , right side. Microscopy observations of GFP tagged cells stained with propidium iodide S1 Fig indicated that fiber fluorescence is due to the presence of eDNA and not caused by bacterial cell aggregation.

In all assays bacterial cells in a bacillary shape were distinguishable from the containing DNA fibers. These results confirm that most of the extracellular fibers have a high eDNA content.



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.



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.



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