ABSTRACT
Iron is an essential element for living organisms that plays critical roles in the process of bacterial growth and metabolism. However, it remains to be elucidated whether piuB encoding iron-uptake factor is involved in iron uptake and pathogenicity of Xanthomonas axonopodis pv. glycines (Xag). To investigate the function of piuB, we firstly generated a piuB deletion mutant (ΔpiuB) by homologous recombination. Compared with the wild-type, the piuB mutant exhibited significantly reduced growth and virulence in host soybean. The mutant displayed markedly increased siderophore secretory volume, and its sensitivity to Fe3+, Cu2+, Zn2+ and Mn2+ was significantly enhanced. Additionally, the H2O2 resistance, exopolysaccharide yield, biofilm formation, and cell mobility of ΔpiuB were significantly diminished compared to that of the wild-type. The addition of exogenous Fe3+ cannot effectively restore the above characteristics of ΔpiuB. However, expressing piuB in trans rescued the properties lost by ΔpiuB to the levels in the wild-type. Taken together, our results demonstrated that PiuB is a potential factor for Xag to assimilate Fe3+, and is necessary for Xag to be pathogenic in host soybean.
Subject(s)
Iron , Glycine max , Virulence , Xanthomonas axonopodis/genetics , Hydrogen PeroxideABSTRACT
Guggal is tapped for extraction of medicinally important oleo–gum–resin (guggul) by inoculating the stem bark with natural gum suspension containing pathogenic bacterium Xanthomonas axonopodis pv. commiphorae (Xac). The tree dies in the process. In absence of any specific medium for isolation of Xac, it is difficult to assess spread of the pathogen within the plant. A PCR based molecular detection technique using fyuA and rpoD gene specific primers is described here. The primers amplified products only from Xac and not from host tissues or common saprophytes. The method was sensitive enough to produce positive signals for up to 4.4 bacterial cells or 2 pg target DNA per reaction mixture. However, PCR inhibitors present in plant tissues drastically reduced the limit of detection. A simple overnight incubation of surface sterilised plant tissues in nutrient medium was introduced to increase pathogen titre and to overcome this problem. This technique was successfully used to measure spread of Xac in plant tissues away from the site of inoculation. The pathogen showed preference for acropetal movement and did not spread to 7–8 cm below the site of inoculation till 15 days after inoculation. This suggests possibility to manage the disease through plant surgery.
Subject(s)
DNA Primers/chemistry , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/microbiology , Limit of Detection , Plant Diseases/genetics , Plant Diseases/microbiology , Polymerase Chain Reaction , Resins, Plant/chemistry , Resins, Plant/metabolism , Triterpenes/chemistry , Triterpenes/metabolism , Xanthomonas axonopodis/genetics , Xanthomonas axonopodis/pathogenicityABSTRACT
Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of 14 strains of Xanthomonas arboricola pv. pruni and seven strains of X. axonopodis pv. phaseoli, which are used in xanthan production studies. Relationships identified by the AFLP profiles were assessed for xanthan production capacity, geographical location and host plant. Strains were isolated from 10 different geographic regions in South and Southeast States in Brazil. Data were analyzed for genetic similarity using the Dice coefficient and subjected to UPGMA cluster analysis. A total of 128 AFLP fragments were generated from four primer combinations: EcoRI+C/MseI+0, EcoRI+A/MseI+0, EcoRI+G/MseI+T and EcoRI+G/MseI+A. Of these, 96.1 percent were polymorphic. X. axonopodis pv. phaseoli (S D = 0.27) was shown to be more polymorphic than X. arboricola pv. pruni (S D = 0.58). All 14 pathovar pruni strains were included in a single main group (S D = 0.58), while the pathovar phaseoli strains were divided into three separate groups, with one group containing five strains (S D = 0.38) and two isolated groups (S D = 0.31 and 0.27) composed of only one strain each. Species were distinguished by three and eight specific AFLP markers present in the pathovar phaseoli and the pathovar pruni, respectively. For the unique strain without xanthan production capacity (X. axonopodis pv. phaseoli str. 48), nine specific AFLP bands were found. There was no evidence that geographic area or host plant influenced genetic heterogeneity. Correlations between AFLP patterns and xanthan production capacity were found in some strains, but were not consistent enough to establish a relationship.
Subject(s)
Amplified Fragment Length Polymorphism Analysis , DNA Fingerprinting , Genetic Variation , Xanthomonas axonopodis/genetics , Xanthomonas axonopodis/isolation & purification , Xanthomonas/genetics , Xanthomonas/isolation & purification , Methods , Methods , VirulenceABSTRACT
The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.