Transcription of the genome of the filamentous bacteriophage cf from both plus and minus DNA strands.
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The filamentous bacteriophage cf infects the bacterium Xanthomonas campestris pv. citri. Northern blot analysis with probes derived from various restriction fragments of cf replicative form (RF) DNA has revealed the presence of five major phage-specific transcripts in infected cells. Four of these transcripts were shown to be derived from the region of the cf genome extending from gene II to gene VIII and are consistent with the cascade model of transcription proposed for Ff coliphages. These transcripts overlap with each other and terminate upstream of an efficient Rho-independent transcription terminator. Unlike the well-characterized Ff phages, in which only the minus strand of viral DNA serves as a transcription template, both strands of the RF DNA of phage cf appeared to be transcribed. Thus one of the five major cf transcripts was shown to be derived from a region of the viral minus strand that contains an open reading frame encoding a putative polypeptide of 165 amino acids. Primer extension analysis mapped the transcriptional initiation site of this RNA to a cytosine residue at position 870. A partial transcription map of phage cf revealed two independent regions of transcriptional activity. The region with the highest activity coincides with that encoding the polypeptides required in the largest amounts during the cf infection cycle. (+info)
The adsorption protein genes of Xanthomonas campestris filamentous phages determining host specificity.
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Gene III (gIII) of phiLf, a filamentous phage specifically infecting Xanthomonas campestris pv. campestris, was previously shown to encode a virion-associated protein (pIII) required for phage adsorption. In this study, the transcription start site for the gene and the N-terminal sequence of the protein were determined, resulting in the revision of the translation initiation site from the one previously predicted for this gene. For comparative study, the gIII of phiXv, a filamentous phage specifically infecting X. campestris pv. vesicatoria, was cloned and sequenced. The deduced amino acid sequences of these two pIIIs exhibit a high degree of identity in their C-terminal halves and possess the structural features typical of the adsorption proteins of filamentous phages: a signal sequence in the N terminus, a long glycine-rich region near the center, and a hydrophobic membrane anchorage domain in the C terminus. The regions between gIII and the upstream gVIII, 128 nucleotides in both phages, are larger than those of other filamentous phages. A hybrid phage of phiXv, consisting of the phiLf pIII and all the other components derived from phiXv, was able to infect X. campestris pv. campestris but not X. campestris pv. vesicatoria, indicating that gIII is the gene specifying host specificity and demonstrating the interchangeability of the pIIIs. (+info)
Identification of a novel beta-lactamase produced by Xanthomonas campestris, a phytopathogenic bacterium.
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The Xanthomonas campestris pv. campestris 11 chromosome encodes a periplasmic beta-lactamase of 30 kDa. Gene replacement and complementation confirmed the presence of this enzyme. Its deduced amino acid sequence shows identity and conserved domains between it and Stenotrophomonas maltophilia L2 and other Ambler class A/Bush group 2 beta-lactamases. Southern hybridization detected a single homologous fragment in each of 12 other Xanthomonas strains, indicating that the presence of a beta-lactamase gene is common among xanthomonads. (+info)
The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens.
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Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen Xanthomonas campestris pv. vesicatoria have been suggested to be part of a type III machinery. Here, we report the hrp-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by X. campestris pv. vesicatoria strains that constitutively express hrp genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly hrp-dependent because a mutant carrying a deletion in hrcV, a conserved hrp gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of X. campestris pv. vesicatoria was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in Ralstonia solanacearum, and AvrB, an avirulence protein from Pseudomonas syringae pv. glycinea. Interestingly, X. campestris pv. vesicatoria also secreted YopE, a type III-secreted cytotoxin of the mammalian pathogen Yersinia pseudotuberculosis in a hrp-dependent manner. YerA, a YopE-specific chaperone, was required for YopE stability but not for secretion in X. campestris pv. vesicatoria. Our results demonstrate the functional conservation of the type III system of X. campestris for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals. (+info)
An hrcU-homologous gene mutant of Xanthomonas campestris pv. glycines 8ra that lost pathogenicity on the host plant but was able to elicit the hypersensitive response on nonhosts.
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Transposon mutagenesis was used to isolate nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra, which causes bacterial pustule disease in soybean. A 6.1-kb DNA region in which a mutation gave loss of pathogenicity was isolated and found to carry six open reading frames (ORFs). Four ORFs had homology with hrcU, hrcV, hrcR, and hrcS genes of Ralstonia solanacearum and X. campestris pv. vesicatoria. One nonpathogenic mutant, X. campestris pv. glycines H80, lost pathogenicity on soybean but was able to elicit the hypersensitive response (HR) on nonhost pepper and tomato plants. This mutant still multiplied as well as the wild type in the leaves or cotyledons of soybean. Although the DNA and amino acid sequences showed high homology with known hrp genes, the hrcU-homolog ORF is not required for HR induction on nonhost plants, pepper and tomato, or for the multiplication of bacteria in the host plant. This gene was only required for the pathogenic symptoms of X. campestris pv. glycines 8ra on soybean. (+info)
Identification of new early markers of the hypersensitive response in Arabidopsis thaliana(1).
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New molecular markers of the hypersensitive response (HR) of Arabidopsis thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris (X.c.c.) have been identified by differential screening of a cDNA library constructed from suspension cells inoculated by an HR-inducing strain in the presence of cycloheximide. Seven families of genes (called Athsr) have been isolated, show similarities to voltage-dependent anion channels (VDAC) and alternative oxidases, or are novel proteins. Athsr genes have shown to be specifically or preferentially expressed during the HR. These data suggest that Athsr genes might be involved in early events conditioning the establishment of the HR. (+info)
Use of an internal control for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of bacteria.
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A method to aid in the analysis of bacterial samples of unknown concentration by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is demonstrated. It is shown that in MALDI analysis of bacteria, the intensities of resulting peaks in spectra are sensitive to the microbial concentration. At the high and low ends of the concentration range, no signal can be obtained, leaving very concentrated or very dilute samples indistinguishable. The addition of cytochrome c as an internal control allows the differentiation of these concentrated and dilute samples. The presence of the internal control causes only a 20% to 30% decrease in signal intensity when the bacterial concentration is optimum. However, the signal quality is improved when the internal control is added to some low concentrations of bacteria. (+info)
Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes.
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hrpG is a key regulatory gene for transcriptional activation of pathogenicity genes (hrp) of Xanthomonas campestris pv. vesicatoria. We identified three mutations in hrpG which render hrp gene expression constitutive in normally suppressing medium. The mutations in hrpG result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutated hrpG enhances the timing and intensity of plant reactions in infection assays. (+info)