RESUMO
Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen Xanthomonas campestris pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase-beta-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the Yersinia homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from Yersinia, YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Xanthomonas vesicatoria/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/genética , Doenças das Plantas/microbiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Xanthomonas vesicatoria/química , Xanthomonas vesicatoria/genéticaRESUMO
The Streptomyces phage phiC31 integrase was tested for its ability to excise transgenic DNA from the wheat genome by site-specific recombination. Plants that stably express phiC31 integrase were crossed to plants carrying a target construct bearing the phiC31 recognition sites, attP and attB. In the progeny, phiC31 recombinase mediates recombination between the att sites of the target locus, which results in excision of the intervening DNA. Recombination events could be identified in 34 independent wheat lines by PCR and Southern blot analysis and by sequencing of the excision footprints. Recombinant loci were inherited to the subsequent generation. The results presented here establish the integrase-att system as a tool for catalysing the precise elimination of DNA sequences from wheat chromosomes.
Assuntos
Cromossomos de Plantas , Integrases/fisiologia , Transgenes , Triticum/genética , Proteínas Virais/fisiologia , Bacteriófagos/enzimologia , Engenharia Genética/métodos , Hibridização Genética , Recombinação Genética , Análise de Sequência de DNARESUMO
The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (T3S) system which is encoded in the 23-kb hypersensitive response and pathogenicity (hrp) gene cluster. The T3S system is essential for pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. In this study, we revisited the operon structure of the right part of the hrp gene cluster. Based on complementation experiments of transposon insertions and reverse-transcription polymerase chain reaction analyses, the hrpD operon contains hrcQ, hrcR, hrcS, and hpaA, whereas hrcD, hrpD6, and hrpE belong to the hrpE operon. We determined the transcriptional start site of the hrpE operon and showed that there is a promoter upstream of hrcD containing a plant-inducible promoter box. Conserved secondary mRNA structures in the intergenic region between hrpD6 and hrpE suggest a posttranscriptional regulated expression of hrpE. Based on comparisons of different hrp gene clusters and the analysis of evolutionary rates, we propose that the hrpE transcriptional unit was integrated into the hrp gene cluster at a later time.
Assuntos
Proteínas de Bactérias/genética , Óperon , Xanthomonas campestris/genética , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis/genética , DNA Intergênico/química , DNA Intergênico/genética , Ordem dos Genes , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Virulência/genéticaRESUMO
The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.