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The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.  相似文献   

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Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.  相似文献   

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Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1.3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41,004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.  相似文献   

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A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.  相似文献   

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The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 microM (at pH 7.0); xylitol, 25 mM; NAD, 100 microM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells.  相似文献   

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Pseudomonas solanacearum is an important phytopathogen that produces a variety of extracellular enzymes. Previous reports suggested that one of these, a 43-kDa beta-1,4-endoglucanase (EGL), is initially synthesized with a 45-residue leader sequence that is removed during export. Experiments with globomycin presented here also suggest that the primary precursor of EGL (ppEGL) has a 45-residue leader sequence but that only the first 19 residues of the leader sequence are removed by signal peptidase II during initial export across the inner membrane. Further analysis suggested that the resultant 46-kDa intermediate precursor (pEGL) is a transient fatty acylated lipoprotein and is located on the periplasmic side of the inner membrane of P. solanacearum. Although Escherichia coli could synthesize ppEGL, modify it with palmitate, and remove the first 19 residues of the leader sequence during export across the inner membrane, only P. solanacearum could export pEGL across the outer membrane and remove the remaining 26 residues of the leader sequence producing the mature, extracellular EGL. The second step of the export process requires export machinery not present in E. coli. To our knowledge this represents the first example of a leader sequence with two distinct parts, one removed during export across the inner membrane and the other removed during export across the outer membrane.  相似文献   

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hrp genes, encoding type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium Ralstonia solanacearum GMI1000. Here, we show phenotypes of R. solanacearum mutant strains disrupted in the prhJ, hrpG, or hrpB regulatory genes with respect to root infection and vascular colonization in tomato plants. Tests of bacterial colonization and enumeration in tomato plants, together with microscopic observations of tomato root sections, revealed that these strains display different phenotypes in planta. The phenotype of a prhJ mutant resembles that of the wild-type strain. An hrpB mutant shows reduced infection, colonization, and multiplication ability in planta, and induces a defense reaction similar to a vascular hypersensitive response at one protoxylem pole of invaded plants. In contrast, the hrpG mutant exhibited a wild-type level of infection at secondary root axils, but the ability of the infecting bacteria to penetrate into the vascular cylinder was significantly impaired. This indicates that bacterial multiplication at root infection sites and transit through the endodermis constitute critical stages in the infection process, in which hrpB and hrpG genes are involved. Moreover, our results suggest that the hrpG gene might control, in addition to hrp genes, other functions required for vascular colonization.  相似文献   

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Excretion of the egl gene product of Pseudomonas solanacearum.   总被引:2,自引:6,他引:2       下载免费PDF全文
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Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.  相似文献   

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