Assuntos
Proteínas de Bactérias/química , Dickeya chrysanthemi/enzimologia , Ressonância Magnética Nuclear Biomolecular , Oxirredutases/química , Carbono/química , Hidrogênio/química , Metionina Sulfóxido Redutases , Nitrogênio/química , Fragmentos de Peptídeos/química , Conformação Proteica , Proteínas Recombinantes de Fusão/químicaRESUMO
Peptide methionine sulfoxide reductase (MsrA), which repairs oxidized proteins, is present in most living organisms, and the cognate structural gene belongs to the so-called minimum gene set [Mushegian, A. R. & Koonin, E. V., (1996) Proc. Natl. Acad. Sci. USA 93, 10268-10273]. In this work, we report that MsrA is required for full virulence of the plant pathogen Erwinia chrysanthemi. The following differences were observed between the wild-type and a MsrA- mutant: (i) the MsrA- mutant was more sensitive to oxidative stress; (ii) the MsrA- mutant was less motile on solid surface; (iii) the MsrA- mutant exhibited reduced virulence on chicory leaves; and (iv) no systemic invasion was observed when the MsrA- mutant was inoculated into whole Saintpaulia ionantha plants. These results suggest that plants respond to virulent pathogens by producing active oxygen species, and that enzymes repairing oxidative damage allow virulent pathogens to survive the host environment, thereby supporting the theory that active oxygen species play a key role in plant defense.
Assuntos
Dickeya chrysanthemi/genética , Dickeya chrysanthemi/patogenicidade , Genes de Plantas , Oxirredutases/genética , Oxirredutases/metabolismo , Sequência de Bases , Clonagem Molecular , Dickeya chrysanthemi/enzimologia , Escherichia coli , Metionina Sulfóxido Redutases , Dados de Sequência Molecular , Estresse Oxidativo , Oxirredutases/isolamento & purificação , Plantas/microbiologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Virulência/genéticaRESUMO
In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.
Assuntos
Dano ao DNA , Erwinia/genética , Genes Bacterianos , Polissacarídeo-Liases/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Deleção Cromossômica , DNA Bacteriano/genética , Indução Enzimática , Erwinia/enzimologia , Dados de Sequência Molecular , Peso Molecular , Polissacarídeo-Liases/biossíntese , Polissacarídeo-Liases/isolamento & purificação , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Mapeamento por Restrição , Transcrição GênicaRESUMO
The structural gene coding for a new endo-beta-1,4-glucanase of Erwinia chrysanthemi strain 3665, previously identified in a cosmid library, was subcloned into pUC18. The gene is expressed from a 1.9 X 10(3)-base-pair insert and its direction of transcription was determined. The properties of the gene product purified from cell-free extracts of Escherichia coli have been studied. The purified protein has an endoglucanase activity but is significantly different from the major endoglucanase Z secreted by E. chrysanthemi strain 3665. The new enzyme was designated as endoglucanase Y and the related gene celY. In E. coli, most of the endoglucanase activity was found in the periplasmic space.
Assuntos
Celulase/genética , Erwinia/enzimologia , Genes Bacterianos , Genes , Celulase/metabolismo , Enzimas de Restrição do DNA , Erwinia/genética , Escherichia coli/genética , Hibridização de Ácido Nucleico , Plasmídeos , Transcrição GênicaRESUMO
Erwinia chrysanthemi produced several pectate lyases (EC 4.2.2.2) and endocellulases (EC 3.2.1.4) which were largely secreted into the culture medium. Mutants deficient in the secretion mechanism for these enzymes were obtained by chemical and insertion mutagenesis. Further study of one such mutant revealed that both enzyme activities were retained simultaneously within the periplasmic space.
Assuntos
Celulase/genética , Erwinia/enzimologia , Glicosídeo Hidrolases/genética , Mutação , Poligalacturonase/genética , Erwinia/genética , Erwinia/crescimento & desenvolvimento , Escherichia coli/genética , Genótipo , Cinética , Fenótipo , Especificidade da EspécieRESUMO
The study of mutants of Erwinia specifically unable to ferment cellobiose indicates that the mutations are clustered between arg and ile on the chromosome of this organism. In vivo cloning of the genes responsible for cellobiose utilization lead to a plasmid, pBEC2, which complements all Erwinia Clb- specific mutants. When introduced into wild-type E. coli it allows this organism to use cellobiose, arbutin and salicin; it also complements bglB and bglC mutants of Escherichia coli indicating that arbutin and salicin utilization is due to the products of the pBEC2 cloned genes. From the characterization of mutants pleiotropically affected in the utilization of various carbon sources, including cellobiose, arbutin and salicin, it is proposed that the three beta-glucosides are substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS).
Assuntos
Celobiose/metabolismo , Dissacarídeos/metabolismo , Erwinia/genética , DNA Bacteriano/genética , Erwinia/metabolismo , Escherichia coli/genética , Genes Bacterianos , Teste de Complementação Genética , Mutação , Especificidade da EspécieRESUMO
To assess the "futile cycle" fructose-6-P leads to fructose-1,6-P2 leads to fructose-6-P in Escherichia coli we have grown the cells on [6-14C]glucose and determined label in the 1-position of glucose obtained from glycogen. In a variety of strains, including a wild type and a mutant without fructose diphosphatase, 1-position labeling was negligible. But there was little label in the 1-position of fructose-1,6-P2 either, which shows that hexose diphosphate and triose-P are not in equilibrium in this organism. Therefore, the lack of 1-position labeling in glycogen does not necessarily indicate lack of futile cycling. One strain, however, a temperature-sensitive glyceraldehyde-3-P dehydrogenase mutant grown at permissive temperature, gave substantial labeling of the 1-position of fructose-1,6-P2. In this strain 1-position labeling in glycogen was low, indicating minimal futile cycling.
