RESUMO
The rhizosphere nitrogen-fixing bacterium Azospirillum irakense KBC1 is able to grow on pectin and beta-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring beta-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl beta-glucosides. A Delta(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB' subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this beta-glucosidase family in the utilization of beta-glucosides for microbial growth.
Assuntos
Azospirillum/metabolismo , Álcoois Benzílicos/metabolismo , Glucosídeos/metabolismo , beta-Glucosidase/genética , Sequência de Aminoácidos , Arbutina/metabolismo , Azospirillum/enzimologia , Azospirillum/genética , Azospirillum/crescimento & desenvolvimento , Celobiose/metabolismo , Clonagem Molecular , Deleção de Genes , Teste de Complementação Genética , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Dados de Sequência Molecular , Filogenia , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura , beta-Glucosidase/química , beta-Glucosidase/isolamento & purificação , beta-Glucosidase/metabolismoRESUMO
The pelA gene from the N2-fixing plant-associated bacterium Azospirillum irakense, encoding a pectate lyase, was isolated by heterologous expression in Escherichia coli. Nucleotide sequence analysis of the region containing pelA indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in E. coli at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca2+ for its activity. Of several divalent cations tested, none could substitute for Ca2+. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of pelA expression was studied by means of a translational pelA-gusA fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition, pelA expression was found to be induced by pectin. An A. irakense pelA::Tn5 mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in A. irakense.
Assuntos
Azospirillum/genética , Genes Bacterianos , Pectinas/metabolismo , Polissacarídeo-Liases/genética , Azospirillum/enzimologia , Azospirillum/patogenicidade , Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Cromossomos Bacterianos , Clonagem Molecular , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Polissacarídeo-Liases/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The early steps of symbiotic nodule formation by Rhizobium spp. on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Rhizobium sp. strain BR816, isolated from Leucaena leucocephala, contains four nodD genes which differ in their interaction with flavonoids. Two of the four NodD proteins, namely, NodD1 and NodD2, obey the LysR rule regarding the need of a coinducer. NodD3 shows hardly any inducing activity, and NodD4 contains a high basal activity and no response to any of the flavonoids tested. Complementation experiments with the NGR234 nodD mutant by the different nodD genes of BR816, as well as the analysis of the nodulation phenotype of different nodD mutants of BR816, revealed that all the nodD genes of BR816 are functional, but differences can be noticed when different host plants are tested. Whereas the nodD2 and nodD4 genes of BR816 have a great impact on the nodulation of L. leucocephala, nodD3 and nodD4 appear to be important for the nodulation of Phaseolus vulgaris. It appears that NodD1 of BR816 can function as a transcriptional activator in bean nodulation but not in nodulation of L. leucocephala.
Assuntos
Proteínas de Bactérias/metabolismo , Plantas/microbiologia , Rhizobium/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese , Fenótipo , Rhizobium/genética , Rhizobium/fisiologia , Homologia de Sequência de Aminoácidos , Simbiose/genética , Simbiose/fisiologia , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologiaRESUMO
The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA. IS427 does not show homology with previously characterized IS elements of A. tumefaciens, based on hybridization experiments and/or sequence comparison.
Assuntos
Elementos de DNA Transponíveis , Genes Bacterianos , Genes Reguladores , Plasmídeos , Rhizobium/genética , Bacillus subtilis/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
We have identified and determined the nucleotide sequence of an IS element (IS136) of Agrobacterium tumefaciens. This is the first IS element isolated and sequenced from a nopaline type Ti-plasmid. Our IS element has 32/30 bp inverted repeats with 6 mismatches, is 1,313 bp long and generates 9 bp direct repeats upon integration. IS136 has 3 main open reading frames (ORF's). Only ORF1 (159 codons) is preceded by sequences that are proposed to serve functional roles in transcriptional and translational initiation. No DNA sequence homology was found between IS136 and IS66, an IS element isolated from an octopine type Ti-plasmid.
Assuntos
Elementos de DNA Transponíveis , Genes Bacterianos , Plasmídeos , Rhizobium/genética , Sequência de Bases , Clonagem Molecular , Colífagos/genética , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Conformação de Ácido NucleicoRESUMO
The nucleotide sequence of the T-DNA region encoding transcripts 6a and 6b of the pTiT37 nopaline Ti plasmid has been determined. Analysis of this sequence allowed us to find two open reading frames in opposite orientation and separated by 482 bp. Comparison with previous published nucleotide sequences of the homologous regions of the pTiAch5 and the pTi15955 octopine Ti plasmids reveals that the coding sequences as well as the 5'- and 3' 3-untranslated regions of both genes are highly homologous.