RESUMO
The population of bacteria ofSelenomonas ruminantium species in the rumen of fallow-deer was analyzed using endonucleolytic activity assay and plasmid profiles. This analysis indicated a high diversity within the population ofS. ruminantium. At least 12 different restriction profiles, indicating the presence of the different specificity nucleases, have been observed. Site-specific endonucleases were detected in 17 out of 45 strains tested. In other strains a various level of nonspecific activity was detected. Plasmid DNAs ranging in size from 0.9 to more than 25 kbp were detected in 60% of strains analyzed. No or little correlation was observed between the endonuclease activity and the plasmid content. The presence of different specificity endonucleases, as well as differences of plasmid profiles of isolates possessing identical specific activity indicate that the population ofS. ruminantium in the rumen of an individual animal consists of at least 10 different clones.
RESUMO
A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.
Assuntos
Bacteroides/enzimologia , Celulase/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Prevotella/enzimologia , Xilosidases/genética , Bacteroides/genética , Carboximetilcelulose Sódica/metabolismo , Celulase/biossíntese , Celulase/metabolismo , Clonagem Molecular , Genes Bacterianos/genética , Prevotella/genética , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/biossíntese , Xilosidases/metabolismoRESUMO
The restriction endonuclease (ENase) Sru30DI, an isoschizomer of StuI, which recognizes the sequence 5'-AGG/CCT-3', was purified from a natural isolate of Selenomonas ruminatinum. The ENase was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. Activity of Sru30DI is inhibited by overlapping Dcm methylation. The ENase is extremely stable at 37 degrees C and is active over a wide range of pH, temperature and salt concentrations.
Assuntos
Bacteroidaceae/enzimologia , Animais , Cromatografia por Troca Iônica , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Poliacrilamida , Rúmen/microbiologiaRESUMO
Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI. This enzyme was isolated from cell extracts by phosphocellulose chromatography. Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5'-AT/TAAT-3' generating 5'dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of vspI, a restriction enzyme isolated from Vibrio sp.
Assuntos
Enzimas de Restrição do DNA/metabolismo , Bactérias Anaeróbias Gram-Negativas/enzimologia , Rúmen/microbiologia , Animais , Sequência de Bases , Enzimas de Restrição do DNA/genética , Dados de Sequência MolecularRESUMO
Restriction endonuclease SbvI, an isoschizomer of HaeIII, has been isolated from rumen amylolytic bacterium Streptococcus bovis II/1. SbvI was purified from cell extract by phosphocellulose chromatography and heparin-Sepharose chromatography. The recognition sequence of SbvI was identified by digestion of pBR322, pUC9 and lambda-DNA and comparing the cleavage patterns obtained with computer-derived data. SbvI recognizes the 4-bp palindrome, 5'-GGCC-3' and cleaves DNA after the second G in the sequence, producing blunt ends.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Streptococcus bovis/enzimologia , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismoRESUMO
SruI, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT decreases AAA-3'. The recognition sequence of SruI was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and lambda DNA. The cleavage patterns obtained were compared with computer-derived data. SruI recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease SruI presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism possesses a DNA-cleaving enzyme with the same specificity as DraI or AhaIII.
