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Bakterije rodu Prevotella so po Gramu negativni, nesporogeni in anaerobni mikroorganizmi, ki so del prebavne ter ustne mikrobiote živali in ljudi. Vrste so izolirane iz ustne votline sesalcev, iz vampa prežvekovalcev, debelega črevesa, urogenitalnega trakta in kliničnih vzorcev. Vampne prevotele predstavljajo največji delež po Gramu negativnih bakterij v populaciji in prispevajo k razgradnji polisaharidov v rastlinski celični steni in metabolizmu proteinov. Orodja za genetsko manipulacijo prevotel so maloštevilna, prav tako ni na voljo orodja za tarčno inaktivacijo genov. Poznamo samo en sprejemni sev Prevotella bryantii TC1-1, za katerega je na voljo plazmidni vektor, ki nam omogoča kontrolirano izražanje genov. Za vnos v ta sev prenosljivi vektor pRH3 najprej in vitro zaščitimo pred restrikcijskimi endonukleazami in ga vnesemo v sev z elektroporacijo. Na enak način smo poskušali elektrotransformirati še tri druge seve P. bryantii in P. brevis, vendar nam ni uspelo, saj imajo sevi izraženo močno nespecifično nukleazno aktivnost. Nadeljevali smo z ugotavljanjem optimalnih pogojev elektrotransformacije P. bryantii TC1-1 in ugotovili najbolj primerne pogoje zaščite in elektroporacije. Z vnosom gena za metilazo PbrTI (locus_tag CIK91_RS00145) iz P. bryantii v sev TC1-1 smo želeli izboljšati metodo zaščite DNK s celičnim ekstraktom, a zaščita ni bila boljša. Z izolacijo proteina z afinitetno Ni-NTA kromatografijo smo želeli nadomestiti pripravo celičnega ekstrakta. Učinkovitost zaščite DNK z izoliranim proteinom prav tako ni bila uspešna, saj je med njegovo izolacijo potekla proteoliza. Bacteria from genus Prevotella are Gram negative, non-spore forming, obligate anaerobes that represent important part of gastrointestinal and oral microbiota of animal and humans. Members of the genus have been isolated from oral cavity of mammals, rumen of cattle, from large intestine, urogenital tract and clinical samples. They are the most abundant Gram negative bacteria in rumen and are contributing to the degradation of plant cell-wall polysaccharides and protein metabolism. The genetic background of the genus is poorly studied because there are not many developed genetic tools. As far as we know only one recipient strain Prevotella bryantii TC1-1 can be electrotransformed. In vitro shuttle vector pRH3 must be protected from restriction endonuclease with cell extract before electroporation. Electrotransformation of three strain P. bryantii and P. brevis was performed, although it wasn’t successful, mainly because of their strong nonspecific nuclease activity. We continued with finding optimal conditions for protection and electroporation in process of electrotransformation of strain P. bryantii TC1-1. With insertion of methylase gene PbrTI (locus_tag CIK91_RS00145) from P. bryantii into strain TC1-1 we tried to improve in vitro protection of DNA with cell extract. Protection of plasmid DNA wasn't successful. By isolation of the protein with Ni-NTA agarose affinity chromatography we tried to substitute preparation of cell extract, but the efficiency of protection of plasmid DNA wasn't successful, because proteolysis occurred.

The restriction endonuclease BliAI, an isoschizomer of ClaI, which recognizes the sequence 5'- AT¯CGAT - 3', was purified from a natural isolate identified as Bacillus licheniformis. The restriction endonuclease was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. The restriction endonuclease is active at 37ºC and over a wide range of pH and salt concentration. The molecular weight of the purified restriction enzyme is consistent with a value of 39 kDa.A endonuclease de restrição BliAI, um isoesquisômero de ClaI, que reconhece a seqüência 5'- AT¯CGAT - 3', foi purificada de um isolado natural identificado como Bacillus licheniformis. A endonuclease de restrição em questão foi isolada a partir de um extrato celular em um único passo cromatográfico utilizando uma coluna contendo a resina fosfocelulose. A endonuclease de restrição é ativa à 37ºC e em uma ampla escala de pH e concentração de sais. O peso molecular da enzima purificada corresponde a um valor de kDa 39.

A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG¯CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.Uma linhagem de Bacillus pumilus foi isolada e identificada de amostras de águas coletadas em um pequeno Igarapé do Rio Amazonas. Foi detectada atividade de restrição do tipo II nesta bactéria. A enzima foi purificada e o peso molecular da proteína nativa foi estimado por gel filtração e por eletroforese em gel de poliacrilamida. Foram determinados, o pH e temperatura ótimos e as necessidades de sais. Os ensaios do controle de qualidade mostraram uma ausência completa de "nucleases não específicas". As analises das clivagens e o seqüenciamento do DNA dos fragmentos de restrição permitiram uma demonstração inequívoca de que 5´GAG¯CTC 3´ é a seqüência de reconhecimento da enzima. Esta enzima foi denominada de BpuAmI e aparentemente é um neoesquisômero da enzima protótipo SacI. Este é o primeiro relato de um isoesquisômero e/ou neoesquisômero da enzima protótipo SacI identificada no gênero Bacillus.

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The restriction endonuclease BliAI, an isoschizomer of ClaI, which recognizes the sequence 5'- AT¯CGAT - 3', was purified from a natural isolate identified as Bacillus licheniformis. The restriction endonuclease was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. The restriction endonuclease is active at 37ºC and over a wide range of pH and salt concentration. The molecular weight of the purified restriction enzyme is consistent with a value of 39 kDa.

The restriction endonuclease BliAI, an isoschizomer of ClaI, which recognizes the sequence 5'- AT¯CGAT - 3', was purified from a natural isolate identified as Bacillus licheniformis. The restriction endonuclease was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. The restriction endonuclease is active at 37ºC and over a wide range of pH and salt concentration. The molecular weight of the purified restriction enzyme is consistent with a value of 39 kDa. A endonuclease de restrição BliAI, um isoesquisômero de ClaI, que reconhece a seqüência 5'- AT¯CGAT - 3', foi purificada de um isolado natural identificado como Bacillus licheniformis. A endonuclease de restrição em questão foi isolada a partir de um extrato celular em um único passo cromatográfico utilizando uma coluna contendo a resina fosfocelulose. A endonuclease de restrição é ativa à 37ºC e em uma ampla escala de pH e concentração de sais. O peso molecular da enzima purificada corresponde a um valor de kDa 39.

A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG¯CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.

A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG¯CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus. Uma linhagem de Bacillus pumilus foi isolada e identificada de amostras de águas coletadas em um pequeno Igarapé do Rio Amazonas. Foi detectada atividade de restrição do tipo II nesta bactéria. A enzima foi purificada e o peso molecular da proteína nativa foi estimado por gel filtração e por eletroforese em gel de poliacrilamida. Foram determinados, o pH e temperatura ótimos e as necessidades de sais. Os ensaios do controle de qualidade mostraram uma ausência completa de "nucleases não específicas". As analises das clivagens e o seqüenciamento do DNA dos fragmentos de restrição permitiram uma demonstração inequívoca de que 5´GAG¯CTC 3´ é a seqüência de reconhecimento da enzima. Esta enzima foi denominada de BpuAmI e aparentemente é um neoesquisômero da enzima protótipo SacI. Este é o primeiro relato de um isoesquisômero e/ou neoesquisômero da enzima protótipo SacI identificada no gênero Bacillus.

Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis. In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5′-GmeATC-3′. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5′-GATC-3′, similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.