Type II restriction modification systems ofPrevotella bryantiiTC1–1 andPrevotella ruminicola23 strains and their effect on the efficiency of DNA introduction via electroporation

Abstract

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

“…Cells were broken with a beadbeater apparatus (BioSpec Products), the DNA was removed with streptomycin (Accetto et al, 2005), and the resulting extracts were used for determination of nuclease activity by means of the quick nuclease assay or DNA protection as described previously (Accetto et al, 2005).…”

“…The orientation of nucB in the resulting clones was determined by restriction analysis. The constructs having the same orientation as tetQ gene were isolated, verified by sequencing using the primer kontrnnuc, protected against the action of PbrTI restriction of P. bryantii TC1-1, and transferred to P. bryantii TC1-1 by electroporation (Accetto et al, 2005). The culture supernatants of the resulting P. bryantii TC1-1-derived strains, which were grown with 2.5 mg tetracycline ml 21 unless stated otherwise, were assayed for nuclease activity either directly or were concentrated using Amicon Ultra4 10 000 MWCO centrifugal filter devices from Millipore.…”

“…In the second study, a shuttle vector pRH3, constructed on the basis of Prevotella sp. 223/M2/7 plasmid pRRI2 (Daniel et al, 1995), and protected against restriction, was successfully transferred to P. bryantii TC1-1 by electroporation (Accetto et al, 2005). These studies highlighted, among other factors, the importance of enzymic barriers preventing the successful establishment of foreign DNA in the microbial cell.…”

“…Most P. bryantii strains have high nucleolytic activity (Avguštin et al, 1997) and strain B 1 4 was shown to posess a 45 kDa non-specific nuclease (Accetto & Avguštin 2001). The electrotransformation frequency of P. bryantii TC1-1 with pRH3 (Accetto et al, 2005) could thus be risen considerably if the non-specific nuclease genes in the recipient strain were inactivated. We report here on the cloning of a P. bryantii B 1 4 non-specific nuclease gene named nucB and an attempt to express it in its native background.…”

“…This appears to be primarily due to the large phylogenetic distance between prevotellas and other well-characterized micro-organisms, and as a consequence of the rather specific genetic elements, exemplified by promoters, containing distinct 27/233 consensus sequences that are recognized by unusual primary s factor (Vingadassalom et al, 2005). Nevertheless, in two studies (Shoemaker et al, 1991;Accetto et al, 2005) the plasmid replicons and tetQ selectable marker derived from Prevotella and related colonic Bacteroides strains were used successfully for shuttle-vector introduction into the xylan-degrading ruminal species Prevotella bryantii, which forms a distinct phylogenetic lineage apparently somewhat closer to oral prevotellas than the other ruminal Prevotella species (Avguštin et al, 2001). Based on the conjugal transfer protocol developed in the first study, a P. bryantii B 1 4 carboxymethylcellulase gene, fused to a cellulose-binding domain of Thermomonospora fusca cellulase, and driven by a Prevotella ruminicola 23 xylanase promoter, was introduced into P. bryantii B 1 4.…”