1 a Percentage identity/similarity, the number in parenthesis is the number of amino acids used in the calculations. b The organism, with associated bacteriophage in parenthesis where applicable. cAccession number Selleck Inhibitor Library of the highest scoring BLAST hit with an annotated function. The regions flanking the C10 loci in a range of Bacteroidetes (B. thetaiotaomicron (AE015928), B. uniformis (AAYH00000000), B. ovatus (AAXF00000000), B. intestinalis (ABJL00000000), Parabacteroides distasonis (CP000140), Porphyromonas gingivalis (AP009380, AE015924) and Prevotella intermedia
(ID: 246198) were examined for the presence of markers for mobile genetic elements (e.g. the Tra functional module, or phage structural modules for instance tail, and capsid). The GenBank accession code or JCVI taxon numbers are given in parenthesis. A cassette of Tra genes (A through O, locus tags PG1473-1486) was found 35.3 Kb away from MK 8931 prtT in Porphyromonas gingivalis strain W83 (locus tag 1427) and again in strain ATCC 33277 Tra
I to Q were found (locus tags PGN_592 to PGN_599) 40.5 Kb away from PrtT (PGN_0561) in that strain. However, no www.selleckchem.com/MEK.html complete CTn or phage could be found adjacent to these or any other C10 protease gene. The Bfgi2 element harbouring the bfp3 gene is capable of excision The putative att sequence for the integration of Bfgi2 was identified by analysis of the sequence at the boundaries of the inserted DNA in strain 638R compared with NCTC9343. A short 16 bp direct repeat sequence was identified flanking the Bfgi2 insertion (Fig. 6, panel A). PCR primers Bfgi2_attB_F and Bfgi2_attB_R (Table 4) were used in a PCR reaction to detect the excision of the Bfgi2 prophage from mitomycin C treated B. fragilis 638R cells. The resulting 595 bp PCR product is consistent with excision of Bfgi2 from the B. fragilis 638R genome (Fig. 6, panel B, Lane 2), and reconstruction of an intact tRNAArg gene (Fig. 6, panel C). Sequencing of this PCR product indicated the presence of a
single copy of the 16 bp repeat region, the proposed attB site for Bfgi2 (Fig. 6, panel C). Figure 6 The prophage carrying bfp3 is capable of excision. Panel A. The Bfgi2 prophage (grey bar) is flanked by the B. fragilis Low-density-lipoprotein receptor kinase 638R genome (black bar). The bfp3 gene (open white arrow), tRNA Arg (white arrowhead) and genes flanking Bfgi2 (mid-grey) are shown. The attR and attL sequences (underlined) are shown in the expanded sequence. The locations of primers used in these studies are shown by small black arrows (see Table 4). Panel B. Agarose gel electrophoretic analysis of PCR reactions to test for excision of the prophage (Lane 2) and for the circular intermediate of the ‘phage (Lane 3). Lane 1 contains DNA size markers. Panel C. Schematic representation of the 638R genome, after excision of the Bfgi2 element. Colour scheme is as for panel A. The regenerated attB site (underlined) is shown in the expanded sequence.