Structural organization of pBC1, a cryptic plasmid from Bacillus coagulans

Abstract

The complete nucleotide sequence of the Bacillus coagulans plasmid pBC1 was determined. The sequence revealed an open reading frame encoding a polypeptide of 259 amino acids. This open reading frame shows sequence similarity to genes coding for replication-associated proteins in a group of gram-positive bacterial plasmids known to replicate via single-stranded intermediates. A region required for replication in cis, when the intact replicon is supplied in trans, was identified as well. (Difco Laboratories) was supplemented with antibiotics as indicated. Erythromycin (5 ,ug/ml for B. subtilis) and chloramphenicol (12.5 and 5 ,ug/ml for E. coli and B. subtilis, respectively) were used. Transformation of B. subtilis or E. coli competent cells with plasmid DNA and all manipulations of DNA were performed as previously described (3,20). pBC1 plasmid was extracted from B. coagulans Zu196I as previously described (5). The DNA sequence was determined by the dideoxy-chain termination method (26) after different DNA restriction fragments were subcloned in the pGEM4Z plasmid (Promega Biotec, Madison, Wis.) by using the strategy shown in Fig. 1. A T7 DNA polymerase sequencing kit (Pharmacia, Uppsala, Sweden) and SP6 and T7 promoters (Promega) were used for sequencing and as primers, respectively.By examination of possible ORFs, we found only one large frame (Fig. 2) Fig. 2) complementary to the 3' end of 16S rRNA (3'-UCUUUCCUCCAGUAG-5') of B. subtilis (23), at position -9 to -13 from the ATG initiation codon. Potential promoter regions following consensus sequences pointed out by Graves and Rabinowitz (10) were found. The most plausible area for transcriptional initiation seems to be the 5'-TATATC-3' sequence (at positions -33 to -38 from the ATG codon) for the -10 region and the 5'-GTGTTT-3' sequence (at positions -64 to -69 from the ATG codon) for the -35 region. Translation of ORF E resulted in a polypeptide of 259 amino acids with-a predicted molecular mass of 30.3 kDa. The amino acid sequence of ORF E was compared with those of the replication proteins of gram-positive bacterial plasmids such as pFTB14 from Bacillus amyloliquefaciens (24), pC194 (16) and pUB110 from Staphylococcus aureus (21), pLP1 from Lactobacillus plantarum (2k, and pBAA1 from B. subtilis (7) and also with the phage PX174 replication protein by using the CLUSTAL homology search system (14). As shown in Fig. 3, significant homologies were observed between the ORF E product and the proteins involved in gram-positive bacterial plasmid replication. This result strongly suggests that the ORF E-encoded protein is implicated in pBC1 replication; thus, this protein was named RepE. As in other published replication protein sequences, a tyrosine residue is present at position 171. This amino acid was described as the linkage site of the pX174 protein A and the DNA when nicking occurs at the plus-strand origin (29). At position 161 began a sequence (5'-TTTCTTATCTTGATA-3') ( Fig. 2) which was identical to the known plus-strand origin of replication sequences o...

Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I 4

“…Amino acid sequencing enabled the partial determination of the first 39 amino acid (aa) residues. Seven positions (9,14,19,24,29,33, and 34) could not be identified. Comparisons with protein sequences contained in the data banks demonstrated a total identity of the 32 identified amino acid residues with the sequence of pediocin PA-1 (22).…”

“…Recent comparative studies of the sequences of the S. aureus antimicrobial multiresistance plasmid pSK41 (16) and the Lactococcus lactis bacteriocin production plasmid pMRC01 (11) suggested that insertion sequence-mediated cointegrate formation may play an important role in the evolution of these plasmids, allowing bacteria to collect and subsequently disseminate advantageous traits. To date, little information is available on plasmids replicating in B. coagulans, except for the report of a cryptic 1.6-kb plasmid called pBC1 (9). This rolling-circle type plasmid (RCR) was classified in the pC194/pUB110 family (19,27).…”

Genetic engineering of Geobacillus spp.

“…The pBC1 plasmid is a small cryptic RC-plasmid isolated from a thermophilic strain of B. coagulans. The replicon of pBC1 ensures the maintenance of plasmids in a variety of Gram-positive bacteria (De Rossi et al, 1992) and was used for the construction of a widely used Geobacillus-E. coli shuttle vector pNW33N (Zeigler, 2001).…”

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

“…This plasmid is constructed on the basis of a cryptic plasmid pBC1 isolated from B. coagulans (De Rossi et al, 1989). The sequence analysis of pBC1 reveals that this plasmid belongs to the pC194 family (De Rossi et al, 1992). Therefore, the replicon of pRP9 is cvidently distinct from that of pSTK1.…”