Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model

Abstract

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pUJ101, pSB24, and pJVl.pBL1 is a multicopy plasmid isolated from Brevibacterium lactofermentum ATCC 13869 (19) and later described as pAM330 (14) Streptomyces lividans. pUL61 was unstable in B. lactofermentum, and two stable deletion derivatives (pUL330 and pUL340) appeared after B. lactofermentum had been transformed with pUL61. A similar instability has also been observed in most other small plasmids from gram-positive bacteria. The main characteristic of plasmids showing instability is their mode of replication via the rolling circle replication (RCR) mechanism. RCR plasmids were classified into four families according to homologies of their replication proteins (Rep) and the position of the double-stranded origin (DSO) (7). RCR plasmids have the information to synthesize their own Rep protein. The Rep protein initiates replication by nicking the DNA at the DSO sequence, after which replication of the plus strand begins. The newly generated single-stranded DNA (ssDNA) intermediates (plus strand) serve as a template for minus strand synthesis. The conversion of the ssDNA intermediates to duplex forms requires the recognition of another sequence, different and separated from the DSO sequence, called single-stranded origin (SSO). The absence or nonfunctionality of SSO results in the accumulation of ssDNA. The SSO sequence is rather specific, and it is usually recognized inefficiently in hosts different from the parental one (7, 15).We describe in this paper the identification and characterization of the region involved in replication functions of pBL1. Our results indicate that pBL1 belongs to the group of plasmids which replicate via the RCR mechanism. MATERIALS AND METHODSPlasmids, bacterial strains, and transformation conditions. Plasmids used in this study are listed in Table 1. B. lactofermentum BL31 (20), a pBL1-cured strain, and C. glutamicum ATCC 13032 were grown in Trypticase soy broth (TSB) medium and transformed by electroporation as described by Dunican and Shivnan (5). E. coli DH5ao was grown in L broth and transformed by the method of Hanahan (8).

ACorynebacterium glutamicum rnhA recGDouble Mutant Showing Lysozyme- sensitivity, Temperature-sensitive Growth, and UV-Sensitivity

“…and 5?-TTTGCCGGATCAAGAGATCTCAACT-3?. The region of pBL1 containing the replication origin and the rep gene 14) was also PCR-ampliˆed using pC2 15) as a template and a set of primers, 5?-AACA-GCATGACAGATCTATTCCTGA-3? and 5?-TCG-GACGGACTTGCAGATCTTGTAT-3?.…”

Plasmid Rolling-Circle Replication

“…The CS-6-containing terminal loop seems to function as a terminator of the primer RNA synthesis, and changes in this region lead to a moderate increase in the intracellular number of ssDNA plasmid forms without impairing ssoA binding by RNAP (121)(122)(123). Sequences similar to CS-6 have been identified in the terminal loops of hairpins located in DNA regions that have been either proposed or shown to act as ssos in plasmids from Actinobacteria (124)(125)(126)(127)(128). In addition to the RS B and CS-6 conserved motifs, sequences resembling the consensus -35 and -10 promoter regions have been identified in various ssoAs (129).…”

Sinorhizobium meliloti Plasmid pRm1132f Replicates by a Rolling-Circle Mechanism

“…Pretreatment of the DNA with S1 nuclease resulted in a loss of the hybridizing bands and confirmed that they consisted of ssDNA. The membrane-bound DNA was also hybridized to the 32 P-labeled strand-specific riboprobes (3), and pBL1 (12). The conserved GG dinucleotide (underlined) indicates the position of the nick site in pSN22, pJV1, and pIJ101 (23,25).…”