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).

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The alpha-amylase gene (amy) from Streptomyces griseus IMRU 3570 and the beta-galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P2 or P3. The alpha-amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum.

Two plasmid cloning vectors (pULMJ55 and pULMJ95) were constructed for Brevibacterium lactofermentum using the origin of replication of the endogenous plasmid pBL1. Plasmid pULMJ55 is a replacement vector with transcriptional terminators from the B. lactofermentum trp operon flanking the BglII cloning sites. Religation of the BglII digested vector without insert creates a 376 bp perfect palindrome that is not tolerated in B. lactofermentum, giving positive selection for recombinant plasmids with inserts. Plasmid pULMJ95 contains the promoter-less alpha-amylase gene from Streptomyces griseus downstream of the trp terminator and is particularly suitable for the detection of promoters which are activated late during the growth phase. alpha-Amylase is secreted and its activity can be detected using simple plate tests.