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The plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15, was sequenced and characterized as a circular, double-stranded DNA molecule of 1592 base pairs with a GC content of 40.01 %. Despite its cryptic nature and small genome, bioinformatic analyses identified conserved motifs associated with replication-related proteins, though BLAST searches revealed no significant homology with other plasmids. The plasmid genome contains five putative Open Reading Frames (ORFs), four palindromic sequences, and two direct repeats on both strands, suggesting regulatory roles. Electron microscopy and Southern hybridization studies confirmed that pAnox1 follows a Rolling Circle (RC) replication mode. The study further demonstrated that the plasmid encodes three distinct transcripts: ORF-1 and ORF-3 are oriented in the same direction, while ORF-5 is on the opposite strand. RACE and LACE analyses revealed transcript lengths of 903 bp for ORF1, 499 bp for ORF3, and 211 bp for ORF5. Quantitative real-time PCR estimated the relative copy number of pAnox1 at 127 ± 2 copies per chromosomal equivalent. This novel RC-type plasmid in the Anoxybacillus genome holds promise as a cloning and expression vector for biotechnological applications and in vivo protein engineering. • A novel and cryptic plasmid from Anoxybacillus gonensis 05S15. • pAnox1 is a small, double stranded and 1562 bp circular molecule with 40.01 % GC content. • pAnox1 replicates via Rolling Circle (RC) Replication mechanism. • The copy number of pAnox1 was calculated by qPCR as 127 ± 2 copies per chromosome. [ABSTRACT FROM AUTHOR]

Abstract: Bacillus subtilis and related species (Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus mojavensis) represent a group of bacteria largely studied and widely employed by industry. Small rolling circle replicating plasmids of this group of bacteria have been intensively studied as they represent a convenient model for genetic research and for the construction of molecular tools for the genetic modification of their hosts. Through the computational analysis of the available plasmid sequences to date, the first part of this review focuses on the main stages that the present model for rolling circle replication involves, citing the research data which helped to elucidate the mechanism by which these molecules replicate. Analysis of the distribution and phylogeny of the small RC plasmids inside the Bacillus genus is then considered, emphasizing the low level of diversity observed among these plasmids through the in silico analysis of their organization and the sequence divergence of their replication module. Finally, the parasitic vs. mutualistic nature of small rolling circle plasmids is briefly discussed. [Copyright &y& Elsevier]

The complete nucleotide sequences for pNAC1 (3538 bp) from strain RW048 as well as for pNAC2 (3684 bp) and pNAC3 (10,224 bp) from strain RW041 of Bifidobacterium longum were determined. The largest ORF (repB) of pNAC1 encodes a putative protein similar to those involved in a rolling-circle (RC) replication mechanism, which was confirmed by demonstration of single-strand intermediates in the host cell. The putative RepB gene product of pNAC2 is most similar to the replication protein of pDOJH10L and pKJ36. A second gene (mob) is similar to mobilization proteins involved in conjugation. Plasmid pNAC3 is the largest bifidobacterial plasmid to be sequenced to date. Of the eight putative gene products coded by pNAC3, one is similar to replication proteins (RepB), and another (Orf2) to putative transfer proteins (Tra). Bifidobacterial plasmids were divided into five groups based on Rep amino acid sequence homology and the results suggest a new plasmid family for B. longum. [Copyright &y& Elsevier]

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6–8 or ∼90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them. [Copyright &y& Elsevier]