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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-borne genes and the activity of the corresponding proteins are severely affected by changes in the numbers of plasmid copies. We studied the impact of growth rate on the dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected, and these varied in size (3 to 39 kb), in replication mechanism (theta or rolling circle), and in putative (dairy-associated) functions. The copy numbers ranged from 1.5 to 40.5, and the copy number of theta-type replicating plasmids was negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), the copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates, showing that the plasmid replication rate was strictly controlled. One low-copy-number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations, reflected in a complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation, or the presence of citrate (maximum 2.2-fold), signifying the stability in copy number of the plasmids. IMPORTANCE Lactococcus lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers of L. lactis at near-zero growth rates, characteristic for cheese ripening. Moreover, we analyzed the effects of pH, nutrient limitation, and the presence of citrate. This showed that the plasmid copy numbers were stable, giving insight into plasmid copy number dynamics in dairy fermentations., (Copyright © 2018 American Society for Microbiology.)

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]

pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter. [ABSTRACT FROM AUTHOR]