Identification, sequencing and comparative analysis of pBp15.S plasmid from the newly described entomopathogen Bacillus pumilus 15.1

Abstract

The Bacillus pumilus 15.1 strain, a recently described entomopathogenic strain active against Ceratitis capitata, contains at least two extrachromosomal elements, pBp15.1S and pBp15.1B. Given that B. pumilus is not a typical entomopathogenic bacterium, the acquisition of this extrachromosomal DNA may explain why B. pumilus 15.1 is toxic to an insect. One of the plasmids present in the strain, the pBp15.1S plasmid, was sub-cloned, sequenced and analyzed using bioinformatics to identify any potential virulence factor. The pBp15.1S plasmid was found to be 7785 bp in size with a GC content of 35.7% and 11 putative ORFs. A replication module typical of a small rolling circle plasmid and a sensing and regulatory system specific for plasmids was found in pBp15.1S. Additionally, we demonstrated the existence of ssDNA in plasmid preparations suggesting that pBp15.1S replicates by the small rolling circle mechanism. A gene cluster present in plasmid pPZZ84 from a distantly isolated B. pumilus strain was also present in pBp15.1S. The plasmid copy number of pBp15.1S in exponentially growing B. pumilus cells was determined to be 33 copies per chromosome. After an extensive plasmid characterization, no known virulence factor was found so a search in the other extrachromosomal elements of the bacteria is needed.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

“…The acridine orange strain was selected for further studies and named B. pumilus 15.1C (cured from plasmid (pBp15.1S) and megaplasmid (pBp15.1B)). As, in contrast to the megaplasmid, the smaller pBp15.1S plasmid has been completely characterized, and its copy number was found to be 33 (Garcia‐Ramon et al ., ), we were able to verify its absence by PCR since, using the same methodology: no amplification was obtained from B. pumilus strain 15.1C (data not shown). Southern blot analysis using a Dig‐labelled probe designed in the orf 7 of the plasmid pBp15.1S was also carried out.…”

“…The majority of crystal toxin genes of Bt are encoded on extrachromosomal elements, and we decided to investigate the location of the oxdD gene. We have recently shown that the B. pumilus 15.1 strain bears one plasmid of 7785 bp named pBp15.1S (Contig 38) and one megaplasmid of unknown size named pBp15.1B (Garcia‐Ramon et al ., ). The oxdD gene was found in Contig 4 (Accesion number ), a contig of 57 329 bp that encodes 51 predicted proteins.…”

“…Electrophoresis was performed in a 1% agarose gel and stained with ethidium bromide. Sothern blot was performed with a DIG ‐labelled probe designed in the orf 7 of the plasmid pB p15.1S (Garcia‐Ramon et al ., ). M: Molecular weight marker (HyperLadder I from Bioline) in base pairs.…”

“…A 727 bp product was detected only when DNA from the wild‐type strain was used as template, but not when total DNA from B. pumilus 15.1C was used (data not shown). As the yuaB gene is not present in the known sequence of pBp15.1S (Garcia‐Ramon et al ., ) and as the strain contains only one plasmid and one megaplasmid, we must conclude that yuaB gene is present in the megaplasmid pBp15.1B. The 24 079 bp Contig 48 (LBDK01000048), where the yuaB gene is present must therefore, be part of this megaplasmid and contains 27 CDSs, most of them encoding hypothetical proteins.…”

“…To establish the location of the gene encoding the parasporal crystals of B. pumilus 15.1, both plasmids of the strain (Garcia‐Ramon et al ., ) were removed. The conventional plasmid curing methods, involving culture at high temperature and/or in the presence of replication‐interfering chemical compounds, have been applied to many bacteria (Hara et al ., ; Ward and Ellar, ; Mahillon et al ., ; Sivropoulou et al ., ).…”