Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from Gram-positive bacteria

Abstract

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain plasmids, several industrial strains and natural soil isolates do contain rolling-circle replicating (RCR) plasmids. So far, knowledge about these plasmids was mainly limited to: (i) a classification into seven groups, based on size and restriction patterns; and (ii) DNA sequences of the replication region of a limited number of them. To increase the knowledge, also with respect to other functions specified by these plasmids, we have determined the complete DNA sequence of four plasmids, representing different groups, and performed computer-assisted and experimental analyses on the possible function of their genes. The plasmids analyzed are pTA1015 (5.8 kbp), pTA1040 (7.8 kbp), pTA1050 (8.4 kbp), and pTA1060 (8.7 kbp). These plasmids have a structural organization similar to most other known RCR plasmids. They contain highly related replication functions, both for leading and lagging strand synthesis. pTA1015 and pTA1060 contain a mobilization gene enabling their conjugative transfer. Strikingly, in addition to the conserved replication modules, these plasmids contain unique module(s) with genes which are not present on known RCR plasmids of other Gram-positive bacteria. Examples are genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases. The latter are likely to be involved in the regulation of post-exponential phase processes. The presence of these modules on plasmids may reflect an adaptation to the special conditions to which the host cells were exposed.

A plasmid-borne Rap-Phr system of Bacillus subtilis can mediate cell-density controlled production of extracellular proteases

“…2), the export and reimport of this peptide from and back into the cell remained unproven. To investigate whether the Phr60 peptide could be imported and antagonize Rap60 activity, the deduced active Phr60 pentapeptide (SRNAT; see Meijer et al, 1998) was synthetically produced. Next, transcription of aprE-lacZ was monitored in B. subtilis ALR (xylose-inducible Rap60 expression) grown in TY medium with xylose to which 1, 5 or 10 mM Phr60 pentapeptide was added.…”

“…While the Rap proteins remain in the cytoplasm, Phr peptides contain an amino-terminal signal peptide and have the potential to be exported as propeptides, most likely via the Sec pathway (Tjalsma et al, 2000). Further extracellular processing results in active Phr pentapeptides with a weakly conserved XRXXT sequence (Meijer et al, 1998;Perego, 1997). After reimport via the oligopeptide permease (Opp) system, Phr peptides specifically inhibit the activity of their cognate Rap protein (Perego, 1997(Perego, , 1998Meijer et al, 1998).…”

“…Further extracellular processing results in active Phr pentapeptides with a weakly conserved XRXXT sequence (Meijer et al, 1998;Perego, 1997). After reimport via the oligopeptide permease (Opp) system, Phr peptides specifically inhibit the activity of their cognate Rap protein (Perego, 1997(Perego, , 1998Meijer et al, 1998). However, four of the eleven known rap genes are not followed by functional phr genes, suggesting that these are not subject to quorum sensing.…”

“…On: Sat, 12 May 2018 17:04:33 that can be regulated by this class of extra-chromosomally encoded Rap-Phr systems (Uozumi et al, 1980;Meijer et al, 1998).…”

Construction of novel shuttle expression vectors for gene expression in <i>Bacillus subtilis</i> and <i>Bacillus pumilus</i>

“…The sequence analysis demonstrated that pSU01 possessed the replication initiation gene (rep) and its cognate target site single-strand origin (SSO) and doublestrand origin (DSO), stress response-like protein gene (hsp), mobilization protein gene (mob), putative DNAbinding protein gene (ORF1), and putative transmembrane protein gene (ORF2) (Meijer et al, 1998). The nucleotide sequence of replication elements for pSU01 is highly identical with pBAA1 (99%) and pLS30 (100%) from B. pumilus and B. subtilis (Devine et al, 1989;Sakaya et al, 2006), respectively, which belong to the rolling-circle replicating plasmid that has been characterized by the synthesis of single-stranded DNA intermediates (Meijer et al, 1998;Sakaya et al, 2006). In addition, pSU01 might have an unexpectedly high resistance to segregational loss because the homologous plasmid pLS30 was proven to be highly resistant to segregational loss (Sakaya et al, 2006).…”