Theta replication of the lactococcal plasmid pWVO2.

pWVO2 is a 3.8 kb narrow-host-range plasmid from Lactococcus lactis ssp. cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli. Single-stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling-circle mechanism. The sequence of pWVO2 neither showed the structural organization typical for rolling-circle plasmids, nor were sequence similarities with known rolling-circle plasmids present. By 2-D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. This is the first proof for the existence of theta-replicating plasmids in lactococci. The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times. Further upstream is another 10bp direct repeat present in an A/T-rich sequence. This structural organization resembles that of several iteroncontaining theta-type plasmids from E. coli. Derivatives of pWVO2 were stably maintained in L. lactis and are good candidates for the development of stable food-grade cloning vectors for this organism.

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

Bacillus subtilis uses two-component signal transduction systems to sense intra- and extracellular stimuli to adapt to fluctuating environmental situations. Regulator aspartate phosphatases (Raps) have important roles in these processes, as they can dephosphorylate certain response-regulators, and are themselves subject to cell-density-controlled inhibition by secreted Phr (phosphate regulator) peptides. Eleven chromosomal genes encode this family of phosphatases, but in addition, certain strains contain endogenous plasmids with genes for homologous Rap-Phr systems. Plasmid pTA1060 encodes Rap60 and its antagonistic signalling molecule Phr60. Strikingly, expression of Rap60 in B. subtilis 168 strongly repressed the production of proteolytic enzymes. In fact, the transcription of the aprE gene, encoding a major extracellular protease, was shown to be decreased upon Rap60 expression, whereas this effect could be antagonized by the extracellular addition of synthetic Phr60 pentapeptide. Finally, transcription studies suggest that Rap60 dephosphorylates a component of the phosphorelay and is coupled to aprE transcription by the transition-state regulator AbrB. In conclusion, these data show that endogenous plasmids contain functional Rap-Phr systems and for the first time, that Rap-Phr systems can mediate cell-density controlled production of secreted proteases. This quorum-sensing mechanism might enable B. subtilis to suppress protease production under conditions of low cell densities when nutrients are still available in sufficient amounts.

The 172 kb prkA-addAB region from 83 degrees to 97 degrees of the Bacillus subtilis chromosome contains several dysfunctional genes, the glyB marker, many genes encoding transporter proteins, and the ubiquitous hit gene.

A 171812 bp nucleotide sequence between prkA and addAB (83 degrees to 97 degrees) on the genetic map of the Bacillus subtilis 168 chromosome was determined and analysed. An accurate physical/genetic map of this previously poorly described chromosomal region was constructed. One hundred and seventy open reading frames (ORFs) were identified on the DNA fragment. These include the previously described genes cspB, glpPFKD, spoVR, phoAIV, papQ, citRA, sspB, prsA, hpr, pbpF, hemEHY, aprE, comK and addAB. ORF yhaF in this region corresponds to the glyB marker. Among the striking features of this region are: an abundance of genes encoding (putative) transporter proteins, several dysfunctional genes, the ubiquitous hit gene, and five multidrug-resistance-like genes. These analyses have also revealed the existence of numerous paralogues of ORFs in this region: about two-thirds of the putative genes seem to have at least one paralogue in the B. subtilis genome.