Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance

Abstract

In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPGlOOO (5.0kb) which carries both a selectable marker (chloramphenicol resistance from Sfuphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonasputidh expressed from a cloned promoter of Bacillus phage SP02) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPGlOOO insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

Conformational Change-Induced Repeat Domain Expansion Regulates Rap Phosphatase Quorum-Sensing Signal Receptors

“…Rap proteins and their inhibitory oligopeptides, called Phr peptides, have been most extensively studied in B. subtilis , which encodes 11 Rap proteins on its chromosome and another five on plasmids [16]–[18]. The founding members of the Rap family, RapA and RapB, were shown to be response regulator aspartate phosphatases (Rap) [19].…”

Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases

“…In B. subtilis alone there are 11 homologous Rap proteins encoded on the chromosome, and there are at least four additional Rap proteins encoded on B. subtilis plasmids [10],[11].…”

Structural Basis of Response Regulator Inhibition by a Bacterial Anti-Activator Protein

“…B. subtilis encodes 11 Rap proteins on its chromosome and another five on plasmids [10]–[12]. Rap proteins were named after the founding members of the family, which were shown to be r esponse regulator a spartate p hosphatases [13].…”