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Bacillus anthracis pXO1 minireplicon (MR) plasmid consisting of open reading frames (ORFs) GBAA_pXO1_0020 toGBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions. Two genetic tools developed for DNA manipulation in B. anthracis (Cre-loxP and Flp-FRT systems) were used to identify pXO1 regions important for plasmid stability. We localized a large segment of pXO1 that enables stable plasmid maintenance during vegetative growth. Further genetic analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Analysis of conserved domains in the corresponding proteins indicated that only AmsP (activator of maintenance system of pXO1) is predicted to bind DNA, due to its strong helix-turn-helix domain. Two conserved domains were found in the

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We previously identified three noncontiguous regions on Bacillus anthracis plasmid pXO1 that comprise a system for accurate plasmid partitioning and maintenance. However, deletion of these regions did not decrease retention of certain shortened pXO1 plasmids during vegetative growth. Using two genetic tools developed for DNA manipulation in B. anthracis (the Cre-loxP and Flp-FRT systems), we found two other noncontiguous pXO1 regions that together are sufficient for plasmid stability. This second pXO1 maintenance system includes the tubZ and tubR genes, characteristic of a type III partitioning system, and the IntXO recombinase gene (GBAA_RS29165), encoding a tyrosine recombinase, along with its adjacent 37-bp perfect stem-loop (PSL) target. Insertion of either the tubZ and tubR genes or the IntXO-PSL system into an unstable mini-pXO1 plasmid did not restore plasmid stability. The need for the two components of the second pXO1 maintenance system follows from the sequential roles of IntXO-PSL in generating monomeric circular daughter pXO1 molecules (thereby presumably preventing dimer catastrophe) and of TubZ/TubR in partitioning the monomers during cell division. We show that the IntXO recombinase deletes DNA regions located between two PSL sites in a manner similar to the actions of the Cre-loxP and Flp-FRT systems. IMPORTANCETyrosine recombinases catalyze cutting and joining reactions between short specific DNA sequences. Three types of reactions occur: integration and excision of DNA segments, inversion of DNA segments, and separation of monomeric forms from replicating circular DNA molecules. Here we show that the newly discovered site-specific IntXO-PSL recombinase system that contributes to the maintenance of the B. anthracis plasmid pXO1 can be used for genome engineering in a manner similar to that of the Cre-loxP or Flp-FRT system. T he large low-copy-number pXO1 plasmid (181,677 bp) of Bacillus anthracis encodes the anthrax toxin proteins and other virulence-related factors. A pXO1 minireplicon plasmid (pMR) comprised of two essential open reading frames (ORFs) (GBAA_RS28535 and GBAA_RS28545) (Fig. 1A) replicates but is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (carrying 217 ORFs) is extremely stable under the same growth conditions (1). (Table S1 in the supplemental material lists all genes and proteins discussed in this work.) Recently we found that retention of pMR can be stabilized by insertion of several noncontiguous pXO1 regions containing three genes which work cooperatively to achieve plasmid maintenance: amsP (GBAA_RS28725), minP (GBAA_RS28775), and sojP (GBAA_ RS28785) (2). The minP and sojP genes encode proteins belonging to the ParA/MinD family described by Lutkenhaus (3). MinD is involved in spatial regulation of the cytokinetic Z ring, and ParA proteins are involved in chromosome and plasmid segregation (3).The amsP-, minP-, and sojP-encoded proteins, comprising the plasmid maintenance system (maintenance system I [MSI]) identified in our pr...