The IntXO-PSL Recombination System Is a Key Component of the Second Maintenance System for Bacillus anthracis Plasmid pXO1

Abstract

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...

Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases

“…To resolve this problem, we brought into use two additional T-SSR systems: Flp- FRT and IntXO- PSL . The functionality of both systems was demonstrated previously [ 6 , 8 ]. These enabled the inactivation of four additional proteases that were identified by proteomic analysis of the BH460 and BH480 culture supernatants.…”

“…A new IntXO- PSL recombinase system based on the recently identified IntXO recombinase of B . anthracis [ 8 ] was created for use in a manner analogous to that of the Flp- FRT system described above. The plasmid pSCP is a shuttle plasmid with all the characteristics of pSCF, except that it contains repeats of the 37-bp PSL sequence instead of the FRT sequence.…”

“…Black triangles represent target sequences loxP , FRT or PSL for Cre, Flp or IntXO T-SSR, respectively. The detailed description of the application of Cre- loxP , Flp- FRT and IntXO- PSL were described previously [ 6 – 8 ] and in the Results section. (B) PCR verification of deletions in cysP1 , nprC , vpR and s41 protease genes with corresponding primer pairs 1995seqF/R, 2183 seqF/R, 4584 seqF/R and 5414 seqF/R.…”

“…Accordingly, in the work continued here, we have created three T-SSR systems for performing genetic modifications in Bacillus anthracis : the Escherichia coli Cre- loxP and Saccharomyces cerevisiae Flp- FRT systems [ 5 – 7 ] and a newly developed B . anthracis IntXO- PSL system [ 8 ]. We previously used the Cre- loxP system to create the BH460 strain [ 9 ] having the following phenotypic characteristics: Spo0A-, pXO1-, pXO2-, NprB-, TasA-, Cam-, InhA1-, InhA2-, MmpZ-.…”

Avaliação de toxicidade de Bacillus thuringiensis em diferentes condições de armazenamento

“…Já a toxicidade, após o período de manutenção, manteve uma constante de efetividade ao longo dos meses com leves alterações, apresentando uma taxa de mortalidade de 90,45% e 91,03% para Btk e Btb, respectivamente (Tabela 4), e consistindo em um resultado excelente a partir da metodologia utilizada. O gerenciamento da Bt em aglomerações laboratoriais vem afirmando a subsistência, a permanência e a autenticidade de estirpes no decorrer de períodos, usando vários métodos de manutenção e, principalmente, disponibilizando-as para fins experimentais, didáticos, industriais ou estudos comparativos (POMERANTSEV et al, 2016).…”