XerD unloads bacterial SMC complexes at the replication terminus.

Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.

Evolution of Acinetobacter baumannii plasmids carrying the oxa58 carbapenemase resistance gene via plasmid fusion, IS26-mediated events and dif module shuffling.

Acinetobacter baumannii RepAci1-RepAci10 plasmids pA388 from a global clone 1 (GC1) isolate from Greece, and pACICU1 and variant pACICU1b from an Italian GC2 isolate were found to share a common ancestor. The ancestor formed via recombination between pdif sites in the widely-distributed RepAci1 plasmid pA1-1 and in a RepAci10 plasmid carrying the oxa58 carbapenem-resistance gene in a dif module. Each plasmid includes copies of IS26 and multiple dif modules surrounded by 28 bp pdif sites resembling the chromosomal dif site, including one carrying the oxa58 gene. Plasmid sequences were compared to identify factors driving their evolution and divergence. IS26-mediated events, recombination between pdif sites and homologous recombination have all contributed. A translocatable unit that includes oxa58, generated by an IS26-mediated adjacent deletion, had been re-inserted by IS26 adjacent to an IS26 in pACICU1b to create the oxa58 gene duplication previously found in pACICU1. The oxa58 duplication has been lost from pACICU1b and the Tn6020 variant carrying the aphA1 (kanamycin, neomycin resistance) gene in pA388 has been lost from pACICU1/1b via recombination between directly-oriented IS26 copies. Two dif modules located between directly-oriented pdif sites in pA388 have been lost from pACICU1/1b and the product of this and other deletion events as well as inversion of dif modules located between inversely-oriented pdif sites were detected experimentally in pA388 DNA by PCR. Also, the new junctions were detected in a minority of reads in pA388 long-read sequence data. Inversion and deletion were only detected when the spacers in the pdif sites were identical and equivalent events involving mismatched spacers were not detected.

Mobilization of pdif modules in Acinetobacter: A novel mechanism for antibiotic resistance gene shuffling?

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.

Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate.

Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired ß-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of blaOXA-58 class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a blaAmpC-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.

A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution.

Unlike eukaryotes and archaea, which have multiple replication origins on their chromosomes, bacterial chromosomes usually contain a single replication origin.1 Here, we discovered a dicentric bacterial chromosome with two replication origins, which has resulted from the fusion of the circular and linear chromosomes in Agrobacterium tumefaciens. The fused chromosome is well tolerated, stably maintained, and retains similar subcellular organization and genome-wide DNA interactions found for the bipartite chromosomes. Strikingly, the two replication origins and their partitioning systems are both functional and necessary for cell survival. Finally, we discovered that the site-specific recombinases XerC and XerD2 are essential in cells harboring the fused chromosome but not in cells with bipartite chromosomes. Analysis of actively dividing cells suggests a model in which XerC/D are required to recombine the sister fusion chromosomes when the two centromeres on the same chromosome are segregated to opposite cell poles. Thus, faithful segregation of dicentric chromosomes in bacteria can occur because of site-specific recombination between the sister chromatids during chromosome partitioning. Our study provides a natural comparative platform to examine a bacterial chromosome with multiple origins and a possible explanation for the fundamental difference in bacterial genome architecture relative to eukaryotes and archaea.1.

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species.

Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D ß-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most ß-lactam antibiotics in the fight against these resistant isolates.

Mechanistic insights into filamentous phage integration in Vibrio cholerae.

Vibrio cholerae, the etiological agent of acute diarrhoeal disease cholera, harbors large numbers of lysogenic filamentous phages, contribute significantly to the host pathogenesis and provide fitness factors to the pathogen that help the bacterium to survive in natural environment. Most of the vibriophage genomes are not equipped with integrase and thus exploit two host-encoded tyrosine recombinases, XerC and XerD, for lysogenic conversion. Integration is site-specific and it occurs at dimer resolution site (dif) of either one or both chromosomes of V. cholerae. Each dif sequence contains two recombinase-binding sequences flanking a central region. The integration follows a sequential strand exchanges between dif and attP sites within a DNA-protein complex consisting of one pair of each recombinase and two DNA fragments. During entire process of recombination, both the DNA components and recombinases of the synaptic complex keep transiently interconnected. Within the context of synaptic complex, both of the actuated enzymes mediate cleavage of phosphodiester bonds. First cleavage generates a phosphotyrosyl-linked recombinase-DNA complex at the recombinase binding sequence and free 5'-hydroxyl end at the first base of the central region. Following the cleavage, the exposed bases with 5'-hydroxyl ends of the central region of dif and attP sites melt from their complementary strands and react with the recombinase-DNA phosphotyrosyl linkage of their recombining partner. Subsequent ligation between dif and attP strands requires complementary base pair interactions at the site of phosphodiester bond formation. Integration mechanism is mostly influenced by the compatibility of dif and attP sequences. dif sites are highly conserved across bacterial phyla. Different phage genomes have different attP sequences; therefore they rely on different mechanisms for integration. Here, I review our current understanding of integration mechanisms used by the vibriophages.