GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes

Carbapenem resistance and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter, but many plasmid types in this genus have not been well-characterised. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains blaNDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognised by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1086 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin-antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus.

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

ABSTRACTMany mobile elements take advantage of the highly-conserved chromosome dimer resolution system of bacteria, Xer. They participate in the transmission of antibiotic resistance and pathogenicity determinants. In particular, the toxin-linked cryptic satellite phage (TLCΦ) plays an essential role in the continuous emergence of new toxigenic clones of the Vibrio cholerae strain at the origin of the ongoing 7th cholera pandemic. The Xer machinery is composed of two chromosomally-encoded tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific 28 base pair site of bacterial chromosomes, dif. The activity of XerD depends on a direct contact with a cell division protein, FtsK, which spatially and temporally constrains the process. TLCΦ encodes for a XerD-activation factor (XafT), which drives the integration of the phage into the dif site of the primary chromosome of V. cholerae independently of FtsK. However, XerD does not bind to the attachment site (attP) of TLCΦ, which raised questions on the integration process. Here, we compared the integration efficiency of thousands of synthetic mini-TLCΦ plasmids harbouring different attP sites and assessed their stability in vivo. In addition, we compared the efficiency with which XafT and the XerD activation domain of FtsK drive recombination reactions in vitro. Taken together, our results suggest that XafT promotes the formation of synaptic complexes between canonical Xer recombination sites and imperfect sites.

Diversity and evolution of pigment types and the phycobilisome rod gene region of marine Synechococcus cyanobacteria

Synechococcus picocyanobacteria are ubiquitous and abundant photosynthetic organisms in the marine environment and contribute for an estimated 16% of the ocean net primary productivity. Their light-harvesting complexes, called phycobilisomes (PBS), are composed of a conserved allophycocyanin core from which radiates six to eight rods with variable phycobiliprotein and chromophore content. This variability allows Synechococcus to optimally exploit the wide variety of spectral niches existing in marine ecosystems. Seven distinct pigment types or subtypes have been identified so far in this taxon, based on the phycobiliprotein composition and/or the proportion of the different chromophores in PBS rods. Most genes involved in their biosynthesis and regulation are located in a dedicated genomic region called the PBS rod region. Here, we examined the variability of gene sequences and organization of this genomic region in a large set of sequenced isolates and natural populations of Synechococcus representative of all known pigment types. All regions start with a tRNA-PheGAA and some possess mobile elements including tyrosine recombinases, suggesting that their genomic plasticity relies on a tycheposon-like mechanism. Comparison of the phylogenies obtained for PBS and core genes revealed that the evolutionary history of PBS rod genes differs from the rest of the genome and is characterized by the co-existence of different alleles and frequent allelic exchange. We propose a scenario for the evolution of the different pigment types and highlight the importance of population-scale mechanisms in maintaining a wide diversity of pigment types in different Synechococcus lineages despite multiple speciation events.

Molecular and Functional Characterization of MobK Protein—A Novel-Type Relaxase Involved in Mobilization for Conjugational Transfer of Klebsiella pneumoniae Plasmid pIGRK

Conjugation, besides transformation and transduction, is one of the main mechanisms of horizontal transmission of genetic information among bacteria. Conjugational transfer, due to its essential role in shaping bacterial genomes and spreading of antibiotics resistance genes, has been widely studied for more than 70 years. However, new and intriguing facts concerning the molecular basis of this process are still being revealed. Most recently, a novel family of conjugative relaxases (Mob proteins) was distinguished. The characteristic feature of these proteins is that they are not related to any of Mobs described so far. Instead of this, they share significant similarity to tyrosine recombinases. In this study MobK—a tyrosine recombinase-like Mob protein, encoded by pIGRK cryptic plasmid from the Klebsiella pneumoniae clinical strain, was characterized. This study revealed that MobK is a site-specific nuclease and its relaxase activity is dependent on both a conserved catalytic tyrosine residue (Y179) that is characteristic of tyrosine recombinases and the presence of Mg2+ divalent cations. The pIGRK minimal origin of transfer sequence (oriT) was also characterized. This is one of the first reports presenting tyrosine recombinase-like conjugative relaxase protein. It also demonstrates that MobK is a convenient model for studying this new protein family.

Archaeal tyrosine recombinases

ABSTRACT The integration of mobile genetic elements into their host chromosome influences the immediate fate of cellular organisms and gradually shapes their evolution. Site-specific recombinases catalyzing this integration have been extensively characterized both in bacteria and eukarya. More recently, a number of reports provided the in-depth characterization of archaeal tyrosine recombinases and highlighted new particular features not observed in the other two domains. In addition to being active in extreme environments, archaeal integrases catalyze reactions beyond site-specific recombination. Some of these integrases can catalyze low-sequence specificity recombination reactions with the same outcome as homologous recombination events generating deep rearrangements of their host genome. A large proportion of archaeal integrases are termed suicidal due to the presence of a specific recombination target within their own gene. The paradoxical maintenance of integrases that disrupt their gene upon integration implies novel mechanisms for their evolution. In this review, we assess the diversity of the archaeal tyrosine recombinases using a phylogenomic analysis based on an exhaustive similarity network. We outline the biochemical, ecological and evolutionary properties of these enzymes in the context of the families we identified and emphasize similarities and differences between archaeal recombinases and their bacterial and eukaryal counterparts.

The TLCΦ satellite phage harbors a Xer recombination activation factor

The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site,dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage ofVibrio cholerae, TLCΦ, which integrates into and excises from thedifsite of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens.

The plasmid diversity of Acinetobacter bereziniae HPC229 provides clues on the ability of the species to thrive on both clinical and environmental habitats

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two radically different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Sequence analysis allows functional annotation of tyrosine recombinases in prokaryotic genomes

Background: Tyrosine recombinases perform site-specific genetic recombination in bacteria and archaea. They safeguard genome integrity by resolving chromosome multimers, as well as mobilize transposons, phages and integrons, driving dissemination of genetic traits and antibiotic resistance. Despite their abundance and genetic impact, tyrosine recombinase diversity and evolution has not been thoroughly characterized, which greatly hampers their functional classification. Results: Here, we conducted a comprehensive search and comparative analysis of diverse tyrosine recombinases from bacterial, archaeal and phage genomes. We characterized their major phylogenetic groups and show that recombinases of integrons and insertion sequences are closely related to the chromosomal Xer proteins, while integrases of integrative and conjugative elements (ICEs) and phages are more distant. We find that proteins in distinct phylogenetic groups share specific structural features and have characteristic taxonomic distribution. We further trace tyrosine recombinase evolution and propose that phage and ICE integrases originated by acquisition of an N-terminal arm-binding domain. Based on this phylogeny, we classify numerous known ICEs and predict new ones. Conclusions: This work provides a new resource for comparative analysis and functional annotation of tyrosine recombinases. We reconstitute protein evolution and show that adaptation for a role in gene transfer involved acquisition of a specific protein domain, which allows precise regulation of excision and integration.

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT

ABSTRACT CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and amino-terminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation. IMPORTANCE CTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.