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.


Introduction
DDBJ/ENA/GenBank under the accession LKDJ02000000. Newly assembled genome 127 sequences were annotated using the pipeline available at National Center for 128 Biotechnology Information (NCBI). 129 Suspected plasmid sequences were analysed and assembled into putative replicons 130 in silico, and the gaps inferred in the sequences were closed by PCR conducted on 131 plasmid extracts with the aid of specifically designed primer pairs (S1 Table). The DNA  Table 1). The ORFs predicted by the NCBI pipeline in these plasmids were compared 136 with the protein sequences deposited in the GenBank database using BlastP [31]. The   Salto et al. [30], and subsequently assigned to the corresponding 200 Acinetobacter AR3G groups following the criterium described by these authors.    (Table 1; GenBank accession numbers are provided in S1 Table).

258
Conventional plasmid extraction combined with S1 nuclease treatment and agarose gel 259 electrophoresis analysis of the digested material (S1 Fig) indicated  other Acinetobacter species (Fig 1, S2 Table).   Table) reported to be involved in defense against bacteriophages [57], was 333 detected in pAbe229-114 displaying higher than 92% identity with a homologous  Table). Remarkably, a region of this locus of around 1 kbp encompassing the central 336 part of pglX (S4 Table) is not conserved between these plasmids. pglX encodes a protein 337 containing an adenine-specific DNA methyltransferase motif, and variability in this 338 gene has been documented and linked to a possible phase variation playing regulatory 339 functions [57].

340
Of note, the overall observations above uncovered extensive regions of identity 341 between the plasmid backbones of A. bereziniae pAbe229-114 and A. venetianus VE-342 C3 pAV3 (Fig 1, S4 Table). Comparative nucleotide sequence analyses between these 343 two plasmids indicated in fact that they share around 50% equivalent sequences, 344 including the replication and stability regions and part of the adaptive region. A further 345 example is represented by region 4, in which these two plasmids share higher than 99% nucleotide identity (Fig 1, S4  probably acquired by HGT. Besides the similarities between pAbe229-114 and other 350 plasmids summarized above, unique regions were also identified in this plasmid 351 including the conjugal transfer region as well as some segments involving genes 352 contiguous to IS elements corresponding to the adaptive regions (Fig 1, S2 Table).  Tn6637 (Table 2). We also identified in pAbe229-114 an incomplete Tn6018 390 transposon carrying cadmium and zinc resistance genes (Fig 1). Notably, the Tn6018  Table 1 for details). The putative oriT located within the conjugal/transfer verify the structure of pAbe229-114 (S1 Table) are also indicated.  Fig 2A), a region showing 100% nucleotide  Table) suggests that they 447 shared a common metabolic pathway.  , Fig 2A, and S4 Table). In 452 turn, the stability region including a TA system was homologous to an equivalent region 453 from a plasmid found in A. baumannii AR_0052 (region 3, Fig 2A, and S4 Table).   Table).  Fig 2B). The whole replication region, including repB 470 and the iterons, shows 79% nucleotide identity with a homologous segment of A.

471
venetianus VE-C3 pAV1 (region 1, Fig 2B, and S4 Table). The stability region carries 472 genes for a SplT/SplA TA system [64] and a toxin of the Doc-type. splT/splA homologous genes were identified also in pAV1 (region 3) and in pD36-4 of A. 474 baumannii D36 (partial region 4, Fig 2B, and S4 Table). The latter plasmid also shares 475 with pAbe229-9 a downstream segment (region 4, Fig 2B), which includes two ORFs of 476 unknown function, and a doc gene encoding an orphan toxin of the Phd-Doc system (S2 477   Table). Concerning the transfer region, a 2,637 bp-homologous segment including a 478 mobA gene was found in A. baumannii pMMD (region 6, Fig 2B, and S4 Table).

525
pAbe229-1, with only 1,329 bp ( Fig 4B and S1 Fig), represents the smallest plasmid 526 reported so far in an Acinetobacter genus member. It harbors 2 predicted ORFs with no 527 homology in databases, but BlastN homology searches against the nucleotide GenBank 528 database revealed significant homology to a 465 bp region of unknown function found 529 in plasmid pM131-11 from an Acinetobacter sp. isolate (Fig 4B, S4 Table).    in sequence and in length (Table 3). Thus, while ten of the above 12 XerC/D sites show 571 cr of an usual length of 6 nucleotides (cr6) displaying high sequence variability, one site (XerC/D_8, located in pAbe229-9) contained a cr of 5 nucleotides while other 573 (XerC/D_5, located in pAbe229-15) a cr of 7 nucleotides in length (Table 3).

574
The above XerC/D sites were bracketing discrete regions in the corresponding 575 HPC229 plasmid sequences (Figs 1 and 2). BlastN-homology searches using as query 576 each of these regions indicated high levels of identity with similar regions carried by 577 different Acinetobacter plasmids for some of them, including region 1 and 2 of 578 pAbe229-15 (Fig 2A, S4 Table), and region 3 of pAbe229-9 (Fig 2B, S4 Table). It is 579 worth noting also that a region of around 1 kbp in pAbe229-114, that includes the 580 XerC/D_11 site and a downstream higA2/higB2 TA system (region 9, Fig 1) showed 581 high sequence identity with a homologous segment found in pOXA58-AP_882 of A. 582 pittii AP_882 (S4 Table). This TA system is bracketed by a XerC/D pair in the latter

Analysis of plasmid idiosyncratic sequences (plasmid markers) in A. bereziniae
604 genomes 605 To obtain further clues on the diversity of the plasmids housed by the A. bereziniae 606 population, we conducted a comparative genomic analysis of genome sequences 607 obtained from all strains assigned to this species available at GenBank (NCBI) database.

608
For this purpose, we retrieved and analysed the available genomic sequence data 609 corresponding to all seven strains classified as A. bereziniae in the database (S6 Table, 610 top seven strains). We also conducted a BlastN search among the currently available 611 Acinetobacter sp. genome sequences (GenBank-WGS database) using as query the A. 612 bereziniae type strain CIP 70.12 rpoB gene sequence (GenBank accession number species. Two strains showing rpoB sequences identities higher than 99% with CIP 70.12 615 rpoB emerged from this analysis, Acinetobacter sp. Ag2 and Acinetobacter sp. WC-743 616 (S6 Table), which prompted us to further delimitate their species assignations. We 617 therefore calculated the percentage of average nucleotide identities (ANI) of these two 618 strains when compared to the A. bereziniae type strain CIP 70.12 [22]. We also included 619 in these comparisons other 6 strains previously assigned to A. bereziniae by other 620 authors (S6 Table). As seen in this Table,  showed ANI values of 83 and 76%, respectively (S6 Table), thus validating the species 626 assignations made above.  Table). A notable case was represented by A. bereziniae CHI-40-1, in which 7 Rep candidates were found distributed among 7 different AR3G groups (S3 Table). This 639 supports the proposed existence of several different plasmids in this strain [28]. 640 The remaining Rep candidate found in an A. bereziniae strain, KCTC 23199 641 (GenBank accession number WP_010591570.1; S3 Table), showed no significant Acinetobacter plasmids (S3 Table) but also enterobacterial plasmids including a  Table), which was assigned to AR3G14 [30]. However, 653 these proteins contain a RepC (pfam06504) rather than a Rep_3 (pfam01051) 654 superfamily domain, and were thus assigned to a novel group tentatively denominated 655 ARCG1 (S3 Table). RepC superfamily members are found among IncQ-like group

Comparative sequence analyses between HPC229 plasmids and other A. bereziniae
674 genomes 675 We next analyzed whether the plasmids identified here in HPC229 share significant 676 regions of identity with sequences of other A. bereziniae genomes (S6 Table, Table; results obtained for sequences present in A. bereziniae genomes are shown in the 683 lower part). The presence of this genomic island among these genomes suggests that it 684 may have been acquired by HGT, although its presence in an A. bereziniae ancestor 685 followed by differential losses in some lineages cannot be ruled out at this stage.

686
Other regions that deserve consideration in HPC229 plasmids are those encoding 687 oxidative stress resistance mechanisms, including a region of Abe5 and the whole Abe10 region (S1A Fig). The corresponding genes were also identified in the CIP 70.12 689 and KCTC 23199 strains (S4 Table). Also, those regions encoding heavy metal 690 (cadmium, cobalt, nickel and zinc) detoxification systems (region Abe6, S1A Fig) were 691 also identified in strains NIPH 3, WC-743 and CHI-40-1 (S4 Table). It is worth noting 692 that similar systems bearing strong adaptive significance have been described in  Abe7 and Abe8 regions of pAbe229-114 (S1A Fig, S4 Table) encoding the type 1-698 BREX system (see above) were also found in sequences derived from the NIPH 3 and  Table) share between them a significant number of 708 DNA regions, most of them likely encoding adaptive functions. Still, pAbe229-114 709 carries a unique set of replication, stability and transfer genes as compared to other A. 710 bereziniae strains (S4 Table). This suggests a high genetic plasticity for this species and  Table). This plasmid shares transfer and replication genes with 717 507_ABAU (region Abe1, S1B Fig), a creatine degradation pathway (region Abe2, S1B  Table). This segment in CHI-40-1 also encodes a Rep_3 722 member (CDEL01000195.1; S4 Table), thus suggesting that this contig corresponds to a  Table). This region, also present in several A.  Table). Finally, pAbe229-1 homologous sequences 740 could not be detected among A. bereziniae genomes other than HPC229.  Table). Furthermore, the modules 747 conforming the HPC229 plasmid backbone are not widely represented in other members 748 of this species, suggesting they were partially acquired from A. non-bereziniae species 749 by HGT.
A total of 55 putative XerC/D sites were identified by this procedure in the eight A. 763 bereziniae strains analyzed (S7 Table). Expectedly, only one XerC/D site was identified 764 in strain XH901 lacking plasmid sequences (see above), probably reflecting the single 765 chromosomal dif site. In most of the other strains the number of XerC/D sites varied 766 between 2 and 7, with the exception of strains HPC229 (10 sites, Table 3) and CHI-40-1 767 (31 sites; S7 Table). This is in line with the above observations indicating that the latter  Table, see also Table 3). This consensus resulted very similar to that reported  pAbe229-4 and pAbe229-1 plasmids lack replication initiator protein genes. However, 799 while the pAbe229-4 plasmid may use a ColE1-type replicon as suggested by sequence 800 analysis, the replication mechanism of pAbe229-1 remains obscure.

801
The overall analysis suggest that A. bereziniae HPC229 plasmids could be regarded 802 as chimeras of diverse origins. An outstanding example is pAbe229-114, which shares 803 extensive backbone sequence similarity to pAV3 from A. venetianus VE-C3 isolated 804 from polluted waters [78]. This correlation was not totally unexpected, providing that A. 805 bereziniae is also isolated from wastewater products of human activities [25].