A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene

Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3’-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.


Introduction
Plasmids are an essential factor driving bacterial evolution. Plasmid-encoded functions such as virulence, resistance, metabolism and/or other advantageous functions can promote bacterial fitness [1]. Pseudomonas aeruginosa is an important opportunistic pathogen responsible primarily for hospital-and cystic fibrosis-associated infections. Plasmids have been shown to confer advantageous traits upon P. aeruginosa clinical isolates [2].
Certain plasmids can insert into chromosomes site-specifically [3], contributing to chromosomal mosaicism together with other integrative mobile elements such as pathogenicity islands, integrative and conjugative elements, and prophages in bacteria [1]. Many of the integrative elements have been found to be incorporated into tRNA gene sites of the host chromosome. Plasmid pKLK106 and pKLC102 that coexist as free plasmids and genomic islands are among the very few cases reported of integrative plasmids in P. aeruginosa [4]. Plasmid pKLC102 that can recombine within the 3 0 end of the tRNA Lys gene has been shown to be phage-related and carry determinants for host tropism and virulence which may allow evolution of the chromosome and genomic island to be traced [4].
Antibiotic resistance is one of the critical issues threatening public health. Plasmids are the most common carriers of antibiotic resistance genes. It has been commonly seen that antibiotic resistance in P. aeruginosa was correlated with the presence of IncP group conjugative plasmids [5,6]. Moreover, many resistance plasmids are being detected in P. aeruginosa [2]. Many of the resistance genes are found embedded in or associated with mobile elements such as transposons, integrons, and IS elements [7][8][9][10][11][12][13][14][15]. In addition, apart from the genomic island-encoded virulence [16], P. aeruginosa plasmid-mediated pathogenicity was revealed [17]. Plasmids in P. aeruginosa also are involved in other fitness traits [18]. However, to our knowledge, no plasmid has been seen in completely sequenced P. aeruginosa genomes to date (http://www.ncbi.nlm. nih.gov/genome), and only a limited number of P. aeruginosa plasmids have been fully sequenced and analysed.
Here we sequenced two novel, native plasmids found in a P. aeruginosa clinical isolate. One is a site-specific integrative plasmid with the size of 11 kb that may drive the plasticity of the genome and has potential to serve as a genetic tool, while another with the size of 26 kb carries mobile genetic elements and an aminoglycoside-resistant gene that may enhance the fitness of the strain. The two plasmids may confer advantageous traits upon and promote evolution of this clinical isolate.

Ethics and consent
Because this study was an observational study by using human sputum samples for the ex vivo experiments and the sampling of patient's sputum was a routine work in hospital treatment, verbal informed consent was obtained from all volunteers. The bacteria samples and data sheets were anonymized. This study protocol including verbal informed consent procedure was approved by the ethics committee of the School of Life Sciences & Biotechnology, Shanghai Jiaotong University, China.
Glimmer 3 [19], GeneMark [20], and the translation initiation sites of certain ORFs were modified with Prodigal [21]. ORFs were annotated by BLAST against NCBI protein database (see S1 Table and S2 Table). Sequences for comparison were retrieved from NCBI. Phylogenetic tree was constructed by MEGA 5.0 [22] with neighbour-joining method and 1,000 bootstrap replicates, based on MUSCLE [23] alignment.

Integration of pHS87b
Homologous attachment sites were detected by Blastn. Presence of genomic island (GI) at the tRNA Thr site was examined by tRIP-PCR (tRNA site interrogation for pathogenicity islands, prophages and other GIs) [24] using primers thr_U and thr_D (S3 Table). Amplification of 16S rRNA gene was performed as a control using universal primers. Attachment site on pHS87b (attP) was amplified using primers attP_U and attP_D (S3 Table). Junctions and orientation of the GI form of pHS87b were examined using the combinations of one of the chromosomal primers (thr_U or thr_D) and one of the plasmid primers (attP_U or attp_D). The GI occupying the tRNA Thr site was amplified via long PCR using thr_U and thr_D. All amplicons were sequenced using Sanger method except the long PCR product which was digested by BamHI, BglII, ClaI or SmaI.
In addition, the integration of pHS87b was further confirmed by Southern blotting assays. Probes specific to the chromosome and the pHS87b plasmid were amplified by PCR, with primers pchG-SBF/R targeting the pchG gene on the chromosome and int-SBF/R targeting the pHS87b integrase gene orf2 (S3 Table), respectively. Southern blotting was performed by using DIG Hybridization Detection Kit I (Mylab Co., Beijing, China), according to its user manual.
Quantitative PCR for examination of pHS87b integration frequency Quantitative real-time PCRs were performed to quantify the abundance of the empty tRNA Thr site and the circular as well as integrated forms of pHS87b with ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) and HieffTM qPCR SYBR1 Green Master Mix (Yeasen). 2 ng of the total DNA of P. aeruginosa HS87 was used as template in 20 μl reaction volume. The empty site was detected by primers attB-qF and attB-qR targeting the unoccupied chromosomal attachment site (attB) (S3 Table). The circular and integrated forms of pHS87b were detected by primers attP-qF and attP-qR targeting the plasmid attachment site (attP) (S3 Table), and primers attB-qR and attP-qR targeting the right direct repeat of the integrated pHS87b (attR) (S3 Table), respectively. The gyrB gene, a single copy chromosomal gene encoding ATPase domain of DNA gyrase, was amplified with primers gyrB-qF and gyrB-qR as the endogenous reference (S3 Table).

Accession numbers
Sequences were deposited in GenBank under the accession numbers KR106190 for pHS87a and KR106191 for pHS87b.

General features of pHS87b
The native plasmid pHS87b has two regions (orf5 -orf8 and orf13 -orf16) that are nearly identical to the pieces located within a prophage (coordinate: 366452 to 386384) of P. aeruginosa PA7 [25] (GenBank accession no. CP000744) (Fig 1), which was predicted by Phage_finder [26]. The counterparts encode phage-related transcriptional factors, a capsid protein and an exonuclease. The rest of the regions show no synteny to any of the characterised sequences to date. Those regions encode a putative integrase, a TOPRIM domain-containing protein and many other function unknown proteins. No known replication genes were found. The TOPRIM domain-containing protein and some function unknown proteins might be involved in replication. The plasmid was mapped with the data from the NCBI and Human Microbiome Project (HMP) (S1 Fig). The plasmid generally possesses no coverage in the HMP resource and limited coverage in the NCBI database. However, a 45-bp region was highly covered in both resources and the whole plasmid was found syntenic to some P. aeruginosa chromosomal regions, which will be discussed in the next section.

Integration of pHS87b
Plasmid pHS87b encodes a putative integrase, significantly related to the bacteriophage P4 integrase family, suggesting pHS87b could be integrative (Fig 2A). Most of the integrase-  mediated recombination has been shown to be site-specific, which requires an attachment site adjacent to the integrase gene on the circular extra-chromosomal element and a homologous attachment site at the insertion locus, usually within the chromosome [27]. The site-specific recombination mediated by P4 integrase could be both integrative and excised. Blast of the nucleotide sequence of pHS87b against the chromosome sequence of the reference strain P. aeruginosa PAO1 [28] (GenBank: AE004091) revealed that a 45-bp region upstream of the putative integrase gene (int) was identical to the 3'-end of a tRNA Thr gene (PA5160.1), which explains the high coverage of this region in the NCBI and HMP resources (S1 Fig). The tRNA Thr gene has only one copy in P. aeruginosa. Moreover, tRIP assay at the tRNA Thr locus resulted in a faint band matching the empty site (Fig 2B), indicating the site was partly occupied by a genomic island (GI) in the population. The tRIP assay is a method to detect tRNA-associated GIs using a pair of primers (e.g. thr_U and thr_D) targeting the upstream and downstream of a tRNA gene [24]. If the tRNA site is empty, there will be only one band with a size corresponding to the empty site. Otherwise, negative or weak amplification of the empty site will be seen depending on the GI integration frequency; meanwhile, a larger band may also appear if the GI size is within the amplification range.
To check whether the GI is the integrated form of pHS87b, we managed to amplify the junctions using the primer pairs with one on the chromosome and another on the plasmid (Fig 2C). The orientation of the integration was consistent with the proposed scenario. Meanwhile, the GI was amplified by long PCR with a band having similar size to pHS87b. The restriction profile of the amplicon generated by BamHI, BglII, ClaI, and SmaI was in accord with the predicted restriction patterns of the integrated form of pHS87b (S2 Fig). Finally, sequencing of the junctions and the unoccupied site confirmed that site-specific integration of pHS87b occurred at the tRNA Thr locus, splitting the attachment site into two direct repeats (Fig 2D). Here, attachment sites (att) simply refer to the 45-bp homologous regions. Sequencing of the faint band mentioned above confirmed the presence of attachment site (attB) at the empty tRNA Thr locus in this strain and that the attB is near-identical to the attachment site (attP) on pHS87b. In addition, direct repeats (attL and attR) flanking the ends of the integrated form of pHS87 were found near-identical to attB and attP, consistent with the integration scenario. The abundance of attB, attP and attR was estimated by quantitative PCR (S3 Fig) Results showed that the tRNA Thr site was~98% occupied by the integrated form of pHS87b. And the abundance of the circular form of pHS87b was~2-fold higher than that of the integrated form, suggesting pHS87b might be capable of self-replication, though the replication genes are currently unknown. In accord to the above result, the Southern blotting assays showed pHS87b-specific probe bind to the plasmid and chromosome (S4 Fig). Besides, a genomic island syntenic to the GI form of pHS87b was found at the same locus of the chromosome of P. aeruginosa NCGM 1900 (GenBank: AP014622) (Fig 2E). In addition, a chromosomal region syntenic to the GI form of pHS87b was also found in P. aeruginosa NCGM 1984 (GenBank: AP014646) (Fig 2E). Interestingly, this relevant region was 36-kb downstream of the tRNA Thr gene but delimited by the same tRNA Thr -associated direct repeats. Phage-related genes lie between the tRNA Thr gene and pHS87b syntenic region, possibly composing another prophage-type GI, suggesting a second insertion at the tRNA Thr locus occurred and the pHS87b-syntenic region does have association with the tRNA Thr locus. This evidence further supports the conclusion that pHS87b is tRNA Thr site-specific integrative. tRNA genes have been shown to be hot spots for GI integration. Few cases of plasmid integration have been reported in P. aeruginosa. Two related plasmids pKLK106 and pKLC102 are known to coexist as a free plasmid and a genomic island in P. aeruginosa [4]. pKLC102 can be incorporated into the tRNA Lys gene under the control of a phage-related XerC-like integrase via targeting the cognate attachment site, showing a similar manner to pHS87b. The integration feature of pHS87b also exhibits a potential to be used in genetic manipulation of P. aeruginosa.

General features of pHS87a
The native plasmid pHS87a contains a backbone region and a variable region. Notably, within the variable region lies an aacA4 gene that may confer resistance to aminoglycoside antibiotics.
Plasmid pHS87a bears a putative replicon nearly identical to that of the cryptic Thiobacillus intermedius plasmid pTiK12 (GenBank: L36865). The pHS87a plasmid possesses a repA gene and an AT-rich region located upstream that potentially can form a stem-loop structure similar to that proposed for pTiK1, but lacks the downstream region containing a series of tandem direct repeats presented in pTiK12. In pTiK12, repA and the upstream region are thought to be involved in replication, while the tandem repeats might have a regulation role in plasmid incompatibility [29]. Blastn search revealed that the replicon also is present in Xanthomonas axonopodis plasmids, including pXAC33 (GenBank: AE008924) and pXAC64 (GenBank: AE008925) [30]. In addition, the putative RepA protein of pHS87a shows significant similarity (60% -70%) to the RepA of well-described pPT23A-family plasmids found in Pseudomonas syringae [31]. pPT23A-family plasmids encode diverse functions such as virulence and fitness. Therefore, those plasmids may be evolutionarily related (S5 Fig). pHS87a carries parA and parC that might be responsible for plasmid stability. Notably, pHS87a encodes a putative relBE toxin-antitoxin (TA) system. Plasmid-borne TA systems has been proved to be capable to maintain plasmid stability via post-segregational killing mechanism [32]. pHS87a also carries other genes likely involved in DNA processing, such as orf19 and orf20 coding for putative resolvase domain-containing protein and adenine-specific DNA methyltransferase, respectively. No conjugative modules were detected, indicating that pHS87a may not be a self-transmissible plasmid.
Plasmid pHS87a has a variable region with mobile elements closely adjacent to each other (Fig 1). It has a Tn402-like class 1 integron, into which an aacA4 gene cassette is inserted. The aacA4 gene encodes aminoglycoside N(6')-acetyltransferase-IIb which has been known to confer resistance to gentamycin, tobramycin and netilmicin, but not to amikacin due to a single amino acid change [33]. The putative integron has the 5'-conserved segment (5'-CS) but lacks the 3'-CS. It resides in a Tn402 transposon which is flanked by 5-bp direct repeats (5'-AAAC C-3'). The transposon encodes putative transposition genes tniABQC and has 25-bp imperfect inverted terminal repeats (IRs, 5'-TGTCgTTTTCAGAAGACGgCTGCAC-3'/5'-GTGCAGtC GTCTTCTGAAAAtGACA-3'). Tn402-like class 1 integron has been one of the major vehicles for a large set of antibiotic-resistant genes [34]. Its frequent detection including here highlights its important role in resistance dissemination. In addition, pHS87a carries a transposon Tn6049 and a truncated IS element ΔIS1071. Tn6049 has 12-bp imperfect inverted terminal repeats (5'-TGTGCaTaAGCA-3'/5'-TGCTcAcGCACA-3') and is flanked by 4-bp direct repeats (5'-GCAG-3'). Tn6049 has been found highly abundant as a promiscuous transposable element in the genome of Cupriavidus metallidurans CH34 [35], a strain widely used to study heavy metal-related cellular processes. The ΔIS1071 element has only one terminus matching the 110-bp IR R of IS1071 and a truncated transposase gene. Interestingly, a genomic island in C. metallidurans CH34, CMGI-3 also has multiple copies of IS1071 [35], further suggesting pHS87a might have acquired genetic materials from sources related to C. metallidurans. However, the segment between Tn6049 and ΔIS1071 has no homology to C. metallidurans CH34. The variable region comprises nearly 57% of the whole plasmid in length, indicating pHS87a is a plastic plasmid and may have evolved with multiple origins.
Furthermore, we mapped the nucleotide sequence data from the NCBI and HMP to that of pHS87a (S6 Fig) to analyse the distribution of the modules of the plasmid. The backbone of pHS87a has limited coverage in the NCBI database and no coverage in the HMP resource, suggesting the backbone has been rarely detected and may not be related to human microbiome.
By contrast, the ΔIS1071, Tn6049 and Tn402-like class 1 integron possess different levels of richness in the NCBI database, indicating genetic exchanges has promoted the amplification of mobile genetic elements. However, the Tn6049 was not found in the HMP resource. In addition, pHS87a only shares a kfrA-like gene and integron-related structures with some of the sequenced P. aeruginosa plasmids and no other similarities.

Conclusions
We completely sequenced two novel plasmids found in P. aeruginosa HS87. pHS87b is a phage-related site-specific integrative plasmid, while pHS87a is a plastic antibiotic-resistant plasmid. The two native plasmids may promote the fitness and evolution of the strain.