The broad-host-range plasmid psfa231 isolated from petroleum-contaminated sediment represents a new member of the proma plasmid family

1,2, 3, Yafei Wang 1, , 1,2, Shan Yang 1, 1 and
1 State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China2 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing, China3 Department of Biological Sciences, Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, ID, USA A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from Holger Heuer, Julius Kühn-Institut, petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), Kok Gan Chan, University of Malaya, 29 of which represent genes involved in replication, partitioning and transfer functions MalaysiaMichael Gillings, Macquarie of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic Oleg Reva, University of Pretoria, analysis shows that pSFA231 shares the common backbone regions with the other PromA South Africa plasmids, i.e., genes involved in replication, maintenance and control, and conjugative *Correspondence:
Hui Li, State Key Laboratory of

transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We Forest and Soil Ecology, Institute of propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) Applied Ecology, Chinese Academy and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of Sciences, No. 72 Wenhua Road, of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA Shenyang 110164, Chinae-mail: and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98,pIPO2T, and pTer331, suggesting these hypothetical orfs may represent "essential"plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also beaccessory genes that were first acquired and then stayed as the plasmid diverged. Ourstudy increases the available collection of complete genome sequences of BHR plasmids,and since pSFA231 is the only characterized PromA plasmid from China, our findings alsoenhance our understanding of the genetic diversity of this plasmid group in different partsof the world.
Keywords: broad-host-range plasmid, PromA plasmid family, complete sequence, plasmid backbone regions,
comparative genomic analysis

metals catabolic functions Plasmids are extra-chromosomal self-replicating DNA elements ), and virulence determinants ), etc.
within the microorganisms They are impor- Conjugative gene transfer mediated by BHR plasmids is gen- tant members of the mobile gene pool, and are among the erally believed to be a common and widespread mechanism for most important contributors to horizontal gene transfer between the transfer of genes across a broad phylogenetic range of bacte- bacteria ). The broad-host-range (BHR) plas- mids have been defined as those plasmids that can self-transfer plays a crucial role in the adaptation of bacteria to environmen- themselves and can stably replicate and maintain in bacterial tal challenges and spread of antibiotic resistance species from at least two subgroups within the Proteobacteria ). Despite the general agreement on the importance of BHR (e.g., between α- and β- Proteobacteria) Sen plasmids in the adaptive evolution of bacteria, the BHR plasmids et al., ). The BHR plasmids typically have mosaic genomes being identified and completely sequenced are still few, initially including two distinct regions The "plasmid limited by the high sequencing cost of first generation (Sanger) backbone" genes encode proteins involved in the replication, sequencing technology. To the best of our knowledge, no more maintenance, control and conjugative transfer of the BHR plas- than 15 BHR plasmids had been fully sequenced until 2006. With mid. Other plasmid regions are comprised of various "accessory" the development of next-generation sequencing methods, such genes conferring important benefits to the host, including resis- as 454 pyrosequencing and Illumina high-throughput sequenc- tance to antibiotics resistance to heavy ing technology, more complete sequences of BHR plasmids have PromA plasmid from contaminated habitat been added to the data pool in recent years, most of which were of the irrigation channel. Surface sediment sample (0∼10 cm) was identified as IncP-1 plasmids ).
collected with a shovel as described by . The In spite of the increasing number of BHR plasmids identified, collected sediment sample was placed in a plastic bag, and then complete sequences of BHR plasmids available in GenBank are was transported to the laboratory on ice. The fresh sediment sam- still not sufficient for systematically analyzing their genetic diver- ple was kept at 4◦C for plasmid isolation, and was air-dried and sity. Thus, isolation and characterization of new BHR plasmids sieved (2 mm) for the analysis of the basic physical and chemical from environmental samples is still required to better understand properties. The total petroleum hydrocarbons (TPH) was deter- the nature and evolutionary history of these important mobile mined as 760.1 mg/kg using gravimetric method genetic elements and their role in horizontal gene transfer.
), and the pH value (soil: water = 1: 5) was 6.5. The organic Among the fully sequenced BHR plasmids, most are classi- matter, total nitrogen and available nitrogen of the sample were fied as the well-known incompatibility groups, such as IncP-1 12.8 g/kg, 0.7 g/kg, 4.0 mg/kg, respectively.
(41 plasmids), IncW (5 plasmids), and IncU (4 plasmids), basedon the backbone genes Norberg STRAINS, PLASMIDS, AND MEDIA
et al., ). Recently, a A list of the strains and plasmids used in this study is pro- novel plasmid group, which could not be classified by the con- vided in Luria-Bertani (LB) broth was generally used ventional inc/rep grouping system, was proposed by Van der to culture the strains. Solid medium was prepared by addition Auwera et al. in terms of phylogenetic analysis of the of 1.7% agar. When necessary for selection, antibiotics were complete genome sequence. This new BHR plasmid group was added to the medium at the following concentrations: rifampicin, defined as PromA family, now including five members, namely 50 μg/ml; gentamicin, 10 μg/ml; kanamycin, 50 μg/ml; strepto- plasmids pMOL98 pIPO2 mycin, 50 μg/ml. Mueller–Hinton (Becton, Dickinson, and Co., ), pSB102 pTer331 Franklin Lakes, NJ) agar was used for detection of antibiotic resis- ), and pMRAD02 ). Their RepA pro- tance. Cycloheximide (300 μg/ml) was added to LB agar (LBA) to tein formed a separate cluster related to but distinct from the prevent growth of fungus during screening of transconjugants.
IncW plasmids, while several plasmid maintenance genes weremost closely related to those of other incompatibility group, and EXOGENOUS ISOLATION OF THE PLASMID
the plasmid mating pair formation genes appeared similar to Triparental exogenous isolation of plasmids was performed as chromosomally encoded Brucella sp. virB genes described by . A rifampicin-resistant strain E. coli ). Additionally, a putative PromA plas- MG1685 (K12 RifR) ) was used as the recip- mid, defined as pMBUI6, was recently identified from freshwater ient to capture the plasmid from the sediment sample. E. coli sample ). Important PromA-like features iden- JM109 (pBBR1MCS-5) was used as tified on pMBUI6 is the presence of topoisomerase gene (topA), the donor, with the mobilizable plasmid pBBR1MCS-5 convey- the relaxase gene (mobB), and the long direct repeats in intergenic ing resistance to gentamicin. Cultures of recipient and donor regions. Nevertheless, some of its backbone genes bear close sim- cells were grown overnight in LB broth containing corresponding ilarity to pXF51 from Xylella fastidiosa but more distantly from antibiotics at 37◦C.
PromA group.
Five grams of air-dried and sieved sediment sample was shaken In our present work, a self-transmissible BHR plasmid for 1 h in 45 ml of sterile saline. The previously reported BHR pSFA231 was isolated from petroleum-contaminated sediment in plasmid pB10 ) was added as a positive con- Shen-fu, China, by using the triparental mating method, selecting trol. In the positive control flask, 5 g of soil sample was mixed with only for the plasmid's ability to mobilize a non-selftransmissible 100 μl of a 10−1 dilution (in saline) of a fully grown E. coli DH5α plasmid. Complete sequencing and phylogenetic analyses of this (pB10) culture (approximately 107 CFU/g soil). The suspension newly isolated plasmid revealed that it fell within the recently was allowed to settle for approximately 30 min. The supernatant defined PromA plasmid group. Isolation of PromA plasmids with was transported to the Eppendorf tube and then centrifuged highly similar backbone sequences from different parts of the (4◦C, 10,000 rpm) for 10 min. After centrifugation, the super- world provides further evidence for global spread of bacteria or natant was discarded and 3 ml of LB solid medium was added to their plasmids, and improves our understanding of the evolution resuspend the pellet. Individually, 500 μl of donor, recipient, and of the PromA plasmid group.
sediment were dispensed into 1.5 ml Eppendorf tubes as controlsfor the mating. For every mating, 500 μl portions of overnight MATERIALS AND METHODS
grown cultures of the donor and the recipient were mixed in an SITE DESCRIPTION AND SAMPLING
Eppendorf tube with 500 μl of sediment supernatant. All the mat- The sampling site was located in Shen-fu wastewater irrigation ing and control preparations were centrifuged (4◦C, 10,000 rpm) zone (123◦35 E, 41◦44 N), the largest petroleum wastewater irri- for 5 min, and the pellet was resuspended in 50 μl of LB broth.
gation zone in Northeast of China. A 70-km irrigation channel Then 30 μl of this cell suspension was spotted onto an LB agar was constructed in 1960's, and the wastewater mainly comes from plate. After overnight of mating at 30◦C, using a sterile loop a an oil refinery. After 50-year exposure to petroleum-containing portion of the biomass (∼1/4–1/3 of the mating "spot") from wastewater, soils in the irrigation zone have been seriously con- each droplet was resuspended in 500 μl of saline, and then agi- taminated ). Plasmid pSFA231 tated vigorously with a Vortex mixer. The cell suspension was was isolated from a sediment sample collected from down-stream serially diluted in saline, and 0.1 ml samples were plated on LB PromA plasmid from contaminated habitat Table 1 Strains and plasmids used in this study.
Strains or plasmids
Genotype and relevant phenotype
E. coli (γ-Proteobacteria)MG1685 (K12 Rif) RifR mutant of MG1655 endA1 recA1 gyrA96 thi hsdR17 (r− m+) relA1 supE44 (lac-proAB) [F' traD36 proAB laqIq Z M15] Agrobacterium tumefaciens C58 (α-Proteobacteria)Cupriavidus necator JMP 228 BHR mobilizable cloning vector; GmR pUT replicon carrying miniTn5::Km1 agar supplemented with rifampicin (50 μg/ml) and gentamicin analysis, pSFA231 was tagged with a miniTn5::Km1 transposon (10 μg/ml). Transconjugant colonies were picked up after 2 days ) using a biparental mating/ mobilization incubation and purified on the same agar medium. Finally, cul- strategy. E. coli MG1685 (pSFA231) served as the recipient and tures of transconjugant cells were grown overnight in LB medium the transposon pUTminiTn5::Km1 was provided by donor strain containing rifampicin (50 μg/ml) and gentamicin (10 μg/ml) at E. coli S17-1. Transconjugants were picked and streaked on LB 37◦C. Physical evidence that mobilizing plasmids were present agar supplemented with kanamycin (50 μg/ml). The tagged plas- was obtained by plasmid extraction by using the alkaline lysis mid (KmR) was eventually transferred to E. coli EC100 (SmR) in method, followed by agarose gel electrophoresis.
a second round of biparental mating, at which point the capturedplasmid was separated from the donor plasmid pBBR1MCS-5.
To test antibiotic resistance of the isolated BHR plasmid, 1.8 ml
overnight culture was centrifuged at 10,000 rpm for 10 min, To determine the self-transferability and the host range, plas- and the cell pellets were washed with sterile saline for three mid pSFA231 was transferred from E. coli EC100 (SmR) to times. The cells were resuspended in 500 μl sterile saline (CFU bacterial species from the other two Proteobacteria subgroups.
approximately 108), and 250 μl bacterial suspension was added Rifampicin-resistant (RifR) strains, Agrobacterium tumefaciens into 150 ml pre-heated Mueller–Hinton agar, mixed by inver- C58 (α-Proteobacteria) and Cupriavidus neca- sion and quickly poured to make an inoculated plate. Paper tor JMP228 (β-Proteobacteria) ), were used as discs (6 mm in diameter) containing different antibiotics of recipients in biparental matings, respectively ).
known amounts (Oxoid Ltd) were placed on the inoculated The plasmids were considered to transfer successfully if colonies plates with sterile forceps. The type and content of antibiotics could grow on medium added with kanamycin and rifampicin.
are the following: kanamycin (K30, 30 μg/slice), chlorampheni- Finally, plasmid was extracted by alkaline lysis method (Feliciello col (C30), Ciprofloxacin (CIP5), erythromycin (E15), amoxi- and Chinali, ) to confirm the presence of the plasmid in cillin (AMC30), rifampicin (RD5), macrodantin(F300), nalidixic acid (NA30), imipenem (IPM10), gentamycin (CN10), carbeni-cillin (CAR100), sulfamethoxazole (W5), ceftazidime (CAZ30), SEQUENCING AND ANNOTATION
polymyxin B (PB300), miramycin (SH100), tetracycline (TE30).
Plasmid DNA for sequencing analyses was prepared using the After 24-h incubation at 37◦C, antibiotic resistance was deter- QIAGEN Plasmid Midi Kit (QIAGEN GmbH, Germany) accord- mined by measuring the inhibition zones around the antibiotic ing to the protocols provided by manufacturer. The whole paper discs compared with those of the recipient strain E. coli genome of plasmid pSFA231 was sequenced by Illumina Hiseq MG1685 and the donor plasmid pBBR1MCS-5. When the diam- 2000 high-throughput sequencing platform at the Majorbio eter of the inhibition zone was 8 mm, the transconjugant strain Bioinformatics Technology Co. Ltd (Shanghai, China). The was considered as resistant to the antibiotic.
paired-end library was generated for high-throughput sequenc-ing. Sequence assembly was primarily done with the SOAPdenovo TAGGING THE PLASMID WITH mini-Tn5 TRANSPOSON
version: v1.05) and GapCloser Antibiotic resistance test revealed that plasmid pSFA231showed software To acquire the complete sequence of no resistance to any antibiotics. To facilitate selection in further plasmid pSFA231, gaps in the plasmid sequence were closed PromA plasmid from contaminated habitat using the general PCR method. Gene prediction was done using rifampicin-resistant strains of Agrobacterium tumefaciens C58 (α-Proteobacteria) and C. necator JMP228 (β-Proteobacteria).
The annotation information of the predicted genes was obtained Results showed that plasmid pSFA231 can self-transfer and repli- through blastp alignment between the amino acid sequences cate in representative strains from three different subgroups of of the predicted genes and the Nr database information using BLAST 2.2.24+. The annotated nucleotide sequence of plasmidpSFA231 was submitted to the GenBank database under the BASIC GENOME SEQUENCE INFORMATION OF THE BHR PLASMID
accession number KJ850907.
The complete nucleotide sequence of the wild-type plasmid
pSFA231 is determined to be 41518 bp with a GC content of GC contents were calculated using the BioXM software. GenBank 60.54%. Annotation of the sequence revealed a total of 50 orfs, was searched for similar sequences using BLAST (Altschul of which 22 are transcribed on one strand and 28 on the other et al., ). The PromA plasmids used in the compara- The sequence has about 84% coding ratio with an tive analysis are listed in Due to the unclear clas- average orf length of 705 bp. The closest relatives (with highest sification, PromA-like plasmid pMBUI6 was not included in amino acid identity scores) of these orfs in GenBank are summa- the comparison. Nucleotide sequences and amino acid (AA) rized in Among the 50 predicted orfs, 29 were attributed sequences translated from the coding sequences were aligned to certain biological functions, 19 orfs coding for conserved hypo- using ClustalX then Mega 6 (Tamura thetical proteins, and the remaining 2 predicted genes do not et al., ) was used to infer the phylogenetic trees using have any known homologs. The putative known coding regions the neighbor joining algorithm with the best-fit model. The of pSFA231 are dominated by essential plasmid backbone genes SplitsTree program was used to infer the phylogenetic net- involved in plasmid replication, maintenance and control, and work The Pairwise genetic distance conjugative transfer based on each backbone protein was calculated by Mega 6 Similarity searches showed that most of the predicted orfs in using the Jones–Taylor–Thornton method plasmid pSFA231 coding for proteins are highly similar to those A circular plasmid map was generated using the SnapGene from plasmid pMOL98, and the remaining orfs were annotated to the proteins from plasmid pTer331 Both pMOL98 Schematic diagrams of multiple alignments of plasmids were and pTer331 are listed as the members of the recently defined produced by manually realigning the linear plasmid maps gen- PromA plasmid family ), leading to erated by the SnapGene Viewer. The identity scores of translated the conclusion that plasmid pSFA231 is a member of the PromA DNA sequences were calculated by the BLAST program, bl2seq PLASMID pSFA231 HARBORS A REPLICATION MODULE UNIQUE TO THE
Before PromA plasmids were recommended as a new fam- ily, their RepA proteins were reported to show some degree A BHR plasmid, named here as pSFA231, was isolated from of identity with the RepA proteins of IncW plasmids. To fur- the petroleum-contaminated sediment. Antibiotic resistance test ther reveal the phylogenetic relationship of the PromA plas- indicated that plasmid pSFA231 carried no additional antibiotic mids with IncW plasmids, a splits network was resistance compared to the recipient strain and donor plasmid.
constructed for 1000 bootstrap replicates of the RepA protein For further analysis, the plasmid was marked by a mini-Tn5 trans- phylogeny of the currently reported PromA members and the poson carrying a kanamycin resistance gene cassette. The tagged selected IncW plasmids, R388 [GenBank accession: BR000038], plasmid was eventually transferred to E. coli EC100 (SmR) by a R7K [GenBank accession: NC_010643.1], pPAES01 [GenBank second round of biparental mating.
accession: CP001109], pSa [GenBank accession: U3071.1], pXV2 To investigate the transferability of the exogenously iso- [GenBank accession: AF201825.1], pIE321 [GenBank accession: lated plasmid pSFA231, the selected clone of E. coli EC100 NC_010716.1], pRM21 [GenBank accession: NC_001755.1], and (pSFA231) was used as donors in biparental matings with pPRO2 [GenBank accession: NC_008608] (Fernández-López Table 2 General features of the PromA plasmids family.
Length (bp)
Hydrocarbon-polluted soil in Essen, Germany Wheat rhizosphere in Wageningen, the Netherlands Rhizosphere of alfalfa in Braunschweig, Germany Dune soil on Wadden Island Terschelling, the Netherlands Strain Methylobacterium radiotolerans JCM2831 isolated from the Unpublished (only available at

PromA plasmid from contaminated habitat FIGURE 1 Circular map of plasmid pSFA231. The 50 orfs identified in the
the transfer functional regions (blue). Hypothetical coding regions are shown nucleotide sequence of pSFA231 are located on a circular map. The orfs are in gray. The genes transcribed in clockwise orientation are in the outer shown by arrows indicating the direction of transcription. Different colors perimeter and those transcribed in anti-clockwise orientation are in the inner indicate replication (orange) and partitioning functional regions (yellow), and perimeter. The restriction enzyme cutting sites are shown as a filled circle.
et al., The network, which presents a combinatorial gener- a putative DnaA-binding site, a potential integration host factor alization of phylogenetic trees, presents a star-like topology with (IHF) site and an AT-rich (86.67%) region were also found in this six main clades It can be visualized that the six PromA plasmids formed a cluster distinct from IncW plasmids, and thePromA-clade was clearly divided into two sub-clades. Plasmids COMPARISON OF THE BACKBONE STRUCTURE OF PromA PLASMIDS
pMRAD02 and pSB102 were clustered separately from the other Comparison of the genome sequence between plasmid pSFA231 four members, and was suggested here as the PromA-α. The ini- and the other five PromA plasmids, pMOL98, pIPO2, pTer331, tiator protein RepA of plasmid pSFA231 had 99, 91, and 90% pSB102, and pMRAD02, revealed a high level of structural similarity to the corresponding protein of pMOL98, pTer331, and similarity. They shared the common backbone regions includ- pIPO2T, respectively, and thus, they all together were proposed to ing functional modules for replication (repA, oriV), conjugative be grouped into the PromA-β.
transfer (tra), and maintenance/control (yacA, parA, korB, incC, Comparison of the oriV region of plasmid pSFA231 and its korA, ssb, kfrA, ardC, and parB) Nevertheless, phy- closest homolog, pMOL98, further verified that the replication logenetic divergences were found in specific loci indicating that module of pSFA231 was similar to PromA plasmids. Like other PromA plasmids may have complex evolutionary histories. For members of the PromA family, pSFA231 was characterized as θ- instance, traO and traO∗ present in pMOL98 provide evidence type mode of replication, with an oriV-like region being located for duplication The putative relaxase gene traS locus at 5.1 kb downstream of the repA gene. Within this region, we on pSFA231, pMOL98, pSB102, and pMRAD02 is not visible on identified four putative iterons (RepA binding site), which are the pTer331 and pIPO2T, additionally, the krfA locus and parB identical to the iteron sequences from pMOL98. Furthermore, locus located on pSFA231, pMOL98, pIPO2T, and pTer331, are PromA plasmid from contaminated habitat Table 3 Location of putative coding regions on plasmid pSFA231 and the closest relatives of the deduced proteins.
Proteins with highest amino acid identity (%)
RepA from pMOL98 (99%) MOL98_2 from pMOL98 (100%) MOL98_3 from pMOL98 (100%) YacA from pTer331 (100%) ParA from pTer331 (98%) MOL98_18 from pMOL98 (99%) hypothetical protein from Pseudomonas sp. HPB0071 (60%) hypothetical 8 kDa protein from uncultured bacterium (100%) ORF41 from pTer331 (100%) ORF40 from pTer331 (100%) hypothetical 8.5 kDa protein from uncultured bacterium (99%) ORF27 from pMOL98 (100%) ORF28 from pMOL98 (100%) ORF29 from pMOL98 (99%) ORF31 from pMOL98 (99%) VirB1 from pTer331 (98%) TraB from pMOL98 (99%) D 12,106–12,414 TraC from pMOL98 (100%) D 12,436–12,747 VirB3 from pTer331 (100%) D 12,754–15,234 TraE from pMOL98 (100%) D 15,239–15,961 TraF from pMOL98 (98%) D 16,065–16,361 TraG from pMOL98 (98%) D 16,373–17,455 TraH from pMOL98 (100%) D 17,592–17,756 TraI from pMOL98 (100%) D 17,762–18,472 TraJ from pMOL98 (100%) D 18,469–19,341 TraK from pMOL98 (100%) D 19,341–20,501 TraL from pMOL98 (100%) D 20,485–21,552 TraM from pMOL98 (99%) D 22,100–24,604 TraN from pMOL98 (99%) D 24,706–25,401 ORF48 from pMOL98 (99%) D 25,413–27,605 TraO from pMOL98 (99%) D 27,602–28,012 TraP from pMOL98 (99%) C 28,048–28,185 D 28,404–28,964 TraQ from pMOL98 (98%) D 28,978–29,556 TraR from pMOL98 (100%) D 29,645–30,124 ORF55 from pMOL98 (99%) C 30,179–31,273 TraS from pMOL98 (100%) C 31,270–31,812 ORF57 from pMOL98 (100%) C 32,079–32,555 ORF11 from pTer331 (99%) C 32,552–33,697 KorB from pTer331 (99%) C 33,698–34,489 IncC from pMOL98 (100%) C 34,486–34,863 KorA from pMOL98 (100%) C 34,888–35,247 Ssb from pMOL98 (100%) C 35,954–36,082 C 36,812–37,846 KfrA from pMOL98 (99%) C 37,949–38,239 ORF64 from pMOL98 (100%) C 38,267–38,659 ORF65 from pMOL98 (100%) C 38,663–38,845 ORF4 from pTer331 (100%) C 38,927–40,387 ArdC from pMOL98 (99%) C 41,055–41,518.1 ParB from pMOL98 (98%)

PromA plasmid from contaminated habitat FIGURE 2 Phylogenetic network of RepA proteins of selected IncW and PromA plasmids, using the neighbor joining algorithm on protein distances
with Poisson correction.
Phylogenetic distance (amino acid difference percentage) was indicated by the length of the tree branches and the scale bars.
almost entirely unrecognizable on pSB102 and pMRAD02. The in pSB102; traG in pMRAD02; traP in pMRAD02; korB in presence and absence of specific backbone genes are most likely pIPO2T; incC in pIPO2T; and korA in pTer331, pIPO2T, and the result of insertions and/or deletions.
pMRAD02) were defined as hypothetical proteins, the corre- To evaluate the evolutionary history of the backbone regions, sponding genes are present in these plasmids, allowing us to the pairwise genetic distances among the PromA plasmids were include them in our comparative genomic analysis. A phylo- calculated based on the amino acid (AA) sequences of each back- genetic tree constructed from the concatenated DNA sequence bone protein using the Jones-Taylor-Thornton method (Table showed that pSFA231 is most similar to pMOL98, then to pIPO2T S1). The AA similarity differed across the 6 plasmids. Among all and pTer331, but is phylogenetically divergent from pSB102 and the 5 known PromA plasmids, pMOL98 had the closest genetic distance to pSFA231 with respect genes of traC, virB3, traE, traH, We also performed a phylogenetic analysis using six con- traI, traJ, traK, traR, incC, korA, and ssb. In contrast, almost all catenated backbone gene products (Figure S1), namely, RepA, the pMRAD02 backbone proteins showed the greatest genetic dis- TraB, TraE, TraN, TraO, and KorB. Not surprisingly, the results tance to our newly isolated plasmid pSFA231, except the traQ and are consistent with those based on the entire backbone regions, traR. As for the plasmid pTer331, genes yacA, parA, virB1, traC, indicating that to simplify the process of comparative genomic traM, traO, korB, and kfrA presented the closest, while genes traP, analysis we can choose gene products with relatively large size and traQ, traR, and parB presented the greatest genetic distance to high level of synteny as targets for comparison.
To further reconstruct their evolutionary history, the phyloge- THE ACCESSORY REGIONS AND TRANSPOSONS OF THE PromA
netic analysis of the whole backbone regions, which are conserved and present in all 6 plasmids, was performed. It was observed that One cluster of hypothetical orfs (orf6-orf15) with unknown func- the gene coding for protein TraS is absent in plasmids pTer331 tions was detected between parA and traA of the PromA mem- and pIPO2T, and plasmids pSB102 and pMRAD02 are lacking in bers. Interestingly, we found that this gene cluster shows high the genes coding for proteins KfrA and ParB (Table S1). Thus, DNA similarity with the corresponding regions on pMOL98 the traS gene sequence and the DNA sequences from kfrA to parB (97%), pIPO2T (95%), and pTer331 (95%), inferring that these were excluded from the alignment, leaving two large backbone genes (or some of them) may also be part of the common back- regions. One region contains 21 continuous genes, organized bone of PromA-β sub-clade, although we still lack direct evidence.
from repA to traR, while the other region includes genes korB, While transposons Tn5178 and Tn6048 were found to be inserted incC, korA, and ssb. Although the amino acid sequences trans- between yacA and parA gene on plasmids pSB102 and pMOL98, lated from the counterparts of backbone genes in selected PromA respectively. No transposon was detected in the corresponding plasmids (gene yacA in pMOL98, pSB102, and pMRAD02; parA region on our plasmid pSFA231, nor on pTer331.

PromA plasmid from contaminated habitat FIGURE 3 Alignment of the pSFA231 replicative origin region with the putative oriV regions of pMOL98. DnaA refers to DnaA boxes, IHF refers to
putative Integration Host Factor binding sites.
opportunities for cell contact and therefore transfer of mobile In the present study, a new BHR plasmid pSFA231 was iso- genetic elements. Moreover, BHR plasmids are frequently cap- lated from a sediment sample collected from a petroleum- tured from contaminated environmental samples wastewater irrigation channel, using the triparental exogenous BHR plasmids play an important role plasmid capture method. It was recommended that choosing in the adaptation of bacterial populations to pollution stress, and donor and recipient strains that are phylogenetically distinct long-term contamination may induce horizontal gene exchange will increase the possibility of obtaining plasmids with a broad and reshuffling of genetic information between phylogenetically host range In our analysis, we also tried the distinct prokaryotes ). With long-term donor/recipient system of E. coli DH5α (pSU4814)/C. necator exposure to petroleum contamination, the Shen-fu wastewater JMP228 (β-Proteobacteria, RifR), but no self-transmissible BHR irrigation zone likely provides a natural pool of BHR plasmids.
plasmids were isolated from the same sediment sample (data not Actually, a set of diverse BHR plasmids were captured from shown). Although the plasmid pSFA231 was isolated by using a dozens of samples collected from Shen-fu irrigation zone in donor and recipient that both belonged to the same subgroup our experiment (unpublished data), including 4 IncP-1ε plas- of Proteobacteria, further host range test showed that pSFA231 mids, 2 unknown plasmids, together with the PromA plasmid could successfully self-transfer and replicate in α-, β-, and γ- reported in this study. We expect an increasing number of phylo- genetically diverse self-transmissible plasmids would be identified Sediments are likely important reservoirs of BHR plasmid from this region by trying different capturing methods, such as biparental mating method or endogenous plasmid isolation The rich biofilm structures in sediments ). Unexpectedly, in contrast to the BHR plas- ) may provide the bacteria more mids isolated from sludge collected from wastewater treatment

PromA plasmid from contaminated habitat FIGURE 4 Schematic diagram of linear alignment of the 6 PromA
are represented by block arrows. Predicted functions are indicated by the plasmids. Phylogenetic tree was constructed based on the DNA sequences
color key featured below the figure. Key backbone genes and accessory of concatenated 25 backbone genes using the Tamura- Nei model. The orfs genes are annotated in the corresponding regions.
backbone regions of PromA family were compared between plas- plasmid pSFA231 carried no antibiotic resistance genes. This mid pSFA231 and five previously reported members. Although phenomenon may not be abnormal. In a previous comparison concatenation of backbone genes is problematic when there are of antibiotic resistance profiles of plasmids captured from non- distinct evolutionary histories of different functional regions of polluted creek and WWTP effluent, no clear difference was found the plasmids ), it is still a recommended method in the proportions of resistant plasmids captured from the two for inferring the evolutionary history of plasmids with higher backbone similarity ). Also, the SplitsTree Based on a similarity search for putative orfs and subse- algorithm allows us to discern the presence of divergent histo- quent comparative analysis, plasmid pSFA231 was proposed as ries. Here, we use gene products for comparison rather than a new member of the recently defined PromA plasmid family.
DNA sequence, because proteins are built from 20 amino acids Compared with the other incompatibility groups, PromA mem- while DNA only contains four different bases, meaning that the bers were symbolized by a distinct replication initiation module, "signal-to-noise ratio" in protein sequence alignments is much which contains a specific oriV-like region and a RepA protein better than in alignments of DNA We found ). While the typi- that all 6 PromA members share the highly conserved backbone cal replication module of the IncP-1 group, into which most BHR regions, comprising replication, maintenance and control, and plasmids have been classified, consists of trfA and ssb genes, and conjugative transfer functions. These plasmids were isolated from also an oriV region Splits network analysis a variety of habitats, such as sediment, rhizosphere and soils, of the RepA protein clearly separated PromA members into two distributed in different locations in The Netherlands, Germany, sub-divisions, illustrating the slight difference in their replication Japan, and in this study, China It is of great interest modules. Thus, in this study, we proposed to divide PromA family that these geographically distinct BHR plasmids harbor backbone into two sub-groups, though there were only six members avail- genes of high similarity. This fact suggests the wide distribution of able. We believe that with more BHR plasmids being added into PromA members.
this recently defined group, new subgroups may be recommended Despite members of PromA family sharing a common back- in the future.
bone structure, phylogenetic analysis of the complete backbone The phylogenetic information of backbone genes provides regions still revealed significant divergence among the PromA fundamental information on the "long-term" evolutionary members. Obviously, pSFA231 is more diverged from plas- history of BHR plasmids. In this study, the concatenated mids pMRAD02 and pSB102 than from pMOL98 and pTer331 PromA plasmid from contaminated habitat which supports our recommendation on divid- (XDB15010103) for Yong Jiang, and also the Idaho INBRE ing the PromA family into two sub-divisions. During the process Program, NIH grants P20RR016454 and P20GM103408 through of evolution, the genetic organization of the backbone regions support for Celeste J. Brown. We also thank for Dr. Hao Sun from can rearrange via inversion, transposition, and duplication/loss Institute of Applied Ecology, Chinese Academy of Sciences for helpful discussion in bioinformatics analysis.
), leading to the presence of diverse plasmids belonging to thesame incompatibility group. For example, plasmid pMOL98 has two traO genes, providing evidence for duplication In The Supplementary Material for this article can be found online addition, lack of the traS genes in the backbone regions of the plasmids pSFA231, pMOL98, pSB102, and pMRAD02 indicated an indel during evolution.
Gains and losses of transposons and other MGEs often hap- pen in BHR plasmid evolution It was Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al.
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