A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation of aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICEbph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICEXTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICEbph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEbph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEbph-salKF716 compared to other catabolic ICEs.


Introduction
Integrative and conjugative elements (ICEs) are mobile genetic elements of bacteria that are excised from the chromosome, transferred to other bacteria via conjugation, and reintegrated into the chromosome. They often carry cargo genes involved in antibiotic resistance, pathogenicity, heavy metal resistance, nitrogen fixation, or aromatic ring catabolism to impart beneficial traits to bacteria [1]. To date, a limited number of ICEs are known to carry cargo genes for the catabolism of aromatic polluting compounds, of which ICEclc is one of the best-characterized [2]. ICEclc contains cargo genes that encode the ortho-cleavage of chlorocatechol (clc genes) and aminophenol catabolism (amn genes). This element was originally identified in the 3-chlorobenzoic acid-degrading bacterium Pseudomonas knackmussii B13. Almost identical copies have been found on the chromosome of Burkholderia xenovorans LB400 (designated ICEclc-LB400) [3] and P. aeruginosa JB2 (ICEclc-JB2) [4]. The structure of ICEclc was compared with other conserved ICEs from five bacterial strains, but none of them contained a gene encoding the aromatic ring degradation pathway [5]. ICE XTD from Azoarcus sp. CIB is another member of the ICEclc family, which is well characterized in terms of the function of cargo genes and the transferability by conjugation [6]. A notable feature of ICE XTD is that it carries gene clusters for both aerobic and anaerobic degradation of xylene and toluene.
The bph genes, which are responsible for the co-metabolic degradation of polychlorinated biphenyls (PCBs), are widely distributed among both Gram-negative and Grampositive bacteria [7]. Some bph genes are known to be horizontally transferred via mobile genetic elements. The first reported ICE carrying bph genes is Tn4371, found in the chromosome of the Gram-negative bacterium Cupriavidus oxalacticus A5 [8], with a total length of 61.8 kb. The chromosome of the Gram-negative bacterium Acidovorax sp. strain KKS102 contains ICE KKS102 4677, which belongs to the Tn4371 family [9]. ICE KKS102 4677 is known to transfer to a wide variety of bacteria across species and genera via a circular intermediate. Recently, we reported the entire genomes of ten PCB-degrading bacteria isolated from biphenyl-contaminated soil in Kitakyushu, Japan [10]. Among them, we detected ICEs carrying the bph gene from nine strains. ICE bph KF708 from Cupriavidus basilensis KF708 and ICE bph KF712 from Comamonas testosteroni KF712 are Tn4371 type ICEs, where ICE bph KF708 is almost identical to ICE KKS102 4677. A 483-kb plasmid pKF715A carrying the bph gene and salicylic acid catabolic sal gene was detected from P. putida KF715, which could be transferred and integrated into the chromosome of P. putida AC30 or KT2440, and then maintained as ICE bph-sal KF715 [11]. Six Pseudomonas strains (P. abietaniphila KF701, P. aeruginosa KF702, P. putida KF703, P. furukawaii KF707 (formerly P. pseudoalcaligenes KF707), P. toyotomiensis KF710, and P. stutzeri KF716) carry an ICE bph-sal element with sizes ranging from 117 kb to 130 kb, and integrate at the 3 end of the tRNA-Gly(CCC) gene. It is obvious from their highly conserved sequences that ICE bph-sal s were generated via horizontal gene transfer. These ICEs carrying bph genes play an important role in the degradation of PCBs in the environment. Precise information on their structure and function will be important to understand the adaptation of their host strains to environmental niches and to design bioremediation processes using PCB-degrading bacteria. Here, we investigated the gene structure of ICE bph-sal KF716 in comparison with other catabolic ICEs and demonstrated that ICE bph-sal KF716 can be transferred via a circular intermediate form.

Bacterial Strains and Culture Conditions
Pseudomonas stutzeri KF716 (NBRC 110668), which has the ability to utilize biphenyl (Bph + ) and salicylic acid (Sal + ), was isolated from the soil in Kitakyushu, Japan [10]. P. aeruginosa PAO1(NBRC 106052), which has kanamycin resistance (Km R ), was obtained from the Biological Resource Center of the National Institute of Technology and Evaluation (NBRC, Tokyo, Japan). For the growth of Pseudomonas strains, a basal salt medium containing (in grams per liter) K 2  O, 0.002 (pH 7.0) was used. The bacterial strains were grown by shaking at 120 rpm at 30 • C. For DNA isolation or freezing stock preparation, Luria-Bertani (LB) medium (Bacto Tryptone, 10 g; yeast extract, 5 g; and NaCl, 10 g/L, pH 7.0) was used.

Detection of Target DNA by Polymerase Chain Reaction
Genomic DNA was extracted using a Genomic-tip 20/G (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Amplification of genes was performed in a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) and PCR conditions were performed in a 25 µL reaction mix containing 12.5 µL Gene RED PCR Mix Plus (Nippon Gene Co. Ltd., Toyama, Japan) at 94 • C for 5 min, followed by 30 cycles of 94 • C for 45 s, 62 • C for 45 s, and 72 • C for 60 s, with a final extension of 5 min at 72 • C. See Table S1 for primer sequences. PCR products were detected by agarose gel electrophoresis according to a standard procedure. The sequences of PCR products were determined using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3500 Genetic Analyzer.

Conjugal Transfer of ICE
Transfer of the Bph + phenotype by conjugation into the recipient cells was carried out through filter mating. Donor and recipient cells were grown overnight in LB agar medium, and both cultures were suspended in 1 mL of LB broth. The cell suspensions (0.5 mL each) were mixed and placed on a nitrocellulose filter (0.45 µm, Merck Millipore, Bedford, MA, USA), and placed on an LB agar plate at 30 • C for 16 h. After incubation, the cells on the filter were suspended in sterilized saline, further diluted, and inoculated onto basal salt medium plates containing 30 µg of kanamycin, providing solid biphenyl in the inverted lid to select transconjugants. Conjugative transfer frequencies were calculated as the number of transconjugant cells per number of donor bacterial cells present in each mating.

Detection of ICE bph-sal KF716
A detailed analysis of the genome sequence of P. stutzeri KF716 revealed a genomic island of 117,300 bp ( Figure 1), named ICE bph-sal KF716. ICE bph-sal KF716 is located at the 3' end (attB site) of tRNAGly(CCC). The left end (attL) of ICE bph-sal KF716 is formed by 18 bp of tRNA-Gly (TTCCCTTCGCCCGCTCCA), and the right end (attR) is formed by a repetition of these 18 bp ( Figure 2). We previously confirmed that this 18 bp direct repeat sequence is located on the border of the conserved ICE region and the non-conserved region of the chromosome [10]. The complete sequence of ICE bph-sal KF716 was submitted to the PGAP and RAST pipeline for annotation, and 112 ORFs were identified. Annotation was refined manually and compared with pKF715A [11], ICEclc [2], and ICE XTD [6]. ICE bph-sal KF716 is a mobile genetic element that shares a core region with ICEclc and ICE XTD , which have been shown to impart aromatic compound degradation genes to bacteria. The genetic maps of ICE bph-sal KF716, ICEclc, and ICE XTD are shown in Figure 1, while their attL and attR are shown in Figure 2. The genes encoded by ICE bph-sal KF716 are listed in Table S2.

Core Region of ICE bph-sal KF716
The attL gene was found to be followed by a phage-related integrase (int) gene, which shares an identity with that of ICEclc or ICE XTD carrying catabolic genes of aromatic compounds. ICE bph-sal KF716 contains core regions that are significantly similar to the core regions of ICEclc [2] and ICE XTD [6] (Figure 1). The ORFs in the core region of ICE bph-sal KF716 were 55-83% identical to those of ICEclc, and 57-93% identical to those of ICE XTD . The ORFs display the same genetic order in the ICE bph-sal KF716 element, and belong to the type IV secretion system (T4SS). Three factors have been recognized as important for DNA transfer by T4SS in Gram-negative and Gram-positive bacteria. These are: murein hydrolase [17], which is involved in controlled local degradation of the peptidoglycan, making space for the formation of a mating channel; the VirB4 ATPase, which provides energy for the translocation: and the VirD4 coupling protein, which links the DNA transfer intermediate to the mating channel [18]. In the ICE bph-sal KF716 element, we identified the virB4 gene (KF716ICE_480; the locus tags are listed in Table S2), virD4 gene (KF716ICE_650), and putative murein hydrolase (KF716ICE_670).
The parA and parB genes (KF716ICE_960, KF716ICE_940) encoding the replication partition proteins present 100 kb and 99 kb downstream of the attL site, respectively, were proposed to act as a stabilization system for the maintenance of mobile elements in the bacterial genomes [19]. Near parB, there is a gene encoding putative integrase regulator (KF716ICE_890) a homolog of InrR involved in the regulation of the expression of integrase on ICEclc [20]. Relaxase (traI, KF716ICE_370) which binds and nicks excised circular ICE at the origin of the transfer [21], TraG protein (traG, KF716ICE_410) [22], a component of the mating pair formation system, and pilin protein (pilL, KF716ICE_700) were also identified in the core region of ICE bph-sal KF716.
The homology of the integrase-encoding int gene between ICE bph-sal and ICEclc was lower (66%) than that in the core region. The integration site of ICE bph-sal was tRNA-Gly(CCC), whereas that of ICEclc was different in tRNA-Gly(GCC). In contrast, the homology of the int gene between ICE XTD and ICE bph-sal KF716 was 70%; the integration site of ICE XTD was tRNA-Gly(CCC), which was identical to that of ICE bph-sal KF716. The sequence of attL and attR at the end of ICE was five bases different between ICEclc and ICE bph-sal KF716, and one base difference between ICE XTD and ICE bph-sal KF716 ( Figure 2). The amino acid sequences of these ICE integrases may reflect different recognition of the integration site.

Variable Region of ICE bph-sal KF716
ICE bph-sal KF716 contains at least four variable regions (VR1-VR4) that are deficient in ICEclc and ICE XTD (Figure 1). Variable region 1 (VR1) located near the attL site contains the biphenyl catabolic bph gene cluster with a total length of 11.2 kb, as well as a salicylate catabolic sal gene cluster with a total length of 11.5 kb. The bph gene cluster was found to be located just downstream of the int gene, followed by the sal gene cluster approximately 6 kb further downstream. The bph and sal genes encode for the degradation of biphenyl and salicylic acid to TCA cycle intermediates via an initial oxygenation step, followed by a meta-cleavage pathway. As shown in Figure 1, the bph gene cluster in VR1 shares 59-79% identity at the nucleotide sequence level with the toluene catabolic tod gene cluster located in the variable region of ICE XTD in the opposite direction. The relationship between the bph gene of P. furukawaii KF707 and the tod gene of P. putida F1 has been reported in our previous paper [23]. The two variable regions (VR2 and VR3) are located at the midst of ICE bph-sal KF716. The GC content of these regions was lower than that of the other regions (Figure 3), and these ORFs (from KF716ICE_570 to KF716ICE_630 and from KF716ICE_710 to KF716ICE_740) were encoded in the opposite direction compared with ORFs in the surrounding core regions (Table S2), suggesting that these regions are inserted from the other genetic elements or chromosomes through horizontal gene transfer. VR2 contained ORFs coding for several hypothetical proteins with unknown functions, whereas VR3 is likely to be an insertion sequence since it includes an ORF identified as transposase (KF716ICE_720). The other variable region (VR4) located near the attR site contained the gene cluster coding for a putative ABC-type multi-drug efflux pump (from KF716ICE_1060 to KF716ICE_1110), with a total length of 6.6 kb that is deficient in ICEclc and ICE XTD . The substrate of this transporter was not identified due to the lack of reliable homologous genes whose functions have been elucidated.

Comparison of ICE bph-sal KF716 and Other Putative ICEs
A search of the public database using BLAST revealed that an almost identical core region to that of ICE bph-sal KF716 was found in other putative ICEs. The identities were higher than those of ICEclc or ICE XTD . Putative ICEs were identified from the genome sequences of P. putida B6-2 (accession number: NZ_CP015202) [24,25], P. alcaliphila JAB1 (CP016162) [26], P. stutzeri AN10 (NC_018028) [27], and P. stutzeri 2A20 (KT935509) [28]. They are flanked by directed repeat sequences corresponding to the attL and attR sites ( Figure 2).
The putative ICE from P. putida B6-2 (tentatively designated as ICE bph-sal B6-2) has the closest relationship with ICE bph-sal KF716; it carries a bph-sal gene cluster that shares 91-100% identity with that of ICE bph-sal KF716, as well as highly conserved core regions ( Figure 4). P. putida B6-2 is capable of degrading various polycyclic aromatic hydrocarbons [25]. Biphenyl (bph), salicylic acid (sal), and ferulic acid (fcs, ech), as well as downstream benzoic acid (ben) and protocatechuic acid (pca) catabolic gene clusters were identified on the genome sequence of P. putida B6-2, of which bph genes and sal genes are located in the ICE (Figure 4). The other putative ICE integrated in the genome of P. alcaliphila JAB1 (tentatively designated as ICE bph-sal JAB1) is the second-closest ICE to ICE bph-sal KF716; in this, the bph-sal gene cluster and benzoate catabolic bza gene cluster are included in the variable region ( Figure 4). It is likely that inversion in the variable region, together with a part of the core region, occurred in the ICE bph-sal JAB1. In addition, sal:bza and bza:sal fusion gene clusters were found, in which parts of the sal genes and the bza genes were replaced with one another. This inversion at the sal-bza locus was also detected in ICE bph-sal KF702 of P. aeruginosa KF702, as described in our previous paper [10]. In addition to the well-characterized Tn4371 and ICE KKS102 4677, ICEs that carry the bph gene cluster included ICE bph-sal KF701, ICE bph-sal KF702, ICE bph-sal KF703, ICE bph-sal KF707, and ICE bph-sal KF710 from five biphenyl/PCB-degrading strains isolated from Kitakyushu, Japan [10]. ICE bph-sal KF716 shares a core region and bph-sal catabolic genes with these ICE bph-sal elements, but lacks the bza gene, which encodes the benzoic acid degradation pathway. The comparison of the overall structure revealed that ICE bph-sal B6-2 from P. putida B6-2, and ICE bph-sal JAB1 from P. alcaliphila JAB1 are also members of the 'ICE bph-sal family' (Figure 4). In particular, ICE bph-sal B6-2 is more closely related to ICE bph-sal KF716 as it lacks the bza gene. Since P. alcaliphila JAB1 was isolated in the Czech Republic, and P. stutzeri KF716 was isolated from Japan [10], it appears that ICE bph-sal s are globally distributed. A BLAST search identified the other putative ICEs carrying a highly conserved core region with that of ICE bph-sal KF716 ( Figure 5). The genome of P. stutzeri AN10 contained the putative ICE (tentatively designated ICE nah AN10) carrying the naphthalene catabolic nah genes [29,30] that share more than 70-90% homology with the nah operon on plasmid NAH7 at the nucleotide sequence level. The aerobic degradation pathway of naphthalene consists of an upper pathway that transforms naphthalene to salicylic acid and pyruvic acid, and a lower pathway that transforms salicylic acid to TCA cycle intermediates. The nah lower operon coding for the lower pathway of ICE nah AN10 shares 97-100% identity with the sal genes of ICE bph-sal KF716. In this context, it is likely that the nah upper operon is replaced by the bph genes in ICE bph-sal KF716, and, conversely, the bph genes are replaced by the nah upper operon in ICE nah AN10. It has been confirmed that many putative mobile protein genes are present on ICE bph-sal s [10], which may act to replace the bph genes and the nah upper operon. The part of ICE nah AN10, including the core region and genes coding for putative multi-drug efflux pumps, is perfectly identical (100%) to that of ICE bph-sal KF716, showing a very close relationship between the two ICEs. In fact, our previous paper described that the nah lower operon of P. stutzeri AN10 and the sal gene of P. furukawaii KF707 are highly conserved [31]. The putative ICE from P. stutzeri 2A20 (tentatively designated lCE phe-xyl 2A20) carried the tou genes coding for multicomponent toluene monooxygenases, phe genes coding for phenol meta-cleavage pathway, and xyl upper and lower operons coding for toluenexylene catabolic genes [28] in the variable regions. The core regions of ICE phe-xyl 2A20, including partial phe genes, overlapped with the sal gene share 91-97% identity with those of ICE bph-sal KF716. A comparison of the variable regions of ICE phe-xyl 2A20 and ICE bph-sal KF716 strongly indicates that the substitution occurred between the biphenyl catabolic bph genes and toluene monooxygenase tou genes together with the part of the phe genes. Although ICE nah AN10 and ICE phe-xyl 2A20 do not carry bph genes, the evolutionary relationship of ICE bph-sal KF716 with ICE nah AN10 or ICE phe-xyl 2A20 was closer than that of ICEclc or ICE XTD , as judged from the nucleotide sequence identity level. Their homologies of the core regions with ICE bph-sal were higher (77-100% identity between ORFs) than those of ICEclc and ICE XTD , indicating that these ICEs are more closely related to ICE bph-sal KF716. The identity of ICE bph-sal KF716 with ICE nah AN10 or ICE phe-xyl 2A20 varies depending on the regions, where major parts including integrase (int) gene were highly conserved (90-100% identity), as shown in Figure 5. Although the variable regions among ICE bph-sal KF716, ICE nah AN10, and ICE phe-xyl 2A20 are different, they commonly possess genes for aromatic compound meta-cleavage pathways. ICE bph-sal KF716, ICE nah AN10, and ICE phe-xyl 2A20 are all ICEs found in P. stutzeri. This species is known to exhibit phenotypical diversity in the ecosystem and can adapt to the environmental niche [32], suggesting that it is particularly preferable as a host for this ICE subfamily.

Excision and Formation of Circular Intermediate Form of ICE bph-sal KF716
ICEs integrated into the chromosome can be excised from the chromosome to produce a circular form, and the host genome was repaired upon excision (Figure 6a). Attempts were made to detect the extrachromosomal circular form of ICE bph-sal KF716 from total DNA isolated from P. stutzeri KF716 grown on biphenyl. The amplicons corresponding to the attP site (R1 in Figure 6a) of excised circular forms of ICE bph-sal KF716, attB sites (R2) of excised closed forms of chromosome, and attL/attR sites (R3 and R4) of the integrated form were detected using PCR (Figure 6b). It has been reported that tRNA-Gly(CCC) locus is the insertion site of ICE bph-sal KF716 [10]. DNA sequences of these amplicons matched the expected sequences compared to the total genome sequence of P. stutzeri KF716 [12].  Table S1) were used to amplify R1 including attP site of ICE bph-sal KF716. Primers attL1 and attL2 were used to amplify R2 including attB site. Primers attL1 and attL2 were used to amplify R3 including attL site. Primers attR1 and attR2 were used to amplify R4 including attR site. (b) Detection of circular form and integrated form of ICE bph-sal KF716 in wild type P. stuzeri KF716 by PCR. Lane 1, DNA ladder marker; Lane 2, R1 (attP); Lane 3, R2 (attB); Lane 4, R3 (attL); Lane 5, R4 (attR).

Transfer and Integration of ICE bph-sal KF716
To demonstrate the autonomous intercellular transfer of ICE bph-sal KF716, we performed mating experiments using P. stutzeri KF716 as a donor strain. P. aeruginosa PAO1 was used as the recipient strain because it carries Km R phenotype, and forms a green colony, which enables it to be distinguished from the donor strain. The donor strain was mated with the recipient strain, and transconjugants were selected based on their Km R and Bph + phenotypes. We observed the appearance of transconjugants, P. aeruginosa PAO1 acquiring Bph + , Sal + and Km R phenotypes. The transfer frequencies (transconjugants per donor cell) ranged from 6.2 × 10 −8 to 1.3 × 10 −6 , with 3.3 × 10 −7 average in eight replicates. The transconjugants grew in a liquid medium containing biphenyl and salicylic acid as the sole source of carbon (data not shown). To confirm the identity of the transconjugant cells, PCR was performed with genomic DNA using primers 27F and 907R that amplify the 16S ribosomal DNA (16S rDNA), and the DNA sequence of the amplicon matched the 16S rDNA of the recipient strain. The amplicons corresponding to the attL/attR site (R6 and R7 in Figure 7a) of the integrated form of ICE bph-sal KF716 were detected from at least three PAO1 transconjugants. (Figure 7b). DNA sequencing these amplicons derived from the integrated form of ICE bph-sal KF716 in the transconjugants confirmed attB site at the 3 end of tRNA-Gly(CCC) overlapping with sequence position 797,721 to 797,738 in genome sequence of P. putida PAO1 (accession number: NC_002516) [33]. The amplicons corresponding to the attP site (R1) of excised circular forms of ICE bph-sal KF716 and the attB site (R5) of excised closed form of chromosome were also detected from the transconjugants. DNA sequences of these amplicons matched the expected sequences compared to the attP site of ICE bph-sal KF716 or its insertion site on the P. aeriginosa PAO1 chromosome. At least two different sized amplicons were observed by electrophoresis (Figure 7b, Lane 3 and Lane 4) and by DNA sequencing when trying to obtain a fragment of R5 corresponding to the attB site or R6 corresponding to the attL site, respectively. This result indicates heterogeneity in the transconjugants, probably due to partial excision of ICE bph-sal KF716 from chromosome. It has been reported that the transfer efficiency of ICEclc is extremely high (1.4 × 10 −2 ) [20], whereas that of ICE KKS102 4677 is extremely low (5.8 × 10 −10 ) [9]. The transfer efficiency of ICE bph-sal KF716 was approximately 3.3 × 10 −7 in the intermediate of ICEclc and ICE KKS102 4677 and comparable with that of ICE XTD . The factors that govern the transfer efficiency of these ICEs remain to be elucidated, however many factors seem to involve the donor and recipient strains. To demonstrate the autonomous intercellular transfer of ICEbph-salKF716, we performed mating experiments using P. stutzeri KF716 as a donor strain. P. aeruginosa PAO1 was used as the recipient strain because it carries Km R phenotype, and forms a green colony, which enables it to be distinguished from the donor strain. The donor strain was mated with the recipient strain, and transconjugants were selected based on their Km R and Bph + phenotypes. We observed the appearance of transconjugants, P. aeruginosa PAO1 acquiring Bph + , Sal + and Km R phenotypes. The transfer frequencies (transconjugants per donor cell) ranged from 6.2 × 10 −8 to 1.3 × 10 −6 , with 3.3 × 10 −7 average in eight replicates. The transconjugants grew in a liquid medium containing biphenyl and salicylic acid as the sole source of carbon (data not shown). To confirm the identity of the transconjugant cells, PCR was performed with genomic DNA using primers 27F and 907R that amplify the 16S ribosomal DNA (16S rDNA), and the DNA sequence of the amplicon matched the 16S rDNA of the recipient strain. The amplicons corresponding to the attL/attR site (R6 and R7 in Figure 7a) of the integrated form of ICEbph-salKF716 were detected from at least three PAO1 transconjugants. (Figure 7b). DNA sequencing these amplicons derived from the integrated form of ICEbph-salKF716 in the transconjugants confirmed attB site at the 3′ end of tRNA-Gly(CCC) overlapping with sequence position 797,721 to 797,738 in genome sequence of P. putida PAO1 (accession number: NC_002516) [33]. The amplicons corresponding to the attP site (R1) of excised circular forms of ICEbph-  Table S1) were used to amplify R5 including the attB site of the recipient strain. Primers attL1 and attL3 were used to amplify R6 including the attL site. Primers attR1 and attR3 were used to amplify R7 including the attR site. Primers bphL1 and bphR1 were used to amplify R8 including the bph gene. Primers salL1 and salR1 were used to amplify R9 including the sal gene. Primers MDL1 and MDR1 was used to amplify R10 including the putative gene for multi-drug efflux pump. (b) Detection of integrated from of ICE bph-sal KF716 in transconjugant P. aeruginosa PAO1 by PCR. Lane 1, DNA ladder marker; Lane 2, R1 (attP); Lane 3, R5 (attB); Lane 4, R6 (attL); Lane 5, R8 (bph); Lane 6, R9 (sal); Lane 7, R10 (multi-drug efflux pump); Lane 8, R7 (attR).

Conclusions
In this study, we investigated the structure and transfer of ICE bph-sal KF716, which is integrated into the chromosome of the biphenyl/PCB-degrading bacterium P. stutzeri KF716. Comparison with putative ICEs from other related Pseudomonas strains suggested the existence of a new ICEclc subfamily that shares nearly identical core regions. Since ICE bph-sal KF716 is the first member of this subfamily found, ICE bph-sal KF716 represents the ICEclc subfamily which is involved in the horizontal transfer of various catabolic genes among Pseudomonas strains. The results presented in this study will provide new insights into the evolution of ICEs in the process of adaptation to environmental niches, as well as a basis for designing bioremediation processes using PCB-degrading bacteria.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.