**3. Results**

#### *3.1. KF Strains and their Genomic Features*

In this study, we found that various types of *bph* genes were present in the ten biphenyl/PCB degrading strains isolated from the biphenyl-contaminated soil. Figure 2 shows the 16S rRNA phylogenetic tree of the ten strains. Among the ten strains, seven belonged to the *Pseudomonas* genus, two strains to *Cupriavidus* spp*.,* and one to *Comamonas* spp. These were all Gram-negative bacteria belonging to β-proteobacteria (*Comamonas* and *Cupriavidus*) and γ-proteobacteria (*Pseudomonas*). The complete genome sequences were determined for *P. furukawaii* KF707 and *P. putida* KF715. KF707 possessed one circular chromosome of 6,242,949 bp and one plasmid (pKF707) of 59,819 bp. On the other hand, *P. putida* KF715 possessed one circular chromosome of 6,583,376 bp and four plasmids as previously reported in Reference [16]. The total length of the contigs (total number of contigs, >500 bp) of the remaining strains were as follows: *P. abietaniphila* KF701, 6,886,250 bp (140) [17]; *P. aeruginosa* KF702, 7,167,540 bp (91) [24]; *P. putida* KF703, 6,434,897 bp (135) [18]; *C. basilensis* KF708, 7,826,077 bp (62) [20]; *C. pauculus* KF709, 6,826,799 bp (227) [24]; *P. toyotomiensis* KF710, 5,596,721 bp (29) [27]; *C. testosteroni* KF712, 5,890,323 bp (97) [21]; and *P. stutzeri* KF716, 4,188,013 bp (30) [29].

**Figure 2.** 16S rRNA phylogenetic trees of the ten KF strains. The multiple alignment outputs were used to generate neighbor-joining phylogenetic trees using MEGA 6.0 [33]. The bar indicates expected nucleotide substitutions per site. Numbers indicate the percentage occurrence of the branch in the bootstrapped trees on 500 replicates.

All ten strains possessed the *bph* genes, while seven strains possessed the salicylate catabolic *sal* genes. The *bph-sal* cluster was localized on the chromosome in KF707. The same cluster was located on the chromosome in the majority of the KF715 cells and also existed as an extrachromosomal circular form (483,376 bp) as well in the minor part of the cells in the stationary phase culture [16]. Based on the features of the *bph* genes, we classified the ten strains into four groups. Group I (five strains) possessed the *bph-sal* cluster that was almost identical to that of KF707, where the *sal* genes were localized approximately 6-kb downstream of the *bph* genes. This group included strains KF702, KF703, KF707, KF710, and KF716. The *bph* gene cluster of this group was composed of *bphRA1A2A3A4BCX0X1X2X3D* (11.2 kb), which encodes the catabolic enzymes that degrade biphenyl into benzoate and acetyl CoA via the *meta*-cleavage pathway. Group II (strains KF701 and KF715) possessed the *bph-sal* cluster that was similar to that of KF707, although the *bphX* region (3.5 kb) that is involved in the metabolism of 2-hydroxypenta-2,6-dienoic acid to acetyl CoA (Figure 1, lower pathway of biphenyl metabolism) was deleted as described in Reference [22]. The structural features of the *bphRABCD* (7.8 kb) and the flanking regions, including the *sal* genes (10.2 kb), were almost identical between KF701 and KF715, indicating that the *bph-sal* clusters of these two strains were horizontally transferred to one another. Group III (KF709) possessed the *bph* genes, but not the adjacent *sal* genes as in the case of *Burkholderia xenovorans* LB400 [35], another well-characterized PCB-degrader. Overall alignment of the *bph* gene cluster in KF709 was similar to that of group I, but each *bph* component of the *bph* genes was relatively low. Group IV (KF708 and KF712) possessed *bph* genes similar to the genes found in Tn*4371* from *C. oxalacticus* A5 [6], and the ones in ICEKKS102*4677* from the *Acidovorax* sp. strain KKS102 [7]. Some rearrangements of the *bph* gene cluster were observed compared to that in groups I–III as described below. This group of strains did not carry the *sal* genes.

All the strains possessed the catabolic genes of benzoate, the lower pathway intermediate of biphenyl catabolism. Strains KF701, KF702, KF703, KF707, KF710, and KF715 possessed multiple benzoate catabolic genes that encoded both the extradiol cleavage *meta*-pathways (*bza*) and intradiol cleavage *ortho-*pathways (*ben*). KF708, KF709, and KF712 possessed the *box* genes encoding the benzoate catabolic pathway via benzoyl-CoA. Besides the *bph*, *sal*, and benzoate catabolic (*bza*, *ben*, and *box*) genes, these ten strains possessed catabolic genes for various aromatic compounds. Eight strains (KF701, KF703, KF707, KF708, KF710, KF712, KF715, and KF716) possessed a phenol-catabolic *dmp* gene cluster [36]. Five strains (KF702, KF703, KF707, KF710 and KF715) had protocatechuate catabolic

*pca* genes. Five strains (KF703, KF707, KF708, KF709 and KF715) possessed phenylacetate catabolic *paa* genes. Other than the aromatic catabolic genes, many strains carried heavy metal-resistant genes. Six strains (KF701, KF702, KF708, KF710, KF712, and KF715) possessed a cluster of *mer* genes that were responsible for the resistance of inorganic mercury [37]. All of the ten strains possessed putative *czc* genes involved in the resistance of cobalt, zinc, and cadmium [38]. In KF708 and KF709, more than ten putative *czc* gene clusters were present. Arsenate-resistant genes were present in all the ten strains. These results, except for the arsenate-resistant genes, are summarized in Table S1.

#### *3.2. Comparison of the bph Genes*

The identities (percent) of the nucleotide sequences of the *bph* genes in groups I–IV strains (named as types I–IV *bph* genes, respectively) are shown in Tables S2–S13. The phylogenetic trees of *bphA1*, *bphB*, *bphC*, and *bphD* belonging to types I and II are shown in Figure 3. A comparative analysis of the nucleotide sequences of each gene revealed that all the *bph* genes were almost identical in types I and II (96.7–100%), except for that of the *bphX* region (3.5 kb) between *bphC* and *bphD*, which was missing in the *bph* gene cluster of type II (Figure 4).

**Figure 3.** Phylogenetic tree of *bphA1*, *bphB*, *bphC*, and *bphD* of KF strains in groups I and II (types I and II *bph* genes). The multiple alignment outputs were used to generate neighbor-joining phylogenetic trees using MEGA 6.0 [33]. The bar indicates expected nucleotide substitutions per site.

**Figure 4.** Comparison of the *bph* gene clusters of KF strains belonging to groups I–III (types I–III). The shading in pink to red shows the identity (65–100%) of the gene clusters as indicated at the bottom of the figure. R, *bphR*; A1, *bphA1*; A2, *bphA2*; A3, *bphA3*; A4, *bphA4*; B, *bphB*; C, *bphC*; D, *bphD*; X0, *bphX0*, X1, *bphX1*; X2, *bphX2*; and X3, *bphX3*.

The *bphA1*, *bphA2*, *bphA3*, *bphA4*, *bphB*, and *bphC* genes of KF716 were almost identical to that of the type I *bph* gene. However, the 3'-terminus of the *bphX3* gene (754 bp) and 5'-terminus of the *bphD* gene (260 bp) of KF716 were different from that of type I (Figure S1a,b), and the 5'-terminus of the *bphD* gene (260 bp) of KF716 was almost identical to that of the type II *bph* gene. These genetic features are reflected in the phylogenetic tree of the *bphD* (Figure 3). Thus, the *bph* genes of KF716 showed the structural features of both groups I and II.

The KF709 (group III strain) possessed a type III *bph* gene similar to the one in group I, but the respective *bph* genes were less conserved and rearranged (Figure 4). The nucleotide sequences of the KF709 *bph* genes had less than 72.7% identities compared to types I and II in the *bphABC* region (5.9 kb). However, the *bphX1, X2, X3* genes and *bphD* gene were more identical (88.6–90.6% in *bphX1* and 74.3–84.9% in *bphX2, bphX2, bphX3,* or *bphD*) (Tables S2–S13). The *bphX0* encoding putative glutathione *S*-transferase was not present between the *bphC* and *bphX1,* but it was located downstream of the tRNA-Gly gene (data not shown). *orf3* was present between the *bphA2* and *bphA3* of types I–III. Despite the conserved feature of *orf3*, its function remains unclear [3].

The genetic features of KF708 and KF712 in the group IV strain *bph* genes (Type IV *bph* gene) were very different to types I and II, ye<sup>t</sup> they were similar to those of the *Acidovorax* sp. strain KKS102 and *C. oxalacticus* A5 (Figure 5). The *bph* gene cluster of this type was composed of *bphSX1X2X3*(*V*)*A1A2A3BCD*(*W*)*A4*. Thus, the *bphX1X2X3* region was located upstream of the *bphA1A2A3BCD* region, and *bphA4* was present just downstream of the *bphD* (Figure 5). The insertion sequence (1190 bp) and the transposase gene (IS/tnp) were present between the *bphS* and *bphX1* of KF708 and KKS102 [39], but these were not present in KF712 and *C. oxalacticus* A5. The identity of the KF708 and KF712 *bph* genes of type IV was compared to types I and II (Tables S2–S13). The nucleotide sequences of *bphA1, bphA2, bphA3, bphA4, bphB, bphC*, and *bphD* were conserved between types I and IV; however, the identities were less than 77%. Two unidentified gene components, *bphV* and *bphW*, were present in all the type IV *bph* genes, but they were not present in types I–III. The type IV *bph* genes possessed a transcriptional regulator, *bphS* [39]. It was reported that *bphS* acts as a repressor, whereas the *bphR* of Type I acts as an activator for biphenyl catabolism. The functions of *bphS* and *bphR* oppose each other, although they belong to the same GntR family [10,39].

**Figure 5.** Comparison of the *bph* genes of *Cupriavidus basilensis* KF708 and *Comamonas testosteroni* KF712 belonging to group IV (type IV) to those of *Acidovorax* sp. KKS102 and *Cupriavidus oxalacticus* A5. The identities (88–100%) between the gene clusters are shown by shading in pink to red as indicated at the right bottom of the figure. E, *bphE*; F, *bphF*; G, *bphG*; S, *bphS*; V, *bphV*; W, *bphW*; IS, insertion sequence; tnp, transposase. The *bphE, bphF, and bphG of* KKS102/Tn*4371* are homologous genes of *bphX1, bphX3,* and *bphX2* of KF708/KF712, respectively. The other signs are defined in the legend in Figure 4.

#### *3.3. ICEbph-sal in KF Strains Belonging to Groups I and II*

The genome sequence analysis of KF701, KF702, KF703, KF707, KF715, and KF716 revealed highly conserved large "genomic islands" that included the *bph-sal* genes adjacent to the tRNA-Gly(CCC) gene (Figure 6). There was an 18 bp direct repeat (5'-TTCCC(T/A)(T/C)(C/T)(G/A)CCCGCTCCA-3') on the border of the conserved region and the non-conserved region (*attL* and *attR*, Figure 7). The *attL* included 18 bp of the 3 end of the tRNA-Gly(CCC) gene. These 18 bp direct repeat sequences could be generated by the integration of genomic islands into the chromosome. In strain KF707, *attL* was followed by a phage-related integrase (*int*) gene. The *bph* cluster was located just downstream of the *int* gene, followed by the *sal* gene cluster approximately 6-kb downstream, and by the *bza* gene approximately 49-kb downstream. The *attR* site was located downstream of the *bza* gene. Thus, the genomic island was estimated to be 122.0 kb in size (Figure 6). Genes identified as the VirB4 component (ATPase) and VirD4 component (coupling protein) of the type IV secretory pathway [40] were located 40-kb and 80-kb downstream of the tRNA-Gly(CCC) gene, respectively. The *parA* and *parB* genes encoding the replication partition proteins were present near the right end, and were proposed to act as a stabilization system for the maintenance of mobile elements in the bacterial genomes [41]. These structures corresponded to the common backbone of many integrative conjugative elements (ICEs) [42], such as ICE*clc* from *Pseudomonas knackmussii* B13 [43], which carries the catabolic genes of chlorocatechols, the tyrosine integrase gene, and type IV secretory machinery. We designated it as ICE*bph-sal*KF707. Within this element, many putative mobile protein genes were present surrounding the *sal* gene cluster. To note, five IS genes at the upstream region and two IS genes at the downstream region were identified. ICE*bph-sal* was observed in other KF strains of group I and KF701 of group II. An ICE including the *bph* and *sal* gene clusters of *P. aeruginosa* KF702, named ICE*bph-sal*KF702, was calculated to be 126.7 kb (Figure 6). Likewise, the ICE*bph-sal*KF703 of *P. putida* KF703, ICE*bph-sal*KF710 of *P. toyotomiensis* KF710, and ICE*bph-sal*KF716 of *P. stutzeri* KF716 were calculated to be 120.8 kb, 130.3 kb, and 117.3 kb, respectively. The ICE*bph-sal*KF701 of *P. abietaniphila* KF701 in the group II strain was 117.4 kb in size. As in the case of ICE*bph-sal*KF707, ICE*bph-sal*KF701, ICE*bph-sal*KF702, ICE*bph-sal*KF703, and ICE*bph-sal*KF710 contained three gene clusters of *bph*, *sal*, and *bza*. ICE*bph-sal*KF716 contained *bph* and *sal* genes, but not the *bza* genes. Sequence comparison revealed an inversion in ICE*bph-sal*KF702. The *sal:bza* and *bza:sal* fusion gene clusters were found, in which half parts of the *sal* genes and the *bza* genes were replaced with each other. These fusion genes were likely generated via homologous recombination between the *sal* and the *bza* genes. ICE*bph-sal*KF710 and ICE*bph-sal*KF716 contained the 5-kb region encoding putative multidrug e fflux pumps, but ICE*bph-sal*<sup>s</sup> from the remaining KF strains was deficient of the corresponding region. Thus, ICE*bph-sal* in groups I and II contained highly conserved nucleotide sequences larger than 110 kb, which were larger than many other ICEs found in bacterial strains to date [44].

We previously reported that KF715 harbors an approximately 90-kb conjugative *bph-sal* gene cluster in the chromosome [22], and that the cluster could be transferred to *P. putida* AC30 and *P. putida* KT2440 with very high frequency. Whole genome sequencing of the KF715 studied here revealed that the *bph-sal* cluster was located on a huge 483-kb extrachromosomal element, previously designated as pKF715A [16]. An 18 bp DNA sequence identical to the 3'- end portion of a tRNA-Gly(CCC) gene as part of its attachment site (*attP*) was present just upstream of the *int* gene in this element. It was also confirmed that the bacterial integration site (*attB*) was present within the 3-end portion of a tRNA-Gly gene in the KF715 chromosome. Furthermore, the two *Spe*I digested bands, approximately 300 kb and 200 kb, hybridized with a *bphA1* probe were observed as reported [16]. Since there was only one *Spe*I site in the element, the large, faint band was matched to 314 kb of the *Spe*I digested extrachromosomal element. The second major band of 220 kb was matched to that of the *Spe*I digested chromosome of strain KF715. This suggested that the majority of the KF715 cells carried an integrated ICE*bph-sal*KF715, whereas fewer cells carried it as an extrachromosomal circular form. Thus, it was likely that the circular form could be obtained by recombining the *attL* site (present at the 3-end of the tRNA-Gly gene) and *attR* site (present far downstream, 483 kb from the *attL* site), forming the *attP* site.

**Figure 6.** Organization of the ICE*bph-sal* in KF strains of group I and KF701 of group II. ICE*bph-sal*KF701, ICE*bph-sal*KF702, ICE*bph-sal*KF703, ICE*bph-sal*KF707, and ICE*bph-sal*KF710 carry the *int* gene, *bph* genes, *sal* genes, and *bza* genes. ICE*bph-sal*KF716 carries the *int* gene, *bph*, and *sal* genes, but not the *bza* genes. The *sal* genes and *bza* genes in ICE*bph-sal*KF702 are recombined. 1, tRNA-Gly(CCC) genes (partial); 2, *int* genes; 3, VirB4 components of the type IV secretory pathway; 4, VirD4 component of the type IV secretory pathway; 5, *parB* genes; 6, *parA* genes; and 7, putative multidrug efflux pumps. The other, undefined gene components are not shown in the figure.

**Figure 7.** Integration sites of ICE*bph-sal* in the KF strains of group I and KF701 of group II. The *attL* (18 bp) site, including 18 bp of the 3' end of tRNA-Gly gene, and *attR* (18 bp) sites are indicated.

#### *3.4. ICEbph in the Group IV Strains*

The *bph* genes of *C. basilensis* KF708 and *C. testosteroni* KF712 were also located on different types of ICEs. ICE*bph*KF708 (61.8 kb), including *bph* genes of KF708, was almost identical to ICEKKS102*4677* of the *Acidovorax* sp. strain KKS102 [7] (Figure 8). ICE*bph*KF708 had covalently bound ends of the conserved 5-GATTTTAAG-3' sequence (*attL1* and *attR1*). This *att* sequence was identical to that of ICEKKS102*4677*. Nine nucleotide substitutions, three nucleotide deletions, and one nucleotide insertion were found in ICE*bph*KF708 when compared to ICEKKS102*4677.* The ICE*bph*KF712 (59.4 kb) carrying the *bph* genes of KF712 was almost identical to the Tn*4371* from *C. oxalaticus* A5, being the first ICE found carrying the *bph* gene cluster [6]. ICE*bph*KF712 had the covalently bound ends of the conserved *attL2* and *attR2* sequence (5'-TTTTCAT-3'). This *att* sequence was identical to that of Tn*4371*, but it was different from the *attL1* and *attR1* sequence (5'-GATTTTAAG-3') of ICEKKS102*4677* and KF708. The core part, including the *bph* and *trb* gene of ICE*bph*KF712 in length of 33 kb, was almost identical to that of Tn*4371*, whereas the remaining part flanked by the *attL2* site was less conserved. Major parts of these ICEs shared common structures, including the *bph* gene cluster; genes encoding the replication and partition proteins; *parA*; VirD2 component (relaxase); as well as two conjugative transfer elements, the *trb* gene cluster, and the *tra* genes (Figure 8). ICE*bph*KF708 and ICEKKS102*4677* possessed a putative arsenate-resistant gene cluster at 15.5–18.5 kb downstream of the *attL* site, which encoded the transcriptional regulator, arsenate reductase, and arsenite efflux transporter. The gene cluster corresponding to them was not found in ICE*bph*KF712 and Tn*4371*.

**Figure 8.** Organization of ICE*bph*KF708 and ICE*bph*KF712 in comparison to ICEKKS102*4677* and Tn*4371*. *attL1* and *attR1* represent integration sites for the ICE*bph*KF708 and ICEKKS102*4677. attL2* and *attR2* represent integration sites for the Tn*4371* or ICE*bph*KF712. 1, integrase (*int*) gene; 2, putative arsenate-resistant gene; 3, *parA* gene; 4, VirD2 component (relaxase); 5, transcriptional regulator *bphS* gene; 6, *traR* gene; and 7, *traG* gene. The shading in pink to red shows the identities (68–100%) of the gene clusters as indicated at the right bottom of the figure.

#### *3.5. Conjugal Transfer of Extrachromosomal ICEbph-salKF715 Into P. putida F39*/*D*

Since *P. putida* AC30 Bph+ and *P. putida* KT2440 Bph+ harbor an extrachromosomal ICE*bph-sal*KF715, from strain KF715, we tried to transfer this element into *P. putida* F39/D through filter mating. The results are shown in Figure 9. Interestingly, the Bph+ transconjugant of F39/D exhibited two bands hybridized to KF715 *bphA1* DNA when KT2440Bph+ was used as a donor strain of pKF715A. On the other hand, only a single band was detected when AC30Bph+ was used as a donor strain. These hybridized bands were all different in size to that of the 310-kb *Spe*I fragment of pKF715A, indicating that the extrachromosomal ICE*bph-sal*KF715 of the two donor strains were transferred into the recipient strain F39/D and then integrated into the chromosomes at different loci.

**Figure 9.** Conjugative transfer of the *bph* genes from *P. putida* AC30Bph+ and *P. putida* KT2440Bph+ into *P. putida* F39/D. Genomic DNA was digested with *Spe*I, applied to pulse field gel electrophoresis, and subjected to Southern blot analysis. The *bphA1* DNA of KF715 was used as a probe. Lanes 1 and 6, F39/D; lanes 2 and 7, F39/DBph+ transconjugant from AC30Bph<sup>+</sup>; Lanes 3 and 8, AC30Bph<sup>+</sup>; Lanes 4 and 9, F39/DBph+ transconjugant from KT2440Bph<sup>+</sup>; and Lanes 5 and 10, KT2440Bph<sup>+</sup>.
