*Article*

## **Biphenyl**/**PCB Degrading** *bph* **Genes of Ten Bacterial Strains Isolated from Biphenyl-Contaminated Soil in Kitakyushu, Japan: Comparative and Dynamic Features as Integrative Conjugative Elements (ICEs)**

**Jun Hirose 1,\*, Hidehiko Fujihara 2, Takahito Watanabe 3, Nobutada Kimura 4, Hikaru Suenaga 5, Taiki Futagami 6, Masatoshi Goto 7, Akiko Suyama 2 and Kensuke Furukawa 2**


Received: 30 March 2019; Accepted: 20 May 2019; Published: 27 May 2019

**Abstract:** We sequenced the entire genomes of ten biphenyl/PCB degrading bacterial strains (KF strains) isolated from biphenyl-contaminated soil in Kitakyushu, Japan. All the strains were Gram-negative bacteria belonging to β- and γ-proteobacteria. Out of the ten strains, nine strains carried a biphenyl catabolic *bph* gene cluster as integrative conjugative elements (ICEs), and they were classified into four groups based on the structural features of the *bph* genes. Group I (five strains) possessed *bph* genes that were very similar to the ones in *Pseudomonas furukawaii* KF707 (formerly *Pseudomonas pseudoalcaligenes* KF707), which is one of the best characterized biphenyl-utilizing strains. This group of strains carried salicylate catabolic *sal* genes that were approximately 6-kb downstream of the *bph* genes. Group II (two strains) possessed *bph* and *sal* genes similar to the ones in KF707, but these strains lacked the *bphX* region between *bphC* and *bphD*, which is involved in the downstream catabolism of biphenyl. These *bph-sal* clusters in groups I and II were located on an integrative conjugative element that was larger than 110 kb, and they were named ICE*bph-sal*. Our previous study demonstrated that the ICE*bph-sal* of *Pseudomonas putida* KF715 in group II existed both in an integrated form in the chromosome (referred to as ICE*bph-sal*KF715 (integrated)) and in a extrachromosomal circular form (referred to as ICE*bph-sal* (circular)) (previously called pKF715A, 483 kb) in the stationary culture. The ICE*bph-sal* was transferred from KF715 into *P. putida* AC30 and *P. putida* KT2440 with high frequency, and it was maintained stably as an extrachromosomal circular form. The ICE*bph-sal*KF715 (circular) in these transconjugants was further transferred to *P*. *putida* F39/D and then integrated into the chromosome in one or two copies. Meanwhile, group III (one strain) possessed *bph* genes, but not *sal* genes. The nucleotide sequences of the *bph* genes in this group were less conserved compared to the genes of the strains belonging to groups I and II. Currently, there is no evidence to indicate that the *bph* genes in group III are carried by a mobile element. Group IV (two strains) carried *bph* genes as ICEs (59–61 kb) that were similar to the genes found in Tn*4371* from *Cupriavidus oxalacticus* A5 and ICEKKS102*4677* from the *Acidovorax* sp. strain KKS102. Our study found that *bph* gene islands have integrative functions, are transferred among soil bacteria, and are diversified through modification.

**Keywords:** biphenyl; *bph* gene; integrative conjugative element; genome sequence
