**1. Introduction**

To date, a number of biphenyl-utilizing bacteria have been isolated and characterized in terms of the degradation of polychlorinated biphenyls (PCBs), which are serious environmental contaminants that are prevalent worldwide [1–3]. These strains include both Gram-negative and Gram-positive bacteria. Biphenyl catabolic enzymes co-metabolize certain PCBs into chlorobenzoic acids. It is well documented that PCB degradation is highly dependent on chlorine substitutions, such as the number and positions of the substituted chlorine [4]. Degradation capabilities are also strain dependent. For the first time, biphenyl catabolic *bph* genes were cloned from *Pseudomonas furukawaii* KF707 [5]. Since then, *bph* genes were cloned from various strains, including both Gram-negative and Gram-positive bacteria, and then they were analyzed in detail [3]. These studies indicated that some strains possessed *bph* genes that were very similar to the ones in KF707 in terms of gene organization and nucleotide sequences, although some strains possessed *bph* gene clusters that were different from KF707 and diversified from each other [3]. Some *bph* genes are located on chromosomes, whereas others are present on plasmids. The *bph* genes of *C. oxalacticus* A5 (formerly *Ralstonia* sp. strain A5) [6] and *Acidovorax* sp. KKS102 [7] are located on the ICEs. Gram-positive *Rhodococcus jostii* RHA1 possesses multiple *bph* genes on large linear plasmids [8,9].

The typical *bph* gene cluster shown in KF707 is composed of *bphRA1A2A3A4BCX0X1X2X3D* (Figure 1) [3]. Briefly, the biphenyl dioxygenase is a multi-component enzyme encoded by *bphA1A2A3A4,* and it catalyzes the initial oxygenation of biphenyl, converting the biphenyl into dihydrodiol, where *bphA1* and *bphA2* encode a large and a small subunit of the terminal dioxygenase, respectively. *bphA3* encodes ferredoxin, and *bphA4* encodes ferredoxin reductase. The dihydrodiol compound is then converted to a dihydroxy-compound by the dehydrogenase encoded by the *bphB*. The dihydroxy-compound is then degraded into 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid by the ring-cleavage dioxygenase encoded by the *bphC*. Then, the ring *meta*-cleavage compound is degraded into benzoic acid and 2-hydroxypenta-2,4-dienoic acid by the hydrolase (encoded by the *bphD*). BphX1X2X3 is responsible for the further degradation of 2-hydroxypenta-2,4-dienoic acid into acetyl CoA. These structural *bph* genes are regulated by the *bphR* located on the *bph* gene cluster [10,11] Among these *bph* genes, *bphA1* is critically important for substrate specificity, i.e., the biodegradation capability for various aromatic compounds, including PCBs [12–14].

Previously, we isolated more than ten biphenyl-utilizing bacterial strains (KF strains) from biphenyl-contaminated soil in Kitakyushu, Japan [15]. Among these KF strains, we determined the complete nucleotide sequence of the *P. putida* KF715 genome [16], which revealed five replicons: one circular chromosome and four plasmids. Southern blot analysis indicated that the majority of the KF715 cell population carried the *bph-sal* cluster on its chromosome. However, a small population of cells carried the cluster on a huge extrachromosomal circular element called pKF715A (483 kb). In addition, this element carried the *oriT* sequence, the *repA* gene involved in replication, the conjugal transfer gene (*tra*), and the partitioning gene (*par*). In this study, we were interested in how the KF strains isolated from the same location carried *bph* gene clusters along with other catabolic genes. We performed whole genome sequencing of these strains, anticipating that their genome information would shed light on the diversity and evolution of biphenyl-utilizing bacteria. Our results indicated that specific DNA blocks, including the *bph* gene cluster, were integrated within glycine tRNA (tRNA-Gly) genes and that some blocks contained an integrase gene, illustrating that certain *bph* gene islands had integrative functions.

**Figure 1.** Catabolic pathway of the biphenyl degradation and organization of the *bph* gene cluster in *P. furukawaii* KF707. Compounds: I, biphenyl; II, 2,3-dihydroxy-4-phenylhexa-4,6-diene (dihydrodiol compound); III, 2,3-dihydroxybiphenyl; IV, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (biphenyl *meta*-cleavage compound: HOPD); V, benzoic acid; VI, 2-hydroxypenta-2,4-dienoic acid; VII, 4-hydroxy-2-oxovalerate. Enzymes: BphA1-BphA2-BphA3-BphA4, biphenyl dioxygenase; BphB, dihydrodiol dehydrogenase; BphC, 2,3-dihydroxybiphenyl dioxygenase; BphX0, glutathione *S*-transferase; BphX1, 2-hydroxypenta-2,4-dienoate hydratase; BphX2, acetaldehyde dehydrogenase (acylating); BphX3, 4-hydoxy-2-oxovalerate aldolase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dieonic acid hydrolase. The BphR protein, belonging to the GntR family, is a transcriptional regulator involved in the expression of *bphR* and *bphX0X1X2X3D*. The function of *orf3* remains unclear.

#### **2. Materials and Methods**

#### *2.1. Bacterial Strains and Cultivation*

The bacterial strains (KF strains) used in this study are presented in Table 1. These biphenyl/PCB degrading strains were isolated at the biphenyl-manufacturing factory in Kitakyushu, Japan [15], and were deposited to the National Biological Resource Center (NBRC). Strains KF701, KF703, KF707, KF708, and KF712 were renamed based on the 16S rRNA sequence [17–21]. *P. putida* AC30Bph+ and *P. putida* KT2440Bph+ were obtained through conjugation with *P. putida* KF715, and these two transconjugants grew on biphenyl as a sole source of carbon and energy, as described in Reference [22]. *P. putida* F39/D is a mutant of toluene utilizing *P. putida* F1 [23], in which the *todD* gene is defective. This strain was used as a recipient for the conjugation experiments. The growth of the KF strains and the transconjugants was examined on a basal salt agar medium with biphenyl and various aromatic compounds as described in Reference [5].

**Table 1.** Biphenyl/PCB degrading KF strains used in this study.


#### *2.2. Genome Sequencing and Computational Analysis*

The whole genome sequences of the KF strains were determined by the National Institute of Technology and Evaluation (NITE), using a combination of shotgun sequencing on a 454 GS FLX+ system (Roche, Basel, Switzerland) and paired-end sequencing on a HiSeq sequencing system (Illumina, San Diego, CA, USA) as previously reported in Reference [17]. The reads obtained by the two systems were assembled using the Newbler version 2.8 (Roche). The draft sequence data of the *P. furukawaii* KF707 and *P. putida* KF715 were further completed using the GenoFinisher computer program (http://www.ige.tohoku.ac.jp/joho/gf\_e/). Remaining gaps between the contigs were closed using polymerase chain reaction (PCR) amplification and DNA sequencing with standard Sanger technology. The genome sequences were annotated using the RAST (Rapid Annotation using Subsystem Technology) server [30]. The identification of the coding genes was checked using a BLAST search (http://www.ncb.gov/BLAST/). Sequence comparison was performed using EasyFig Ver. 2.1 [31], and the map was generated using drawGeneArrows3 (http://www.ige.tohoku.ac.jp/joho/labhome/tool.html). The whole genome sequences of the 10 strains were deposited to the DDBJ/EMBL/GenBank under the accession numbers presented in Table 1. The nucleotide sequences of the integrative conjugative elements in KF701, KF702, KF703, KF707, KF708, KF710, KF712, and KF716 were deposited separately. Their accession numbers were LC469607, LC469608, LC469609, LC469610, LC469611, LC469612, LC469613, and LC469614, respectively.

#### *2.3. Phylogenetic Analysis and Gene Alignment*

The nucleotide sequences were aligned computationally using the ClustalW algorithm as in Reference [32]. Phylogenetic trees were generated using the neighbor-joining method with the Mega 6.0 program [33]. The trees were evaluated through bootstrap resampling (500 replicates).

## *2.4. Conjugation Experiments*

Transfer of the Bph+ phenotype (*P. putida* AC30Bph+and *P. putida* KT2440Bph+) by conjugation into the recipient cells (*P. putida* F39/D) was carried out through filter mating as described in Reference [22].

## *2.5. DNA Manipulation*

DNA isolation, Southern blot analysis, PCR, DNA sequencing, and other DNA manipulations were performed according to standard procedures as described in Reference [34]. Pulsed-field gel electrophoresis was performed in accordance with the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA, USA).
