multivorans cells was sampled at the Ehime Agricultural Experiment Station (Matsuyama, Japan), and sieved and autoclaved before the use according to the

protocol described previously (Nishiyama et al., 2010). The physicochemical properties of the soil have also been described by Wang et al. (2008). Established methods were used for the preparation of plasmid DNAs and their digestion with restriction endonucleases, as well as for ligation and agarose gel electrophoresis (Maniatis et al., 1982). Total genomic DNA was prepared using a Genomic DNA Purification kit (BioRad Laboratories, Hercules, CA). DNA was recovered from agarose gel slices using a Qiagen Gel Extraction kit (Qiagen, Valencia, CA). For plasmid construction, E. coli DH5α or S17-1(λpir) find more was used. The transformation of bacterial cells by electroporation was performed as described previously (Ohtsubo et al., 2006). PCR was performed Baf-A1 with KOD-Plus DNA polymerase (Toyobo,

Osaka, Japan) or ExTaq polymerase (Takara, Kyoto, Japan). The primers used are listed in Supporting Information, Table S1. pEX18Tc (Hoang et al., 1998) was used for the construction of 17616ΔandAc, 17616ΔandR, 17616ΔpdyP, and EN80ΔkynA (Hoang et al., 1998). Three DNA fragments, the upstream and downstream regions of the target gene and the kanamycin resistance (Kmr) gene from pUC4K (Taylor & Rose, 1988), were amplified by PCR and cloned into the multiple-cloning sites (MCS) of pEX18Tc so that the Kmr gene was flanked by the other two fragments. The primers used and their annealing targets are listed in Table S1. The resulting plasmids were conjugally transferred, using E. coli HB101 harboring pRK2013 as a helper, from E. coli DH5αto ATCC 17616 to select the Kmr transconjugants. From the transconjugants, Tc-sensitive, Km-resistant, and sucrose-resistant derivatives were selected.

An 870-bp ATCC 17616 genomic region ranging from nucleotide positions 388, 159–389, 028 on the second (2.6-Mb) chromosome (GenBank accession no. AP009386) covers the promoter and 5′-part of the andAc gene (positions −271 to + 591, taking + 1 as the first position of the andAc start codon). This region was PCR-amplified, digested by BglII and SpeI, and cloned into the MCS of pUIC3 (Rainey, 1999) in a direction such that the inserted andA promoter directs the transcription of the lacZ gene on pUIC3. The resulting plasmid, pEN80, was conjugally transferred from Urease E. coli S17-1(λpir) to ATCC 17616, 17616ΔandR, and DF1 [equal to ATCC 17616Δfur; (Yuhara et al., 2008)] to generate EN80, EN80ΔandR, and EN80Δfur, respectively. Because pUIC3 is incapable of autonomous replication in ATCC 17616, the selection of transconjugants by tetracycline resulted in strains in which pEN80 is integration into the recipient genomes by the single-crossover-mediated homologous recombination event between the cloned DNA region and the corresponding genomic region. This expected recombination was confirmed by PCR using an appropriate set of primers.