603 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2005, The Society for Biotechnology, Japan Vol. 99, No. 6, 603–610. 2005 DOI: 10.1263/jbb.99.603 Organization and Localization of the dnaA and dnaK Gene Regions on the Multichromosomal Genome of Burkholderia multivorans ATCC 17616 YUJI NAGATA, 1 * MUNEAKI MATSUDA, 1 HARUNOBU KOMATSU, 1 YOSHIYUKI IMURA, 1 HIROYUKI SAWADA, 2 YOSHIYUKI OHTSUBO, 1 AND MASATAKA TSUDA 1 Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan 1 and Department of Biological Safety, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan 2 Received 4 January 2005/Accepted 24 March 2005 The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for funda- mental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH- dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating se- quence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE- orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the  32 -de- pendent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome. [Key words: dnaA, DnaA box, replication origin, Burkholderia, dnaN, gyrB, heat shock proteins, dnaK, dnaJ] The bacteria in the ‘Burkholderia cepacia complex (Bcc)’ (1) are members of -Proteobacteria (2), and have attracted attention because of their extraordinary biodegradative abil- ities and potential agents for bioremediation (3, 4). On the other hand, they are notorious as opportunistic pathogens for patients with cystic fibrosis (1, 5). Three genomic fea- tures of the Bcc are supposed to be involved in their extra- ordinary nutritional versatility and adaptability. The first is their large (7 to 8 Mb) genome sizes (6, 7), most probably possessing great genetic potential. Secondly, the genomes of Bcc contain a large number of insertion sequences (ISs), identified on the basis of their abilities to promote genomic rearrangements and to activate the expression of neighbor- ing genes (8–15). Such elements have been implicated in the dynamic rearrangements of genomes and recruitment of foreign genes for novel catabolic functions. The last is their chromosome multiplicity although its advantage for adapta- tion has not been experimentally demonstrated. The members of Bcc have been categorized into at least nine genomovars (1); ATCC 17616, a strain belonging to genomovar II, has been designated Burkholderia multivorans (16). This strain carries three circular chromosomes of 3.4, 2.5, and 0.9 Mb in sizes (hereafter designated Chr I, Chr II, and Chr III, respectively). The genome map of ATCC 17616 has been constructed by use of three rare-cutting restriction enzymes, PacI, PmeI, and SwaI (6), and we recently map- ped five copies of the rrn operons on the three chromo- somes (17). Furthermore, various auxotrophic mutants of ATCC 17616 were isolated using a Tn5 derivative, and the following features of the auxotrophic genes were clarified (17): (i) all nine his genes are clustered on Chr I; (ii) seven trp genes are organized at two distinct clusters, a trpEGDC culster on Chr I and a trpFBA cluster on Chr II; (iii) the leu gene cluster, leuCDB, is also located close to the trpFBA cluster; and (iv) the lysA and argG genes are located on Chr II while the argH gene is on Chr I. Southern hybridization analyses and allelic exchange mutagenesis of ATCC 17616 demonstrated that all of the genes examined are functional and exist as single copies on the genome (17). To understand the organization of the multichromosomal ATCC 17616 genome in more detail, it is necessary to know the distribution of genes for fundamental cell functions. In this study, we cloned and sequenced the dnaA and dnaK gene regions of ATCC 17616. The DnaA protein, which is an essential initiator of chromosome replication, has been encoded in all eubacteria so far analyzed (18). The DnaA protein recognizes the replication origin (oriC) by binding * Corresponding author. e-mail: aynaga@ige.tohoku.ac.jp phone: +81-(0)22-217-5682 fax: +81-(0)22-217-5699