Analysis of the ORFs surrounding the B. cepacia RR10 alkane hydroxylase gene. The 6,237-bp B. cepacia DNA region sequenced was analyzed for the presence of ORFs homologous to described genes of known function at the website services of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and of the European Bioinformatics Institute (http://www2.ebi.ac.uk) using the program BLASTX (19). The 1,158-bp ORF coding for a polypeptide showing high similarity scores to known or predicted alkane hydroxylases was named alkB (see the text). A polypeptide showing 30% identity to the Methanococcus jannaschii hypothetical protein MJ1207, which belongs to the acetyltransferase family of proteins, was found upstream of alkB; it is indicated as orf1. Downstream of orf1, an ORF was present encoding a protein 43% identical to E. coli MinC, followed by part of another ORF coding for a polypeptide homologous to E. coli MinD (80% identity in the region analyzed, which covers just the 112 N-terminal residues). E. coli MinC and MinD are known to play an important role in the process of cell division (reviewed in reference 31). Downstream of alkB and oriented in the opposite direction, an ORF was present encoding a protein 61% identical to the E. coli seryl-tRNA synthetase (serS gene). Upstream from serS and in the same orientation, there is an ORF coding for a polypeptide homologous to part of an E. coli hypothetical protein named YcaJ (84% identity in the region analyzed, which covers only the C-terminal half of the protein), the gene of which is also located upstream of serS in E. coli. It is indicated as orf2. The nucleotide sequence of the analyzed segment was deposited at the EMBL data bank under accession no. AJ293306.

Expression of the B. cepacia RR10 PalkB promoter in cells growing in a minimal salts medium using tetradecanol as the carbon source. B. cepacia strain CPBC2, a derivative of RR10 containing a PalkB::lacZ transcriptional fusion integrated into the chromosome, was grown in minimal salts medium containing tetradecanol as the sole carbon source. Samples were taken at different times, and the amount of β-galactosidase present in the cells was measured (represented as gray circles). Growth was followed in parallel by counting CFU on solid media (dark rectangles).

Similarity of the B. cepacia RR10 alkane hydroxylase to other known or predicted bacterial alkane hydroxylases. Proteins were aligned with ClustalW (55) at the European Bioinformatics Institute web site (http://www2.ebi.ac.uk). The data obtained were used to generate a phylogenetic tree with the Phylodendron application at the IUBio Archive for Biology web site (http://iubio.bio.indiana.edu). The identity of each alkane hydroxylase to that of B. cepacia RR10 is indicated in parentheses.

Identification of the promoter for the B. cepacia alkane hydroxylase gene. (A) B. cepacia RR10 was grown in either M9 minimal salts medium in the presence of tetradecane (indicated as C₁₄) or in LB medium in the absence or presence of tetradecane. At different time points (early exponential phase, eE; exponential phase, E; early stationary phase, eS; or stationary phase, S), samples were collected and processed to obtain total RNA. Transcripts originating upstream of alkB were analyzed by S1 nuclease protection assays using equal amounts of total RNA and an excess of ssDNA probe in all cases. Samples were electrophoresed in parallel with a DNA size ladder obtained by chemical sequencing (35) of the same ssDNA used as a probe (lane M). The transcription start site is indicated by an arrow. (B) Sequence of the promoter for the alkB gene. The transcription start site observed in the S1 nuclease protection assay is indicated with an arrow. Positions at the −10 and −35 regions identical to those recognized by the vegetative RNA polymerase at promoters in most eubacteria (60) are highlighted in bold face. (C) The growth curve of B. cepacia RR10 grown in M9 minimal salts medium with tetradecane. Arrows indicate the time points when samples were collected to obtain the total RNA used for the S1 nuclease protection assays described for panel A. Growth was followed by measuring the increase in CFU on LB plates.

Expression of the B. cepacia RR10 alkB gene in cells growing at the expense of alkanes of different chain lengths. B. cepacia RR10 was grown in minimal salts medium containing either dodecane (C₁₂), tetradecane (C₁₄), hexadecane (C₁₆), octadecane (C₁₈), eicosane (C₂₀), docosane (C₂₂), tetracosane (C₂₄), hexacosane (C₂₆), or triacontane (C₃₀) as the sole carbon source. Samples were taken at the end of the exponential phase, and expression of the PalkB promoter was analyzed as described in the legend to Fig. 3. Lane M corresponds to the DNA size ladder. The two panels correspond to gels containing equivalent amounts of sample exposed for the same period of time.