CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the ؊10 and ؊35 regions in Actinobacteria. Further analysis of the ␤-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria.

Two Gram-stain-negative, rod-shaped bacterial strains, cai42T and b45, were isolated from oil-production water taken from Xinjiang Oilfield, China. Optimum growth was observed at 30 6C, at pH 8 and with 1-3 % (w/v) NaCl. According to phylogenetic analyses, the two strains were members of the genus Defluviimonas, with 16S rRNA gene sequence similarities of 95.5"96.3 % with the type strains of species of the genus. The major cellular fatty acids of strains cai42 T and b45 were C 10 : 0 3-OH, C 16 : 0 and summed feature 8 (C 18 : 1 v7c/C 18 : 1 v6c), and the predominant ubiquinone was Q-10, all of these data being typical for the genus Defluviimonas. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, glycolipid, phosphatidylcholine, two unidentified aminolipids, an unidentified phospholipid and two unidentified lipids. The mean genomic DNA G+C contents of strains cai42 T and b45 were 60.8±1.1 and 60.4±1.0 mol%, respectively. On the basis of phylogenetic, physiological and chemotaxonomic analyses, strains cai42 T and b45 represent a novel species of the genus Defluviimonas, for which the name Defluviimonas alba sp. nov. is proposed. The type strain is cai42 T (5CGMCC

-Alkanes are ubiquitous in nature and are widely used by microorganisms as carbon sources. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of -alkanes, which plays important roles in natural alkane attenuation and is used in industrial and environmental applications. The alkane oxidation operon,, in the alkane-degrading strain sp. strain DQ12-45-1b is negatively autoregulated by the TetR family repressor AlkX via a product positive feedback mechanism. To predict the gene regulation mechanism, we determined the 3.1-Å crystal structure of an AlkX homodimer in a non-DNA-bound state. The structure showed traceable long electron density deep inside a hydrophobic cavity of each monomer along the long axis of the helix bundle, and further gas chromatography-mass spectrometry analysis of AlkX revealed that it contained the-derived long-chain fatty acid molecules as a ligand. Moreover, an unusual structural feature of AlkX is an extra helix, α6', forming a lid-like structure with α6 covering the inducer-binding pocket and occupying the space between the two symmetrical DNA-binding motifs in one dimer, indicating a distinct conformational transition mode in modulating DNA binding. Sequence alignment of AlkX homologs from strains showed that the residues involved in DNA and inducer binding are highly conserved, suggesting that the regulation mechanisms of-alkane hydroxylation are possibly a common characteristic of strains. With -alkanes being ubiquitous in nature, many bacteria from terrestrial and aquatic environments have evolved-alkane oxidation functions. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of -alkanes, which plays important roles in natural alkane attenuation and petroleum-contaminating environment bioremediation. The gene regulation of the most common alkane hydroxylase, AlkB, has been studied widely in Gram-negative bacteria but has been less explored in Gram-positive bacteria. Our previous study showed that the TetR family regulator (TFR) AlkX negatively autoregulated the alkane oxidation operon,, in the Gram-positive strain sp. strain DQ12-45-1b. Although TFRs are one of the most common transcriptional regulator families in bacteria, the TFR involved in-alkane metabolism has been reported only recently. In this study, we determined the crystal structure of AlkX, which implies a distinct DNA/ligand binding mode. Our results shed light upon the regulation mechanism of the common alkane degradation process in nature.