Involvement of the oscA gene in the sulphur starvation response and in Cr(VI) resistance in Pseudomonas corrugata 28

Pseudomonas corrugata 28 is a Cr(VI)-hyper-resistant bacterium. A Cr(VI)-sensitive mutant was obtained by insertional mutagenesis using EZ-Tn5 <R6Kγori/KAN-2>Tnp. The mutant strain was impaired in a gene, here named oscA (organosulphur compounds), which encoded a hypothetical small protein of unknown function. The gene was located upstream of a gene cluster that encodes the components of the sulphate ABC transporter, and it formed a transcriptional unit with sbp, which encoded the periplasmic binding protein of the transporter. The oscA–sbp transcriptional unit was strongly and quickly overexpressed after chromate exposure, suggesting the involvement of oscA in chromate resistance, which was further confirmed by means of a complementation experiment. Phenotype MicroArray (PM) analysis made it possible to assay 1536 phenotypes and also indicated that the oscA gene was involved in the utilization of organosulphur compounds as a sole source of sulphur. This is believed to be the first evidence that oscA plays a role in activating a sulphur starvation response, which is required to cope with oxidative stress induced by chromate.

The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

ABSTRACT Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS 2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS 2 ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS 2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM and k cat of 0.27 ± 0.04 s−1) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.