RESUMEN
Rhodococcus opacus 1CP produces trehalose dinocardiomycolates during growth on long-chained n-alkanes. Trehalose and trehalose-6-phosphate, which are synthesized via the OtsAB pathway, are probable intermediates in the biosynthesis of these biosurfactants. By molecular genetic screening for trehalose-6-phosphate synthases (TPSs and OtsAs), two chromosomal fragments of strain 1CP were obtained. Each contained an ORF whose amino acid sequence showed high similarity to TPSs. To prove the activity of the otsA1 and otsA2 gene product and to detect catalytic differences, both were expressed as His-tagged fusion proteins. Enzyme kinetics of the enriched proteins using several potential glucosyl acceptors showed an exclusive preference for glucose-6-phosphate. In contrast, both enzymes were shown to differ significantly from each other in their activity with different glucosyl nucleotides as glucosyl donors. OtsA1-His10 showed highest activity with ADP-glucose and UDP-glucose, whereas OtsA2-His10 preferred UDP-glucose. In addition, the wild-type OtsA activity of R. opacus 1CP was investigated and compared with recombinant enzymes. Results indicate that OstA2 mainly contributes to the trehalose pool of strain 1CP. OtsA1 seems to be involved in the overproduction of trehalose lipids. For the first time, a physiological role of two different OtsAs obtained of a single Rhodococcus strain was presumed.
Asunto(s)
Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Rhodococcus/enzimología , Rhodococcus/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Expresión Génica , Glucosiltransferasas/química , Cinética , Datos de Secuencia Molecular , Fosfatos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Especificidad por Sustrato , Trehalosa/metabolismoRESUMEN
Rhodococcus opacus 1CP, a potent degrader of (chloro-) aromatic compounds was found to utilise C10-C16 n-alkanes as sole carbon sources. Highest conversion rates were observed with n-tetradecane and n-hexadecane, whereas the utilisation of n-dodecane and n-decane was considerably slower. Thin-layer chromatography of organic extracts of n-alkane-grown 1CP cultures indicated the growth-associated formation of a glycolipid which was characterised as a trehalose dimycolate by 1H-NMR spectroscopy and mass spectrometry. Total chain lengths between 48 and 54 carbons classify the fatty acid residues as nocardiomycolic acids. The presence of two double bonds in each mycolic acid is another feature that distinguishes the corresponding trehalose dinocardiomycolates from trehalose dicorynomycolates reported for Rhodococcus erythropolis DSM43215 and Rhodococcus ruber IEGM231. R. opacus 1CP was not found, even under nitrogen limitation, to produce anionic trehalose tetraesters which have previously been reported for R. erythropolis DSM43215.