, Volume 38, Issue 4, pp 493-501

Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea

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Abstract

From a genomic library of Thiocystis violaceae strain 2311 in λL47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned. The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A. eutrophus. The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis. The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a β-ketothiolase [phbA Tv, relative molecular mass (Mr) 40850], which exhibited 87.3% amino acid identify with the β-ketothiolase from Chromatium vinosum. The amino acid sequences of the putative proteins deduced from ORF2Tv (Mr 41 450) and phbC Tv (Mr 39 550), which were located upstream of and antilinear to phbA Tv, exhibited 74.7% and 87.6% amino acid identify, respectively, with the corresponding gene products of C. vinosum. Downstream of and antilinear to phbC Tv was located ORF5, which encodes for a protein of high relative molecular mass (Mr 76428) of unknown function. With respect to the divergent organisation of ORF2Tv and phbC Tv on one side and of phbA Tv on the other side and from the homologies of the putative gene products, this region of the T. violaceae genome resembled very much the corresponding region of C. vinosum, which was identified recently.

Correspondence to: A. Steinbüchel