Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli.

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RESUMO

From genomic libraries of Alcaligenes eutrophus H16 in lambda L47 and in pVK100, we cloned DNA fragments which restored the wild-type phenotype to poly(beta-hydroxybutyric acid) (PHB)-leaky mutants derived from strains H16 and JMP222. The nucleotide sequence analysis of a 4.5-kb region of one of these fragments revealed two adjacent open reading frames (ORF) which are relevant for the expression of the PHB-leaky phenotype. The 1,799-bp ORF1 represented a gene which was referred to as phbI. The amino acid sequence of the putative protein I (Mr, 65,167), which was deduced from phbI, exhibited 38.9% identity with the primary structure of enzyme I of the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system (PEP-PTS). The upstream 579-bp ORF2 was separated by 50 bp from ORF1. It included the 270-bp phbH gene which encoded protein H (Mr, 9,469). This protein exhibited 34.9% identity to the HPr protein of the E. coli PEP-PTS. Insertions of Tn5 in different PHB-leaky mutants were mapped at eight different positions in phbI and at one position in phbH. Mutants defective in phbH or phbI exhibited no pleiotropic effects and were not altered with respect to the utilization of fructose. However, PHB was degraded at a higher rate in the stationary growth phase. The functions of these HPr- and enzyme I-like proteins in the metabolism of PHB are still unknown. Evidence for the involvement of these proteins in regulation of the metabolism of intracellular PHB was obtained, and a hypothetical model is proposed.

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