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

Abstract

From genomic libraries of Alcaligenes eutrophus H16 in XL47 and in pVK100, we cloned DNA fragments which restored the wild-type phenotype to poly(j3-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 phbl. The amino acid sequence of the putative protein I (Mr,65,167), which was deduced from phbl, 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 (Mrs 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 phbl 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.The ability to accumulate poly(P-hydroxybutyric acid) (PHB), functioning as a carbon and/or energy source or as a sink for reducing equivalents, is widespread among prokaryotic organisms. The hydrogen-oxidizing bacterium Alcaligenes eutrophus accumulates PHB to more than 80% (wt/wt) of the cellular dry weight (48). PHB and related polyesters are already produced industrially on a small scale by A. eutrophus and are distributed under the trade name Biopol (7,30). Recently, two classes of transposon-induced mutants of A. eutrophus, which are affected in the accumulation of PHB, were isolated (55). PHB-negative mutants were completely impaired in the synthesis of PHB; in this class of mutants TnS::mob (56) mapped within the structural gene of PHB synthase. This fragment encodes the complete A. eutrophus PHB-synthetic pathway and harbors the genes for the biosynthetic 3-ketothiolase (phbA), NADPH-dependent acetoacetyl coenzyme A reductase (phbB), and PHB-synthase (phbC) (39,40,55,58). The genes are organized in one operon (phbCAB) which is transcribed from a &r70-dependent promoter (54, 60). DNA fragments harboring this operon conferred the ability to accumulate PHB to Escherichia coli and to many pseudomonads belonging to rRNA homology group 1 (55, 58, 61, 63, 64 DNA sequence analysis and analysis of sequence data. DNA sequencing was performed by the dideoxy-chain termination method of Sanger et al. (46) with alkali-denatured doublestranded plasmid DNA, 7-deazaguanosine 5'-triphosphate instead of dGTP, and [a-35S]...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…The signaling pathway might also include NPr and EIIA Ntr , as genome sequencing has revealed the presence of both ptsN and npr in the A. vinelandii rpoN operon (551). Similarly, "leaky" Ralstonia eutropha phbI and phbH mutants show diminished accumulation of poly-␤-hydroxybutyrate (687). A close relationship between phbI and phbH and the ptsP and npr genes of E. coli was established (680), although the PhbI protein of R. eutropha lacks the NifA-like extension typical of other EI Ntr s. Mutants of P. aeruginosa (854) or Legionella pneumophila (333) affected in ptsP do not show an obvious phenotype when grown in rich medium.…”

Physiological and Proteomic Adaptation of “Aromatoleum aromaticum” EbN1 to Low Growth Rates in Benzoate-Limited, Anoxic Chemostats

“…Generally, PHA synthesis requires high intracellular ratios of NAD(P)H to NAD(P) ϩ and of acetyl-CoA to free CoA (59). However, other factors, which have not been studied in much detail yet, also have an impact on PHA synthesis (52,53). Pseudomonas oleovorans has been demonstrated to accumulate PHA also under carbon-limited conditions (17,58).…”

Comparative Genomic Analyses of the Bacterial Phosphotransferase System

“…Genetic evidence has indicated that in various bacteria, processes regulated by the PTS include (i) transport of non-PTS carbon sources (35), (ii) the net production of carbon and energy storage sources, such as poly-␤-hydroxybutyrate (64,90) and glycogen (91), (iii) the switch between fermentative and respiratory metabolism (36), (iv) flagellar motility (54), and (v) the control of 54 -dependent transcription of carbon and nitrogen metabolic genes (12,46,63,70,74). Some evidence implicates the PTS in the regulation of cell division (44,63).…”