Descripción: Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing E. coli. While PHB accumulation was found to increase the transcription of dnaK and ibpA, the expression of these genes and of groES, groEL, rpoH, dps, and yfiD was reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA. The effect of PhaP was also analyzed in non-PHBsynthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect in E. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins. © 2011, American Society for Microbiology.

Descripción: Asr1, a tomato gene induced by abiotic stress, belongs to a family, composed by at least three members, involved in adaptation to dry climates. To understand the mechanism by which proteins of this family seem to protect cells from water loss in plants, we expressed Asr1 in the heterologous expression system Saccharomyces cerevisiae under the control of a galactose-inducible promoter. In a mutant yeast strain deficient in one component of the stress-responsive high-osmolarity glycerol (HOG) pathway, namely the MAP kinase Hog1, the synthesis of ASR1 protein restores growth under osmotic stress conditions such as 0.5 M NaCl and 1.2 M sorbitol. In contrast, the rescuing of this phenotype was less evident using a wild-type strain or the upstream MAP kinase kinase (Pbs2)-deficient strain. In both knock-out strains impaired in glycerol synthesis because of a dysfunctional HOG pathway, but not in wild-type, ASR1 led to the accumulation of endogenous glycerol in an osmotic stress-independent and unrestrained manner. These data suggest that ASR1 complements yeast HOG-deficient phenotypes by inducing downstream components of the HOG pathway. The results are discussed in terms of the function of ASR proteins in planta at the molecular and cellular level. Copyright © Physiologia Plantarum 2006.

Descripción: We assessed the effects of different arcA mutations on poly(3- hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% ± 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% ± 3%. This strain grew in a simple medium at a specific growth rate of 0.69 ± 0.07 h-1, whereas the deletion mutant needed several nutritional additives and snowed a specific growth rate of 0.56 ± 0.06 h-1. The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Descripción: Pseudomonas sp. 14-3 accumulates polyhydroxybutyrate (PHB) from octanoate, but not from glucose. To elucidate this unusual phenotype, genes responsible for the synthesis of PHB were cloned and analyzed. A PHB polymerase gene (phaC) was found downstream from genes coding for a β-ketothiolase (phaA), an acetoacetyl-coenzyme A reductase (phaB) and a putative transcriptional regulator (phaR). All genes were similar to pha genes from several related species, but differences were observed in the distal region of phaA. Complementation with heterologous β-ketothiolase genes from Azotobacter sp. FA8 or Pseudomonas putida GPp104 restored the capability of Pseudomonas sp. 14-3 to synthesize PHB from glucose, demonstrating that its β-ketothiolase was nonfunctional. Analysis of the genome sequences of other Pseudomonas species has revealed the existence of putative β-ketothiolase genes. The functionality of one of these thiolase genes, belonging to P. putida GPp104, was experimentally demonstrated. Pseudomonas sp. 14-3 is the first natural phaA mutant described, that despite this mutation accumulates high amounts of PHB when growing on fatty acids. © 2006 Federation of European Microbiological Societies.

Descripción: Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation. © 2011 The Author.