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Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum.

ABSTRACT: 1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.

SUBMITTER: Higa A

PROVIDER: S-EPMC1165440 | biostudies-other | 1975 May

REPOSITORIES: biostudies-other

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Project description:Halobacterium salinarum is a bioenergetically flexible, halophilic microorganism that can generate energy by respiration, photosynthesis, and the fermentation of arginine. In a previous study, using a genome-scale metabolic model, we have shown that the archaeon unexpectedly degrades essential amino acids under aerobic conditions, a behavior that can lead to the termination of growth earlier than necessary. Here, we further integratively investigate energy generation, nutrient utilization, and biomass production using an extended methodology that accounts for dynamically changing transport patterns, including those that arise from interactions among the supplied metabolites. Moreover, we widen the scope of our analysis to include phototrophic conditions to explore the interplay between different bioenergetic modes. Surprisingly, we found that cells also degrade essential amino acids even during phototropy, when energy should already be abundant. We also found that under both conditions considerable amounts of nutrients that were taken up were neither incorporated into the biomass nor used as respiratory substrates, implying the considerable production and accumulation of several metabolites in the medium. Some of these are likely the products of forms of overflow metabolism. In addition, our results also show that arginine fermentation, contrary to what is typically assumed, occurs simultaneously with respiration and photosynthesis and can contribute energy in levels that are comparable to the primary bioenergetic modes, if not more. These findings portray a picture that the organism takes an approach toward growth that favors the here and now, even at the cost of longer-term concerns. We believe that the seemingly "greedy" behavior exhibited actually consists of adaptations by the organism to its natural environments, where nutrients are not only irregularly available but may altogether be absent for extended periods that may span several years. Such a setting probably predisposed the cells to grow as much as possible when the conditions become favorable.

Project description:BackgroundHalobacterium sp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment, Halobacterium NRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach.ResultsA modified 2-D gel electrophoretic procedure, employing IPG strips in the range of pH 3-6, enabled improved separation of acidic proteins relative to previous techniques. Combining experimental data from 2-D gel electrophoresis with available genomic information, allowed the identification of at least 30 cellular proteins involved in many cellular functions: stress response and protein folding (CctB, PpiA, DpsA, and MsrA), DNA replication and repair (DNA polymerase A alpha subunit, Orc4/CDC6, and UvrC), transcriptional regulation (Trh5 and ElfA), translation (ribosomal proteins Rps27ae and Rphs6 of the 30 S ribosomal subunit; Rpl31eand Rpl18e of the 50 S ribosomal subunit), transport (YufN), chemotaxis (CheC2), and housekeeping (ThiC, ThiD, FumC, ImD2, GapB, TpiA, and PurE). In addition, four gene products with undetermined function were also identified: Vng1807H, Vng0683C, Vng1300H, and Vng6254. To study the heat shock response of Halobacterium NRC-1, growth conditions for heat shock were determined and the proteomic profiles under normal (42 degrees C), and heat shock (49 degrees C) conditions, were compared. Using a differential proteomic approach in combination with available genomic information, bioinformatic analysis revealed five putative heat shock proteins that were upregulated in cells subjected to heat stress at 49 degrees C, namely DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2.ConclusionThe modified 2-D gel electrophoresis markedly enhanced the resolution of the extremely acidic proteome of Halobacterium NRC-1. Constitutive expression of stress proteins and chaperones help the organism to adapt and survive under extreme salinity and other stress conditions. The upregulated expression pattern of putative chaperones DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2 under elevated temperature clearly suggests that Halobacterium NRC-1 has a sophisticated defense mechanism to survive in extreme environments.

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Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum.

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