Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.     

Progress in pathogenesis research of Ustilago maydis,and the metabolites involved along with their biosynthesis

Ustilago maydis is a pathogenic fungus that causes corn smut. Because of its easy cultivation and genetic transformation, U. maydis has become an important model organism for plant-pathogenic basidiomycetes. U. maydis is able to infect maize by producing effectors and secreted proteins as well as surfactant-like metabolites. In addition, the production of melanin and iron carriers is also associated with its pathogenicity. Here, advances in our understanding of the pathogenicity of U. maydis, the metabolites involved in the pathogenic process, and the biosynthesis of these metabolites, are reviewed and discussed. This summary will provide new insights into the pathogenicity of U. maydis and the functions of associated metabolites, as well as new clues for deciphering the biosynthesis of metabolites.     

Development of a genetic system for the moderately halophilic bacterium Halobacillus halophilus: generation and characterization of mutants defect in the production of the compatible solute proline

A procedure for markerless mutagenesis gene deletions was developed for the moderately halophilic model strain Halobacillus halophilus. Gene transfer was achieved by protoplast fusion and allelic replacement by a two-step procedure. In the first step the non-replicating plasmid integrated over the upstream or the downstream region of the target gene or operon into the chromosome to obtain single-crossover mutants. When cells were grown under non-selective conditions a second homologous recombination happened (segregation). This resulted in either the wild-type or the mutated allele. The method was used to delete the proHJA operon from H. halophilus. The mutant still produced proline and thus was not proline auxotroph but it completely lost the ability to produce proline as a compatible solute. However, growth was not impaired and the loss of the solute proline was compensated for by an increase in glutamate, glutamine and ectoine concentration. Expressions of the genes encoding the biosynthesis enzymes of theses solutes were upregulated and the activity of the key enzyme in glutamine biosynthesis, the glutamine synthetase, was increased. A model for the proline biosynthesis in the ΔproHJA mutant is discussed.     

Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6

Among the 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as strain QW6 showed high capacity in the removal of toxic oxyanions of tellurium in a wide range of culture medium factors including pH (5.5-10.5), temperature (25-45 degrees C), various salts including NaCl, KCl, and Na(2)SO(4) (0.5-4M), selenooxyanions (2-10mM), and at different concentrations of potassium tellurite (0.5-1mM) under aerobic condition. Phenotypic characterization and phylogenetic analyses based on 16S rDNA sequence comparisons indicated that this strain was a member of the genus Salinicoccus. The maximum tellurite removal was exhibited in 1.5M NaCl at 35 degrees C, while the activity reduced by 53% and 47% at 25 and 45 degrees C, respectively. The optimum pH for removal activity was shown to be 7.5, with 90% and 83% reduced removal capacities at the two extreme values of 5.5 and 10, respectively. The impact of different concentrations of selenooxyanions (2-10mM) on tellurite removal by strain QW6 was evaluated. The ability of strain QW6 in the removal of tellurite in the presence of 6mM selenite increased by 25%. The concentration of toxic potassium tellurite in the supernatant of the bacterial culture medium decreased by 99% (from 0.5 to 0.005mM) after 6 days and the color of the medium changed to black due to the formation of less toxic elemental tellurium.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Structure,function and biosynthesis of carotenoids in the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus halophilus</Emphasis>

Inhibitor studies and mutant analysis revealed a C₃₀ pathway via 4,4′-diapophytoene and 4,4′-diaponeurosporene to 4,4′-diaponeursoporene-4-oic acid esters related to staphyloxanthin in Halobacillus halophilus. Six genes may be involved in this biosynthetic pathway and could be found in two adjacent gene clusters. Two genes of this pathway could be functionally assigned by functional pathway complementation as a 4,4′-diapophytoene synthase and a 4,4′-diapophytoene desaturase gene. These genes were organized in two operons together with two putative oxidase genes, a glycosylase and an acyl transferase ortholog. Pigment mutants were obtained by chemical mutagenesis. Carotenoid analysis showed that a white mutant accumulated 4,4′-diapophytoene due to a block in desaturation. In a yellow mutant carotenogenesis was blocked at the stage of 4,4′-diaponeurosporene and in an orange mutant at the stage of 4,4′-diaponeurosporene-4-oic acid. The protective function of these pigments could be demonstrated for H. halophilus after inhibition of carotenoid synthesis by initiation of oxidative stress. A degree of oxidative stress which still allowed 50% growth of carotenogenic cells resulted in the death of the cells devoid of colored carotenoids.     

Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.     

Identification of cold acclimation-responsive Rhododendron genes for lipid metabolism, membrane transport and lignin biosynthesis: importance of moderately abundant ESTs in genomic studies

We have previously analysed expressed sequence tags (ESTs) from non-acclimated (NA) and cold-acclimated (CA) Rhododendron leaves, and identified highly abundant complementary DNAs (cDNAs) possibly involved in cold acclimation. A potentially significant, but relatively unexplored, application of these EST data sets is the study of moderately abundant cDNAs, such as those picked only 1-3 times from each Rhododendron EST library containing approximately 430 ESTs. Using statistical tests and Northern blots, we established that the probability of differential expression of moderately abundant cDNAs based on the EST data is, indeed, a reasonably accurate predictor of their 'true' upregulation or downregulation as 11 out of 13 cDNAs (85%) studied fit this criterion. The analyses also revealed four aspects of cold acclimation in Rhododendron leaf tissues. Firstly, the concomitant upregulation of long-chain acyl-coenzyme A (acyl-CoA) synthetase, CTP:cholinephosphate cytidylyltransferase and delta-12 fatty acid desaturase in CA leaf tissues suggests that phospholipid biosynthesis and desaturation are important components of cold hardening in Rhododendron. Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphatemalic enzyme (NADP-ME) in CA tissues suggests that malate is an important source of acetyl-CoA used for fatty acid biosynthesis during cold acclimation. Thirdly, down-regulation of plasma membrane intrinsic protein (PIP)2-1 aquaporin and upregulation of gated outward rectifying K+ channel (GORK) in CA tissues may be associated with the protection of overwintering leaves from freeze-induced cellular dehydration. Fourthly, upregulation of coumarate 3-hydroxylase may be associated with cell wall thickening in CA tissues. Physiological implications of these results, which reveal potentially novel regulations of cold acclimation in overwintering woody evergreens, are discussed. This work highlights the importance of also investigating low/moderately abundant ESTs (in addition to highly abundant ones) in genomic studies, in that it offers an effective strategy for identifying stress-related genes, especially when large-scale cDNA sequencing/microarray studies are not possible.     

Gene expression of alpha-endosulfine in the rat brain: correlative changes with aging, learning and stress

Endosulfine (EDSF) belongs to a highly conserved cAMP-regulated phosphoprotein (ARPP) family and was first isolated from ovine brain as a possible endogenous ligand for sulfonylurea receptors. To explore its involvement in brain functions, we investigated regional distribution of alpha-EDSF gene expression in the rat brain, and its regulation under physiological and pathological conditions. The majority of alpha-EDSF gene was expressed in the pyramidal neurons, which represent the principal excitatory neurons in various brain regions. Down-regulation of alpha-EDSF mRNA was detected in the rat hippocampus during long-term memory consolidation following a spatial learning experience, whereas swimming-related stress caused persistent up-regulation of alpha-EDSF gene expression in several brain regions. These changes, however, were absent from brains of diabetic rats that were subjected to the same behavioral treatments. Intracerebroventricular injection of streptozocin with a toxic dose induced severe learning deficits and brain structure alteration accompanied by a massive increase of alpha-EDSF mRNA in the somatosensory cortex. These results suggest that alpha-EDSF gene expression is differentially regulated by distinct brain processes involving excitatory neuronal activities.     

Mapping of the Na+, K+-ATPase subunit α 2 (ATP1A2) and muscle phosphofructokinase (PFKM) genes in pig by somatic cell hybrid analysis

Two expressed sequence tags were isolated from a porcine skeletal muscle cDNA library and identified as the putative partial cDNAs of the porcine Na⁺, K⁺-ATPase subunit α 2 ( ATP1A2 ) and muscle phosphofructokinase ( PFKM ) genes after sequencing and homology search. Results of analysis of a pig–rodent somatic cell hybrid panel by PCR allowed the assignments of ATP1A2 to porcine chromosome (chr) 4 and of PFKM to porcine chr 5. These assignments support previously observed conservation of syntenic relationships between human chr 1 and porcine chr 4 and between human chr 12 and porcine chr 5.     

Identification of cytochromes o and a3 as functional terminal oxidases in the thermophilic bacterium, PS3

Low-temperature photodissociation spectra of membranes from the thermophile PS3 reveal cytochromes o and a₃. The latter reacts with O₂ at −103°C to give a light-insensitive compound(s), but the initial stages of O₂ binding to cytochrome o could not be studied under these conditions. Photochemical action spectra identify cytochromes a₃ and o, but not a CO-binding c-type cytochrome, as functional terminal oxidases in this bacterium.     

Identification of three cDNA clones expressed in the leaf extension zone and with altered patterns of expression in theslender mutant of barley: a tonoplast intrinsic protein, a putative structural protein and protochlorophyllide oxidoreductase

Three cDNA clones have been isolated on the basis of altered patterns of expression in the leaf extension zone of the developmental mutant,slender barley, compared with the wild type. mRNAs corresponding to two of the cDNAs, 7s and 8s, are increased inslender compared with normal. 7s encodes a putative -TIP and is expressed throughout the elongation zone. -TIPs form transmembrane channels which allow the passive transfer of water. Although expression of 7s was increased inslender leaf tissue, the increase was much less extreme than that shown by Phillips and Huttly (1994) following the application of GA to an extreme dwarf ofArabidopsis. 8s is maximally expressed in the region of early cell elongation and has 66% encoded protein identity with MFS18, a cDNA encoding a putative cell wall structural protein isolated from male flowers of maize. Both 8s and MFS18 encode small (128 amino acids) basic proteins rich in glycine, alanine, proline and serine. mRNA corresponding to the third cDNA, 24n, is present at a greatly reduced level inslender compared with normal and encodes protochlorophyllide oxidoreductase (POR). POR catalyses the conversion of protochlorophyllide into chlorophyllide. The reduced level of POR mRNA is not correlated with a similar reduction in expanded leaf blade chlorophyll levels. Western analysis identified two POR proteins present in light-grown seedlings. Whilst the larger of the proteins is present throughout most of the leaf, the smaller protein mimics the mRNA results, being both maximally present in the elongation tissue and present at a reduced level inslender. An antagonistic relationship between chlorophyll biosynthesis and extension growth is suggested.     

Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole

When illuminated leaf discs and detached leaves of spinach ( Spinacia oleracea L. cv. Estivato) were exposed to 0.4 and 0.25 μl 1^-1 H₂S, respectively, pool sizes of cysteine and glutathione increased. In the dark, apart from these compounds, the level of γ-glutamyl-cysteine also increased. Incubation of leaf discs with 1.0 m M buthionine sulfoximine (BSO) resulted in the accumulation of cysteine only, both in the light and in darkness. When glycine was supplied to the petioles of detached leaves exposed to H₂S in the dark, the accumulation of glutathione was stimulated, while γ-glutamyl-cysteine accumulation was prevented completely. Glycolate and glyoxylate, precursors of glycine in the glycolate pathway, had nearly the same effect as glycine. Although other amino acids were apparently taken up equally well as glycine when supplied to the petiole, they were much less effective, or not effective at all, in restoring glutathione synthesis in the dark. These results provide evidence, that H₂S-induced glutathione accumulation in spinach leaves in the dark is limited by the availability of glycine, giving rise to the accumulation of the metabolic precursor γ-glutamyl-cysteine.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.