Variants of the Obligate Methanotroph Isolate 761M Capable of Growth on Glucose in the Absence of Methane

Isolate 761M is an unusual type I methanotroph that possesses a complete tricarboxylic acid cycle. Variants of this methanotroph that were capable of growth with methanol (isolate 761AR) or glucose (isolate 761H) have been isolated. Cultures of isolate 761H grown with glucose and casein hydrolysate as the sole carbon and energy sources retained the ability to grow on methane, contained methane monooxygenase and 3-hexulose phosphate synthase, and possessed intracytoplasmic membranes similar to those found in thin sections of isolate 761M grown on methane. Methane monooxygenase was also present in cultures of isolate 761AR grown on methanol and casein hydrolysate.     

A New Labyrinthulid Isolate That Produces Only Docosahexaenoic Acid

We show here that a new labyrinthulid strain, L72, isolated from a fallen leaf in the Seto Inland Sea of Japan, produced only docohexaenoic acid (DHA) among all the long-chain polyunsaturated fatty acids (LCPUFAs). The main fatty acid composition was 16:0 (28.9%), 18:0 (7.2%), 18:1 (5.7%), 18:2 (10.4%), and DHA (45.9%) without any other LCPUFA. The lipid content of the strain was 27.4%. The cells had many lipid bodies, which were densely located in all of the cells. On phylogenetic analysis using the 18S rDNA sequence, the strain was located in the labyrinthulids group, forming a monophyletic group with Labyrinthula sp. (strain s) and Labyrinthuila sp. (strain L59). We further tested the culture optimization of strain L72 to evaluate the ability of the strain to produce DHA. The optimum salt concentration and the temperature of the strain were 100% of artificial seawater and 20°C. Strain L72 could grow well on soybean oil (SBO) or soybean lecithin (SBL) as the carbon source. When 20 g/l of SBL was added to the medium, DHA production reached the maximum amount at 0.67 g/l for 14 d. The two important facts, that the strain can use SBL as the main nutrient and contains only DHA among the LCPUFAs, will be of great advantage for industry.     

Biosynthesis of Glutamic Acid in Saccharomyces: Accumulation of Tricarboxylic Acid Cycle Intermediates in a Glutamate Auxotroph

Aconitaseless glutamic acid auxotroph MO-1-9B of Saccharomyces grew in glutamic acid-supplemented minimal medium, but failed to grow when glutamic acid was substituted by proline, arginine, ornithine, or glutamine. This mutant was also unable to utilize lactate or glycerol as a carbon source. Under a glutamic acid-limiting condition, by using acetate-1-(14)C as tracer, the mutant accumulated rather large amounts of (14)C-citric acid and (14)C-succinic acid when compared with the wild-type strain. Under excess glutamic acid supplementation, accumulation of citric acid and succinic acid was considerably reduced. When (14)C-glutamic acid-(U) was used as tracer, (14)C-alpha-ketoglutaric acid, (14)C-citric acid, and (14)C-succinic acid were accumulated in the mutant. The citric acid peak was the largest, followed by alpha-ketoglutaric acid and succinic acid. In the wild-type strain under similar conditions, only small amounts of (14)C-citric acid and (14)C-succinic acid and no (14)C-alpha-ketoglutaric acid were accumulated.     

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates: DIFFERENTIAL CHEMOTACTIC RESPONSE TOWARDS RECEPTOR LIGANDS*

We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T_m) and unfolding enthalpy (ΔH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.     

Complete Genomic Sequence Analysis of a New SV40 Isolate

The genome of a new SV40 strain (SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

Photosynthesis in the Higher Plant, Vicia faba: III. Serine, a Precursor of the Tricarboxylic Acid Cycle

Evidence is presented to support the hypothesis that serine, rather than 3-phosphoglycerate of the Calvin cycle, is a precursor of the tricarboxylic acid cycle during photosynthesis by the higher plant, Vicia faba. Identification of the serine intermediate is based upon a unique C₁ > C₂ > C₃ isotope distribution for that metabolite following the fixation of ¹⁴CO₂. This labeling pattern, while incompatible with an origin either in the Calvin cycle or the glycolate pathway, satisfies a critical criterion for the 3-carbon precursor of the anomalously labeled organic acids. The predominant carboxyl carbon atom labeling of serine reflects either a mixing of two pools of that metabolite, ie., C₁ = C₂ > C₃ and C₁ > C₂ = C₃, or a higher order of complexity in its synthesis. An anomalous C₁ = C₂ > C₃ < C₄ distribution for aspartate, however, suggests an origin by the carboxylation of a 3-carbon intermediate related to serine which has a C₁ = C₂ > C₃ distribution. The latter distribution has been proposed for the serine intermediate of the postulated formate pathway. This pathway is described by the generalized metabolic sequence: CO₂ → formate → serine → organic acids. Corresponding carbon atom distributions for citrate (C₁ > C₂), aspartate (C₂ > C₃), and serine (C₂ > C₃) belie a precursor-product relationship with alanine (C₂ = C₃), which is a molecular parameter of the Calvin cycle product, 3-phosphoglycerate.     

Photosynthesis in the Higher Plant Vicia faba: V. Role of Malate as a Precursor of the Tricarboxylic Acid Cycle

In the higher plant Vicia faba, anomalous labeling patterns in the organic acids and related amino acids of the tricarboxylic acid cycle which result from photosynthetic ¹⁴CO₂ fixation (in conjunction with an enzyme localization pattern unique to plant mitochondria) suggest that the tricarboxylic acid cycle functions primarily as a pathway leading to glutamic acid biosynthesis during autotrophic growth. The distribution of isotope in citrate indicates little recycling of oxaloacetate for the resynthesis of citrate. Rather, malate appears to provide both the C₂ and C₄ fragments for the synthesis of citrate, and [³H]formate and ¹⁴CO₂-labeling patterns implicate serine as the ultimate C₃ precursor of malate.     

On the Metabolic Relationship between the Calvin Cycle and the Tricarboxylic Acid Cycle: IV. A Plant Survey for Anomalous Acetyl Coenzyme A

Leaves of 10 randomly selected plants representing eight dicotyledonous families were exposed to ¹⁴CO₂ for a 10-minute period in the light. Citrate and alanine were isolated, purified isotopically, and degraded to obtain the ¹⁴C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.     

IGF Type 1 Receptor: A Cell Cycle Progression Factor That Regulates Aging

No abstract available.     

Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle : Evidence for Partial Oxidative and Reductive Pathways during Dark Ammonium Assimilation

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH₄⁺ in the dark under anaerobic conditions. Addition of NH₄⁺ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO₂ efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H¹⁴CO₃⁻ to anaerobic cells assimilating NH₄⁺ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH₄⁺ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH₄⁺ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH₄⁺ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH₄⁺ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.     

Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 ± 27 and 321 ± 16 nmol h^-1 mg of protein ^-1, respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 ± 260 and 995 ± 160 nmol h^-1 mg of protein^-1, respectively. The substrate affinity of 68-1 sMMO to naphthalene (K_m, 70 ± 4 μM) and TCE (K_m, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.     

Mqo,a Tricarboxylic Acid Cycle Enzyme,Is Required for Virulence of Pseudomonas syringae pv. tomato Strain DC3000 on Arabidopsis thaliana

Plant pathogenic bacteria, such as Pseudomonas syringae pv. tomato strain DC3000, the causative agent of tomato bacterial speck disease, grow to high levels in the apoplastic space between plant cells. Colonization of plant tissue requires expression of virulence factors that modify the apoplast to make it more suitable for pathogen growth or facilitate adaptation of the bacteria to the apoplastic environment. To identify new virulence factors involved in these processes, DC3000 Tn5 transposon insertion mutants with reduced virulence on Arabidopsis thaliana were identified. In one of these mutants, the Tn5 insertion disrupted the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the tricarboxylic acid cycle. mqo mutants do not grow to wild-type levels in plant tissue at early time points during infection. Further, plants infected with mqo mutants develop significantly reduced disease symptoms, even when the growth of the mqo mutant reaches wild-type levels at late stages of infection. Mutants lacking mqo function grow more slowly in culture than wild-type bacteria when dicarboxylates are the only available carbon source. To explore whether dicarboxylates are important for growth of DC3000 in the apoplast, we disrupted the dctA1 dicarboxylate transporter gene. DC3000 mutants lacking dctA1 do not grow to wild-type levels in planta, indicating that transport and utilization of dicarboxylates are important for virulence of DC3000. Thus, mqo may be required by DC3000 to meet nutritional requirements in the apoplast and may provide insight into the mechanisms underlying the important, but poorly understood process of adaptation to the host environment.One important aspect of interactions between plant pathogens and their hosts is the ability of the pathogen to obtain nutrients within the plant tissue. Nutrient acquisition is essential for growth within the host, since both cell division and DNA replication can be influenced by nutrient availability. Bacterial plant pathogens differ in the strategies they use to get necessary nutrients during infection. Some pathogens, such as Agrobacterium tumefaciens, elicit production of specific carbon and nitrogen sources by the plant (1). Other pathogens may rely on metabolites that are readily available in the plant apoplast or may stimulate the release of water or nutrients from surrounding plant cells (27).Little is known about how pathogenic Pseudomonas syringae strains acquire nutrients when growing in their hosts. P. syringae strains are gram-negative gammaproteobacteria, which as a group cause disease on many agriculturally important plants. For example, P. syringae pv. tomato strain DC3000 causes disease on tomato, A. thaliana, and several agriculturally important Brassicas, such as turnip, mustard, collard, and cauliflower (8, 51). Initially, DC3000 colonizes plant surfaces and then enters the plant tissue through natural openings (such as stomata) or wounds (34, 38). DC3000 then establishes itself in the plant apoplast, the intercellular space between plant cells (38). Once in the apoplast of susceptible hosts, DC3000 multiplies to high levels, and the infected plants develop disease symptoms, including chlorosis (yellowing) of the leaf tissue and necrotic spots or patches called lesions (38, 49). Pseudomonads, such as P. aeruginosa and P. fluorescens, preferentially utilize tricarboxylic acid (TCA) cycle intermediates (20, 29, 33, 44), and DC3000 utilizes these carbon sources in culture (19). Some studies to investigate nutrient acquisition of DC3000 have been carried out (3, 7, 40); however, it is not clear what carbon sources DC3000 utilizes when growing in plant tissue.Several virulence factors are necessary for DC3000 to enter, grow inside the plant, and cause disease. Like many other bacterial pathogens, DC3000 uses a type III secretion system (TTSS) (15), which is encoded by the hrp/hrc genes, to inject effector proteins into plant cells (16, 27). Many of these effectors suppress host defenses, and it is likely that some may be involved in modulating the apoplastic environment or nutrient acquisition (16). DC3000 also produces the phytotoxin coronatine, which promotes entry of the bacteria into the plant apoplast by stimulating the opening of stomata (34) and is required for bacterial growth in the apoplast by suppressing salicylic acid (SA)-dependent host defenses (4, 45). Coronatine also promotes disease symptom development via an SA-independent mechanism (4). While much emphasis has been placed on exploring how type III-secreted effectors and coronatine promote DC3000 virulence, other factors are also likely to be important during pathogenesis.To identify additional factors involved in pathogenesis, we undertook a genetic screen to identify novel virulence factors (5, 24). DC3000 mutants with reduced virulence were identified by assaying for their ability to elicit disease symptoms on A. thaliana and tomato plants (24, 37). One of these mutants, AK4C9, had reduced virulence on both hosts. The gene disrupted in this mutant is the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the TCA cycle. mqo mutants grow more slowly than wild-type DC3000 in planta and in culture when dicarboxylates are the only carbon source, suggesting that dicarboxylates are important for the growth of DC3000 in the apoplast. In the present study, we explore the role of Mqo and a dicarboxylate transporter, DctA1, in DC3000 pathogenesis.     

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: II. Intermediary Metabolism and the Location of Control Points

Long term feeding of acetate-2-¹⁴C, ¹⁴CO₂, citrate-1,5-¹⁴C, fumarate-2,3-¹⁴C, and succinate-2,3-¹⁴C to mung bean (Phaseolus aureus L. var. Mungo) leaves in the dark gave labeling predominantly in tricarboxylic acid cycle intermediates. Kinetics of the intermediates during dark/light/dark transitions showed a light-induced interchange of ¹⁴C between malate and aspartate, usually resulting in an accumulation of ¹⁴C in malate and a decrease of it in aspartate. ¹⁴C-Phosphoenolpyruvate also showed a marked decrease during illumination. Changes in other intermediates of the tricarboxylic acid cycle were relatively minor. The kinetic data have been analyzed using the Chance crossover theorem to locate control points during the dark/light/dark transitions. The major apparent control points are located at malate and isocitrate dehydrogenases, and less frequently at citrate synthase and fumarase. These findings are explained in terms of the light-induced changes in adenine nucleotides and nicotinamide adenine dinucleotides.     

Regulation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum: Properties Associated with Replenishment of Tricarboxylic Acid Cycle Intermediates during Ammonium Assimilation

Two isoforms of phosphoenolpyruvate carboxylase (PEPC) with very different regulatory properties were partially purified from the green alga Selenastrum minutum. They were designated PEPC₁ and PEPC₂. PEPC₁ showed sigmoidal kinetics with respect to phosphoenolpyruvate (PEP) whereas PEPC₂ exhibited a typical Michaelis-Menten response. The S_0.5(PEP) of PEPC₁ was 2.23 millimolar. This was fourfold greater than the S_0.5(PEP) of PEPC₂, which was 0.57 millimolar. PEPC₁ was activated more than fourfold by 2.0 millimolar glutamine and sixfold by 2.0 millimolar dihydroxyacetone phosphate (DHAP) at a subsaturating PEP concentration of 0.625 millimolar. In contrast, PEPC₂ showed only 8% and 52% activation by glutamine and DHAP, respectively. The effects of glutamine and DHAP were additive. PEPC₁ was more sensitive to inhibition by glutamate, 2-oxoglutarate, and aspartate than PEPC₂. Both isoforms were equally inhibited by malate. All of these metabolites affected only the S_0.5(PEP) not the V_max. The regulatory properties of S. minutum PEPC in vitro are discussed in terms of (a) increased rates of dark carbon fixation (shown to be catalyzed predominantly by PEPC) and (b) changes in metabolite levels in vivo during enhanced NH₄₊ assimilation. Finally, a model is proposed for the regulation of PEPC in vivo in relation to its role in replenishing tricarboxylic acid cycle intermediates consumed in NH₄₊ assimilation.