Enhancement of growth and salt tolerance of rice seedlings by ACC deaminase-producing Burkholderia sp. MTCC 12259.

Increasing soil salinity is often associated with accelerated ethylene production in plants, leading to overall growth reduction. The salt-tolerant 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing PGPR may alleviate salt stress by reducing the production of stress ethylene. In this study, a salt-tolerant ACC deaminase-producing strain named P50 was isolated from a coastal rice field in Odisha, India, which enhanced the growth of rice seedlings under salt stress. The P50 strain was identified as Burkholderia sp. based on phenotypic characteristics, MALDI-TOF MS data for ribosomal proteins and 16S rDNA sequence-based homology. Various PGP traits of strain P50 were characterized, among which the ACC deaminase activity was optimized at different physical conditions and confirmed by enzyme assay, as well as FTIR. The IAA, EPS and proline production of this strain were estimated under increasing NaCl concentrations essential for plant growth promotion under salt stress. Finally, the P50 strain was utilized in a gnotobiotic assay using rice seedlings (cv. Swarnamasuri) under saline stress. Seedlings treated with the P50 strain showed improvement in various morphological and biochemical characteristics, ROS scavenging antioxidant enzymatic activities, and reduced amounts of stress ethylene compared to non-inoculated strains under salinity. In addition, isolation of the ACC deaminase mutant of this strain was not found to reduce stress ethylene, confirming that the P50 strain was associated with a reduction in stress ethylene. Strain P50 was also found to colonize the root surfaces of rice seedlings associated with the plant-microbe interaction process. Thus, as an effective salt-tolerant PGPR, strain P50 can be utilized in salt-affected agricultural fields to improve plant growth in a sustainable manner.

Peroxisomal 2-Hydroxyacyl-CoA Lyase Is Involved in Endogenous Biosynthesis of Heptadecanoic Acid.

Circulating heptadecanoic acid (C17:0) is reported to be a pathology risk/prognosis biomarker and a dietary biomarker. This pathology relationship has been shown to be reliably predictive even when independent of dietary contributions, suggesting that the endogenous biosynthesis of C17:0 is related to the pathological aetiology. Little is known about C17:0 biosynthesis, which tissues contribute to the circulating levels, and how C17:0 is related to pathology. Hacl1+/- mice were mated to obtain Hacl1-/- and Hacl1+/+ control mice. At 14 weeks, they were anesthetized for tissue collection and fatty acid analysis. Compared to Hacl1+/+, C15:0 was not significantly affected in any Hacl1-/- tissues. However, the Hacl1-/- plasma and liver C17:0 levels were significantly lower: ~26% and ~22%, respectively. No significant differences were seen in the different adipose tissues. To conclude, Hacl1 plays a significant role in the liver and plasma levels of C17:0, providing evidence it can be endogenously biosynthesized via alpha-oxidation. The strong inverse association of C17:0 with pathology raises the question whether there is a direct link between α-oxidation and these diseases. Currently, there is no clear evidence, warranting further research into the role of α-oxidation in relation to metabolic diseases.

myo-Inositol catabolism in Bacillus subtilis.

The iolABCDEFGHIJ operon of Bacillus subtilis is responsible for myo-inositol catabolism involving multiple and stepwise reactions. Previous studies demonstrated that IolG and IolE are the enzymes for the first and second reactions, namely dehydrogenation of myo-inositol to give 2-keto-myo-inositol and the subsequent dehydration to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione. In the present studies the third reaction was shown to be the hydrolysis of 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione catalyzed by IolD to yield 5-deoxy-d-glucuronic acid. The fourth reaction was the isomerization of 5-deoxy-D-glucuronic acid by IolB to produce 2-deoxy-5-keto-D-gluconic acid. Next, in the fifth reaction 2-deoxy-5-keto-D-gluconic acid was phosphorylated by IolC kinase to yield 2-deoxy-5-keto-D-gluconic acid 6-phosphate. IolR is known as the repressor controlling transcription of the iol operon. In this reaction 2-deoxy-5-keto-D-gluconic acid 6-phosphate appeared to be the intermediate acting as inducer by antagonizing DNA binding of IolR. Finally, IolJ turned out to be the specific aldolase for the sixth reaction, the cleavage of 2-deoxy-5-keto-D-gluconic acid 6-phosphate into dihydroxyacetone phosphate and malonic semialdehyde. The former is a known glycolytic intermediate, and the latter was previously shown to be converted to acetyl-CoA and CO(2) by a reaction catalyzed by IolA. The net result of the inositol catabolic pathway in B. subtilis is, thus, the conversion of myo-inositol to an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO(2).

Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif.

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.

Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture.

Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into alpha-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.

Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.).

AIMS: This study was conducted to test the hypothesis that the bacterial strains possessing 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity may also promote growth of inoculated plants and could increase nodulation in legumes upon co-inoculation with rhizobia. METHODS AND RESULTS: Several rhizobacteria were isolated from maize rhizosphere through enrichment on ACC as a sole N source. Purified isolates were screened for growth promotion in maize under axenic conditions and for in vitro ACC-deaminase activity. A significant positive correlation was observed between in vitro ACC-deaminase activity of bacterial cells and root elongation. None of the isolates produced auxins. Bradyrhizobium japonicum produced less amount of auxins but did not carry ACC-deaminase activity. Results of pot experiment revealed that co-inoculation with Bradyrhizobium and plant growth promoting rhizobacteria (PGPR) isolates enhanced the nodulation in mung bean compared with inoculation with Bradyrhizobium alone. CONCLUSIONS: It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolysed endogenous ACC into ammonia and alpha-ketobutyrate instead of ethylene. Consequently, root and shoot growth as well as nodulation were promoted. SIGNIFICANCE AND IMPACT OF THE STUDY: The ACC-deaminase trait could be employed as an efficient tool to screen effective PGPR, which could be successfully used as biofertilizers to increase the growth of inoculated plants as well as nodulation in legumes.

Structural and mechanistic studies on carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily catalyzing the first step in carbapenem biosynthesis.

The first step in the biosynthesis of the medicinally important carbapenem family of beta-lactam antibiotics is catalyzed by carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily. CarB catalyzes formation of (2S,5S)-carboxymethylproline [(2S,5S)-t-CMP] from malonyl-CoA and l-glutamate semialdehyde. In addition to using a cosubstrate, CarB catalyzes C-C and C-N bond formation processes as well as an acyl-coenzyme A hydrolysis reaction. We describe the crystal structure of CarB in the presence and absence of acetyl-CoA at 2.24 A and 3.15 A resolution, respectively. The structures reveal that CarB contains a conserved oxy-anion hole probably required for decarboxylation of malonyl-CoA and stabilization of the resultant enolate. Comparison of the structures reveals that conformational changes (involving His(229)) in the cavity predicted to bind l-glutamate semialdehyde occur on (co)substrate binding. Mechanisms for the formation of the carboxymethylproline ring are discussed in the light of the structures and the accompanying studies using isotopically labeled substrates; cyclization via 1,4-addition is consistent with the observed labeling results (providing that hydrogen exchange at the C-6 position of carboxymethylproline does not occur). The side chain of Glu(131) appears to be positioned to be involved in hydrolysis of the carboxymethylproline-CoA ester intermediate. Labeling experiments ruled out the possibility that hydrolysis proceeds via an anhydride in which water attacks a carbonyl derived from Glu(131), as proposed for 3-hydroxyisobutyryl-CoA hydrolase. The structural work will aid in mutagenesis studies directed at altering the selectivity of CarB to provide intermediates for the production of clinically useful carbapenems.

Breakdown of 2-hydroxylated straight chain fatty acids via peroxisomal 2-hydroxyphytanoyl-CoA lyase: a revised pathway for the alpha-oxidation of straight chain fatty acids.

2-Hydroxyfatty acids, constituents of brain cerebrosides and sulfatides, were previously reported to be degraded by an alpha-oxidation system, generating fatty acids shortened by one carbon atom. In the current study we used labeled and unlabeled 2-hydroxyoctadecanoic acid to reinvestigate the degradation of this class of lipids. Both in intact and broken cell systems formate was identified as a main reaction product. Furthermore, the generation of an n-1 aldehyde was demonstrated. In permeabilized rat hepatocytes and liver homogenates, studies on cofactor requirements revealed a dependence on ATP, CoA, Mg(2+), thiamine pyrophosphate, and NAD(+). Together with subcellular fractionation data and studies on recombinant enzymes, this led to the following picture. In a first step, the 2-hydroxyfatty acid is activated to an acyl-CoA; subsequently, the 2-hydroxy fatty acyl-CoA is cleaved by 2-hydroxyphytanoyl-CoA lyase, to formyl-CoA and an n-1 aldehyde. The severe inhibition of formate generation by oxythiamin treatment of intact fibroblasts indicates that cleavage through the thiamine pyrophosphate-dependent 2-hydroxyphytanoyl-CoA lyase is the main pathway for the degradation of 2-hydroxyfatty acids. The latter protein was initially characterized as an essential enzyme in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid. Our findings point to a new role for peroxisomes in mammals, i.e. the breakdown of 2-hydroxyfatty acids, at least the long chain 2-hydroxyfatty acids. Most likely, the more abundant very long chain 2-hydroxyfatty acids are degraded in a similar manner.

Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis.

The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp352-His380-Ser257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser257-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants.

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.

Benzylsuccinate synthase of Azoarcus sp. strain T: cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization.

Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the alpha-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the DeltabssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.