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This project aims to solve the structure of the crenarchaeal Sulfolobus acidocaldarius enzyme malate synthase. Other known malate synthase enzymes have been found to require a magnesium ion in the active site to carry out catalytic activities, but a study reported that S. acidocaldarius malate synthase does not require magnesium. This suggests a novel mechanism for this enzyme. Additionally, the mature S. acidocaldarius protein is approximately 100 residues larger than any other structurally characterized malate synthase. It has also been reported to form a dimer, while previously solved structures have only displayed monomeric, trimeric, and hexameric arrangements. We plan to determine the structure experimentally. However, major advances in the accuracy of protein structure prediction were made recently by AlphaFold, an artificial intelligence system developed by DeepMind, which has revolutionized the field and has largely solved the protein folding problem. A similar AI system, RoseTTAFold, developed by David Baker's lab at the University of Washington, has been made publicly available. Here, we report our analysis of the structure of this protein, predicted using both of these algorithms and of a predicted structural model for the dimeric form of the enzyme using ClusPro. Our results strongly support a conserved catalytic mechanism requiring magnesium, which is common with all previously solved malate synthase isoforms. [ABSTRACT FROM AUTHOR]

A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1 H NMR, 13 C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 μM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC 99 2.55 μg/mL whereas compounds 5d and 5j showed activity with MIC 99 18.91 μg/mL and 25.07 μg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC 99 value of 2.55 μg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target., (© 2024 John Wiley & Sons Ltd.)

The stramenopile alga Nannochloropsis evolved by secondary endosymbiosis of a red alga by a heterotrophic host cell and emerged as a promising organism for biotechnological applications, such as the production of polyunsaturated fatty acids and biodiesel. Peroxisomes play major roles in fatty acid metabolism but experimental analyses of peroxisome biogenesis and metabolism in Nannochloropsis are not reported yet. In fungi, animals, and land plants, soluble proteins of peroxisomes are targeted to the matrix by one of two peroxisome targeting signals (type 1, PTS1, or type 2, PTS2), which are generally conserved across kingdoms and allow the prediction of peroxisomal matrix proteins from nuclear genome sequences. Because diatoms lost the PTS2 pathway secondarily, we investigated its presence in the stramenopile sister group of diatoms, the Eustigmatophyceae, represented by Nannochloropsis. We detected a full-length gene of a putative PEX7 ortholog coding for the cytosolic receptor of PTS2 proteins and demonstrated its expression in Nannochloropsis gaditana. The search for predicted PTS2 cargo proteins in N. gaditana yielded several candidates. In vivo subcellular targeting analyses of representative fusion proteins in different plant expression systems demonstrated that two predicted PTS2 domains were indeed functional and sufficient to direct a reporter protein to peroxisomes. Peroxisome targeting of the predicted PTS2 cargo proteins was further confirmed in Nannochloropsis oceanica by confocal and transmission electron microscopy. Taken together, the results demonstrate for the first time that one group of stramenopile algae maintained the import pathway for PTS2 cargo proteins. To comprehensively map and model the metabolic capabilities of Nannochloropsis peroxisomes, in silico predictions needs to encompass both the PTS1 and the PTS2 matrix proteome.

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism. [ABSTRACT FROM AUTHOR]

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the β-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in β galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Summary: Seed vigour is an imperative trait for the direct seeding of rice. Isopropylmalate synthase (IPMS) catalyses the committed step of leucine (Leu) biosynthesis, but its effect on seed vigour remains unclear. In this study, rice OsIPMS1 and OsIPMS2 was cloned, and the roles of OsIPMS1 in seed vigour were mainly investigated. OsIPMS1 and OsIPMS2 catalyse Leu biosynthesis, and Leu feedback inhibits their IPMS activities. Disruption of OsIPMS1 resulted in low seed vigour under various conditions, which might be tightly associated with the reduction of amino acids in germinating seeds. Eleven amino acids that associated with stress tolerance, GA biosynthesis and tricarboxylic acid (TCA) cycle were significantly reduced in osipms1 mutants compared with those in wide type (WT) during seed germination. Transcriptome analysis indicated that a total of 1209 differentially expressed genes (DEGs) were altered in osipms1a mutant compared with WT at the early germination stage, wherein most of the genes were involved in glycolysis/gluconeogenesis, protein processing, pyruvate, carbon, fructose and mannose metabolism. Further analysis confirmed that the regulation of OsIPMS1 in seed vigour involved in starch hydrolysis, glycolytic activity and energy levels in germinating seeds. The effects of seed priming were tightly associated with the mRNA levels of OsIPMS1 in priming seeds. The OsIPMS1 might be used as a biomarker to determine the best stop time‐point of seed priming in rice. This study provides novel insights into the function of OsIPMS1 on seed vigour and should have practical applications in seed priming of rice. [ABSTRACT FROM AUTHOR]

Highlights • Chloroplastic transgenic C. reinhardtii was developed that stably expressed malate synthase. • This transgenic strain showed a more 19% increase in dry cell weight than wild-type strain. • Transcripts of MDH4 , ACS3 , ICL1 , and MAS1 were increased, and F v /F m was decreased. • Upregulation of heterotrophic metabolism might be involved in biomass increase. • This could serve as a valuable strain for treating wastewater containing acetate and glyoxylate. Abstract High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic Chlamydomonas reinhardtii was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (MDH4) and acetyl-CoA synthetase (ACS3), isocitrate lyase (ICL1) and malate synthase (MAS1), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic F v /F m was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass. [ABSTRACT FROM AUTHOR]

Utilisation of stored lipids plays an important role in germination of oil seeds. In the present study, key enzyme activity (lipase, isocitrate lyase and malate synthase) in lipid utilisation was determined during germination in dimorphic seeds of euhalophyte Suaeda salsa (L.) Pall. The results revealed that the percentage of germination were highest in intertidal brown seeds, followed by inland brown seeds and then inland black seeds moistened with 0 and 300 mM NaCl during early seed germination. The same trend was found in the activity of three enzymes and soluble sugar content when seeds were moistened with 0 and 300 mM NaCl for 3 h. Salinity reduced the activity of three enzymes in inland brown and black seeds in the initial 3 h, except that salinity had no adverse effect on isocitrate lyase activity of brown seeds. Salinity had no adverse effect on three enzymes in inland brown and black seeds in the initial 30 h, except that it decreased malate synthase activity of black seeds. Salinity had no effect on three enzymes in intertidal brown seeds in the initial 3 h and 30 h. In conclusion, high activity of these enzymes in brown seeds may play an important role in utilisation of stored lipids during their rapid seed germination. Suaeda salsa is an economically important euhalophyte because its seed oil is edible and rich in unsaturated fatty acids. Compared with black seeds, certain enzymes in the glyoxylate cycle in brown seeds play a more important role in the utilisation of stored lipids to provide soluble sugar during their rapid seed germination. This work provides new insights to aid understanding of the adaptation of dimorphic seeds to harsh saline environments. [ABSTRACT FROM AUTHOR]

2017 marks the 60th anniversary of Krebs’ seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism. [ABSTRACT FROM AUTHOR]

Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very little is known regarding the proteins containing multiple domains. It has been shown that the high stability of domains, in conjunction with inter-domain interactions, manifests as a frustrated energy landscape, causing complexity in the global folding pathway. However, multidomain proteins despite containing independently foldable, loosely cooperative sections can fold into native states with amazing speed and accuracy. To understand the complexity in mechanism, studies were conducted previously on the multidomain protein malate synthase G (MSG), an enzyme of the glyoxylate pathway with four distinct and adjacent domains. It was shown that the protein refolds to a functionally active intermediate state at a fast rate, which slowly produces the native state. Although experiments decoded the nature of the intermediate, a full description of the folding pathway was not elucidated. In this study, we use a battery of biophysical techniques to examine the protein's folding pathway. By using multiprobe kinetics studies and comparison with the equilibrium behavior of protein against urea, we demonstrate that the unfolded polypeptide undergoes conformational compaction to a misfolded intermediate within milliseconds of refolding. The misfolded product appears to be stabilized under moderate denaturant concentrations. Further folding of the protein produces a stable intermediate, which undergoes partial unfolding-assisted large segmental rearrangements to achieve the native state. This study reveals an evolved folding pathway of the multidomain protein MSG, which involves surpassing the multiple misfolding traps during refolding. [ABSTRACT FROM AUTHOR]

Malate is regarded as one of the key building block chemicals which can potentially be produced from biomass at a large scale. Although glucose has been extensively studied as the substrate for malate production, its high price and potential competition with food production are serious limiting factors. In this study, Escherichia coli was metabolically engineered to effectively produce malate from xylose, the second most abundant sugar component of lignocellulosic biomass. First, the biosynthetic route of malate was constructed by overexpressing D -tagatose 3-epimerase, L-fuculokinase, L-fuculose-phosphate aldolase, and aldehyde dehydrogenase A. Second, genes encoding malic enzyme, malate dehydrogenase, and fumarate hydratase were knocked out to eliminate malate consumption, resulting in a titer of 1.99 g/l malate and a yield of 0.47 g malate/g xylose. Third, glycolate oxidase and malate synthase were overexpressed to strengthen the conversion of glycolate to malate, which led to a titer of 4.33 g/l malate and a yield of 0.83 g malate/g xylose, reaching 93% of the theoretical yield. Finally, catalase HPII was overexpressed to decompose H 2 O 2 and alleviate its toxicity, which improved cell growth and further boosted malate titer to 5.90 g/l with a yield of 0.80 g malate/g xylose. To the best of our knowledge, this is the first study to report efficient malate production from xylose as the carbon source. [ABSTRACT FROM AUTHOR]

Polymalic acid (PMA) is a water-soluble biopolymer produced by the yeast-like fungus Aureobasidium pullulans. In this study, the physiological response of A. pullulans against exogenous alcohols stress was investigated. Interestingly, ethanol stress was an effective inducer of enhanced PMA yield, although cell growth was slightly inhibited. The stress-responsive gene malate synthase ( mls ), which is involved in the glyoxylate shunt, was identified and was found to be regulated by exogenous ethanol stress. Therefore, an engineered strain, YJ-MLS, was constructed by overexpressing the endogenous mls gene, which increased the PMA titer by 16.2% compared with the wild-type strain. Following addition of 1% (v/v) of ethanol, a high PMA titer of 40.0 ± 0.38 g/L was obtained using batch fermentation with the mutant YJ-MLS in a 5-L fermentor, with a strongest PMA productivity of 0.56 g/L h. This study was the interesting report to show strengthening of the carbon metabolic flow from the glyoxylate shunt for PMA synthesis, and also provided a new sight for re-recognizing the regulatory behavior of alcohol stress in eukaryotic microbes. [ABSTRACT FROM AUTHOR]

Glyoxylate is an important chemical building block which is currently produced by chemical or electrochemical oxidation methods We herein described the achievement of microbial glyoxylate production from renewable biomass by metabolic engineering of Escherichia coli . The synthetic ribulose-1-phosphate pathway was expressed to produce glycolate from xylose. Additional genomic modifications including the inactivation of malate synthase genes aceB / glcB and glyoxylate carboligase gene gcl were performed to prevent the consumption of glyoxylate. The constructed strain was found to accumulate 0.13 g/L glyoxylate in shake flask cultivations. Further overexpression of glycolate oxidase and catalase significantly improved glyoxylate production to 0.74 g/L, which is the highest titer reported to date. These results demonstrate that microbial fermentation has a promising potential to manufacture glyoxylate from renewable feedstocks. [ABSTRACT FROM AUTHOR]

Malic acid (MA), one of the major organic acid exudates from roots, plays a significant role in the chemotaxis of beneficial bacteria to the plant's rhizosphere. In this study, the effect of a plant-growth-promoting rhizobacterium, Bacillus subtilis RR4, on the synthesis and exudation of MA from roots is demonstrated in rice. To test the chemotactic ability of strain RR4 towards MA, a capillary chemotaxis assay was performed, which revealed a positive response (relative chemotactic ratio of 6.15 with 10 μmol/L MA); with increasing concentrations of MA, an elevated chemotactic response was observed. Quantitative polymerase chain reaction, performed to analyze the influence of RR4 on the MA biosynthetic gene, malate synthase ( OsMS), and the transporter gene, aluminium-activated malate transporter ( OsALMT), demonstrated significant differential expression, with 1.8- and -0.58-fold changes, respectively, in RR4-treated roots. The gene expression pattern of OsMS corroborated the data obtained by high-performance liquid chromatography, which showed elevated MA levels in roots (1.52-fold), whereas the levels of MA in root exudates were not altered significantly although expression of OsALMT was reduced. Our results demonstrate that B. subtilis RR4 is chemotactic to MA and can induce biosynthesis of MA in rice roots. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While 4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each 4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) in Sinorhizobium meliloti Rm1021 dct mutants unable to transport 4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF with Medicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-type S. meliloti. Rhizobium leguminosarum bv. viciae 3841 dct mutants unable to transport 4-dicarboxylates but expressing the maeP transporter had strong symbiotic properties, with Pisum sativum plants inoculated with these strains appearing similar to plants inoculated with wild-type R. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combined P. sativum-R. leguminosarum nodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses, L-malate is likely the primary 4-dicarboxylate provided to the bacterium. IMPORTANCE Symbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other 4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont. [ABSTRACT FROM AUTHOR]

The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans . We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate. [ABSTRACT FROM AUTHOR]

Defending phagocyte generated oxidants is the key for survival of Salmonella Typhimurium ( S. Typhimurium) inside the host. Met residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO). Methionine sulfoxide reductase (Msr) can repair Met-SO to Met thus restoring the function(s) of Met-SO containing proteins. Using pull down method we have identified several MsrA interacting proteins in the S. Typhimurium, one of them was malate synthase (MS). MS is an enzyme of glyoxylate cycle. This cycle is essential for survival of S. Typhimurium inside the host under nutrient limiting conditions. By employing in vitro cross-linking and blot overlay techniques we showed that purified MsrA interacted with pure MS. Treatment of pure malate synthase with H 2 O 2 resulted in reduction of MS activity. However, MsrA along with thioredoxin-thioredoxin reductase system partially restored the activity of oxidized MS. Our mass spectrometry data demonstrated H 2 O 2 mediated oxidation and MsrA mediated repair of Met residues in MS. Further in comparison to S. Typhimurium, the msrA gene deletion (∆ msrA ) strain showed reduced (60%) malate synthase specific activity. Oral inoculation with wild type, ∆ msrA and ∆ ms strains of S. Typhimurium resulted in colonization of 100, 0 and 40% of the poultry respectively. [ABSTRACT FROM AUTHOR]

Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed. [ABSTRACT FROM AUTHOR]

Malate synthase is a condensing enzyme responsible for conversion of glyoxylate to malate in the presence of acetyl-CoA. This reaction helps in bypassing the TCA cycle reactions involving carbon loss and leads to diverting some of the carbon skeletons to gluconeogenic events while rest can continue to provide TCA cycle intermediates. Malate synthase (GlcB) is encoded by MRA_1848 of Mycobacterium tuberculosis H37Ra ( Mtb -Ra). We developed a knockdown (KD) Mtb -Ra strain by down-regulating GlcB. The survival studies suggested increased susceptibility to oxidative and nitrosative stress as well as to rifampicin. The susceptibility profile was reversed in the presence of free radical scavengers. Also, KD showed reduced biofilm maturation, failed to enter persistent state, and showed reduced growth inside macrophages. The study of post-endocytosis events showed differences in late stage endosomal maturation behavior in macrophages infected with KD compared to WT. Increased iNOS, LAMP1 and cathepsin D expression was observed in macrophages infected with KD compared to WT. [ABSTRACT FROM AUTHOR]

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis (Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and β-hydroxybutyrate. These changes were indicative of a glyoxylate- induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth ofMS-deficient Mtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development. [ABSTRACT FROM AUTHOR]

The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13 C labeling. In the wild-type strain, the malate shunt accounts for about 33±2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. This provides the first direct evidence of the in-vivo function of the malate shunt. [ABSTRACT FROM AUTHOR]

Expression of rice malate synthase (OsMS), one of the two key genes in the glyoxylate cycle, is highly upregulated under salt stress. In this study, we aimed to investigate the role of OsMS in salt stress responses using the Arabidopsis T-DNA insertional mutant line of malate synthase (AtMS), an OsMS orthologous gene, for ectopic expression. Germination of the Atms mutant under salt stress was lower than that of Arabidopsis Col-0 wildtype (WT); furthermore, the two Atms mutant lines ectopically expressing OsMS reversed the salt-sensitive phenotype. Atms mutants salt-treated for 3 days exhibited higher electrolyte leakage, higher Na+/K+ ratio, lower expression of stress-responsive genes, and lower soluble sugar content than WT and the two OsMS -expressing Atms mutant lines. Consistently, Atms mutants salt-treated for 3 days followed by a 5-day recovery period displayed greater fresh-weight reduction. Notably, leaf greenness and chlorophyll and total carotenoid contents were higher in the Atms mutant lines than in the WT under stress. OsMS -expressing Atms mutants exhibited a change in pigment content closer to that of WT. During dark-induced senescence, an Atms mutant, Aticl , mutant (the other key gene in the glyoxylate cycle), and three double mutant lines of Atms and Aticl exhibited lower decreases in leaf greenness than the WT and OsMS -expressing Atms mutant lines. Furthermore, SAG12 expression levels in the Atms mutant, Aticl mutant, and three double mutant lines were lower than those in OsMS -expressing Atms mutant lines. Altogether, our data indicate that OsMS likely plays a key role in salt stress responses, possibly through the induction of leaf senescence. [ABSTRACT FROM AUTHOR]

Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/β domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Fragment screening and high throughput screening are complementary approaches that combine with structural biology to explore the binding capabilities of an active site. We have used a fragment-based approach on malate synthase (GlcB) from Mycobacterium tuberculosis and discovered several novel binding chemotypes. In addition, the crystal structures of GlcB in complex with these fragments indicated conformational changes in the active site that represent the enzyme conformations during catalysis.Additional structures of the complexwith malate and of the apo form of GlcB supported that hypothesis. Comparative analysis of GlcB structures in complex with 18 fragments allowed us to characterize the preferred chemotypes and their binding modes. The fragment structures showed a hydrogen bond to the backbone carbonyl of Met-631. We successfully incorporated an indole group from a fragment into an existing phenyl-diketo acid series. The resulting indole-containing inhibitor was 100-foldmore potent than the parent phenyl-diketo acid with an IC50 value of 20 nM. [ABSTRACT FROM AUTHOR]

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress. [ABSTRACT FROM AUTHOR]

Nesterenkonia sp. AN1 is a polyextremophile isolated from Antarctic desert soil. Genomic analyses and genome comparisons with three mesophilic Nesterenkonia strains indicated that the unique genome fraction of Nesterenkonia sp. AN1 contains adaptive features implicated in the response to cold stress including modulation of membrane fluidity as well as response to cold-associated osmotic and oxidative stress. The core genome also encodes a number of putative cold stress response proteins. RNA-Seq-based transcriptome analyses of Nesterenkonia sp. AN1 grown at 5°C and 21°C showed that there was significant induction of transcripts that code for antioxidants at 5°C, demonstrated by the upregulation of sodA, bcp and bpoA2. There was also overexpression of universal stress protein genes related to uspA, along with genes encoding other characterized cold stress features. Genes encoding the two key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (AceB) were induced at 5oC, suggesting possible adaptation strategies for energy metabolism in cold habitats. These genomic features may contribute to the survival of Nesterenkonia sp. AN1 in arid Antarctic soils. [ABSTRACT FROM AUTHOR]

Elucidation of protein structures at the atomic level using X-ray crystallography is an effective technique for studying metabolic enzymes and defining binding interactions of small molecules and ions. Our research centers on the structure of the malate synthase isoform H (MSH) from Haloferax volcanii, which is a key enzyme in the glyoxylate pathway of cellular metabolism and allows this organism to integrate two carbon compounds for anabolic biosynthetic reactions. We have collected X-ray diffraction data from a protein crystal soaked in a solution containing lead(II) acetate. This heavy atom derivative provided phasing information using the single isomorphous replacement with anomalous scattering (SIRAS) method to determine the structure of the native enzyme, but this structure has not previously been analyzed and fully refined. Here we report the iterative model-building and refinement of this structure at 2.1Å resolution to an overall R-value of 0.1831 and an R-free of 0.2167. This structure allows a detailed analysis of lead(II) ion binding to the protein with implications for lead toxicity and inhibition of the enzyme. In addition to the displacement of the required magnesium ion and accompanying distortions in the local vicinity of the active site, we find three additional binding sites for lead ions. Strong peaks are observed at these lead binding sites in anomalous difference Fourier maps, and there are very high electron-density peaks in the 2Fo-Fc map at these four locations. Lead binding at intersubunit contacts may explain the increased resolution of X-ray diffraction from this derivative versus the native protein. [ABSTRACT FROM AUTHOR]

Glycolic acid is the smallest member of the α-hydroxy acid family. In order to produce glycolate from glucose via the glyoxylate shunt stably, one malate synthase gene aceB in Escherichia coli BW25113 was deleted by homologous recombination; another malate synthase gene glcB was then replaced by a DNA cassette WAK harboring isocitrate lyase gene ( aceA ), glyoxylate reductase gene ( ycdW ) and isocitrate dehydrogenase kinase/phosphatase gene ( aceK ). The above three genes were over-expressed in the chromosome of E. coli EYX-1WAK. This strain was then transferred 20 times on M9 medium to have a mutant strain: EYX-2 with a significantly improved growth rate. The glycolate yields of EYX-2 in the shaken flasks and the 5-L bioreactor using batch fermentation strategy under 2 vvm aeration and 800 rpm stirring speed were 0.33 g/g-glucose and 0.48 g/g-glucose, respectively. The fed-batch fermentation of EYX-2 on 120 g/L glucose had the highest titer of 56.44 g/L with 0.52 g/g-glucose yield in 120 h, and this is the highest reported glycolate yield ever. [ABSTRACT FROM AUTHOR]

Ricinus communis seeds germinate to a high percentage and faster at 35 °C than at lower temperatures, but with compromised seedling establishment. However, seedlings are able to cope with high temperatures at later stages of seedling establishment if germination occurred at lower temperatures. Our objective was to assess the biochemical and molecular requirements of R. communis germination for successful seedling establishment at varying temperatures. For that, we performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways, such as storage oil mobilization, β-oxidation and gluconeogenesis of seeds germinated at three different temperatures. We identified a thermo-sensitive window during seed germination in which high temperatures compromise seedling development, most likely by down-regulating some energy-generating pathways. Overexpression of malate synthase ( MLS ) and glycerol kinase ( GK ) genes resulted in higher starch levels in Nicotiana benthamiana leaves, which highlights the importance of these genes in energy-generating pathways for seedling establishment. Additionally, we showed that GABA, which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition. Herewith, we provide new insights into the molecular requirements for vigorous seedling growth of R. communis under different environmental conditions. [ABSTRACT FROM AUTHOR]

Summary Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens. [ABSTRACT FROM AUTHOR]

We cultured Chlamydomonas reinhardtii cells in a minimal culture medium supplemented with various concentrations of acetate, fatty acids, ethanol, fatty alcohols, or sucrose. The presence of acetate (0.5 or 1.0 %, w/v) was advantageous for cell growth. To determine whether peroxisomes are involved in fatty acid and fatty alcohol metabolism, we investigated the dynamics of peroxisomes, including changes in their number and size, in the presence of acetate, ethanol, and sucrose. The total volume of peroxisomes increased when cells were grown with acetate, but did not change when cells were grown with ethanol or sucrose. We analyzed cell growth on minimal culture medium supplemented with various fatty acids (carbon chain length ranging from one to ten) to investigate which fatty acids are metabolized by C. reinhardtii. Among them, acetate caused the greatest increase in growth when added to minimal culture media. We analyzed the transcript levels of genes encoding putative glyoxysomal enzymes. The transcript levels of genes encoding malate synthase, malate dehydrogenase, isocitrate lyase, and citrate synthase increased when Chlamydomonas cells were grown on minimal culture medium supplemented with acetate. Our results suggest that Chlamydomonas peroxisomes are involved in acetate metabolism via the glyoxylate cycle. [ABSTRACT FROM AUTHOR]

Paracoccidioides species are dimorphic fungi and are the etiologic agents of paracoccidioidomycosis, which is a serious disease that involves multiple organs. The many tissues colonized by this fungus suggest a variety of surface molecules involved in adhesion. A surprising finding is that most enzymes in the glycolytic pathway, tricarboxylic acid (TCA) cycle and glyoxylate cycle in Paracoccidioides spp. have adhesive properties that aid in interacting with the host extracellular matrix and thus act as 'moonlighting' proteins. Moonlighting proteins have multiple functions, which adds a dimension to cellular complexity and benefit cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play a role in bacterial pathogenesis by either acting as proteins secreted in a conventional pathway and/or as cell surface components that facilitate adhesion or adherence. This review outlines the multifunctionality exhibited by many Paracoccidioides spp. enzymes, including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase, and enolase.We discuss the roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host. [ABSTRACT FROM AUTHOR]

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes. [ABSTRACT FROM AUTHOR]

l-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, l-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce l-serine from glucose. To accumulate l-serine, sdaA encoding the l-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious l-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA, serB, and serC genes. The resulting strain YF-7 produced 4.5 g/L l-serine in batch cultivation and 8.34 g/L l-serine in fed-batch cultivation. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisomes play a crucial role in regulating cellular metabolism, providing compartments where metabolic pathways can be contained and controlled. Their importance is underlined by the developmental brain disorders caused by peroxisome malfunction, while disturbances in peroxisome function also contribute to ageing. As peroxisomes do not contain DNA, they rely on an active transport system to obtain the full quota of proteins required for function. Organelle protein transport however, is rarely a one-way process and exciting recent data have demonstrated that peroxisomes can selectively export membrane and matrix proteins to fulfil specific functions. This review will summarise the current knowledge on peroxisomal membrane and matrix protein export, discussing the mechanisms underlying export as well as the role of peroxisomal protein export in peroxisomal and cellular function. [Copyright &y& Elsevier]

The glyoxylate shunt is an anaplerotic bypass of the traditional Krebs cycle. It plays a prominent role in Mycobacterium tuberculosis virulence, so it can be exploited for the development of antitubercular therapeutics. The shunt involves two enzymes: isocitrate lyase (ICL) and malate synthase (GlcB). The shunt bypasses two steps of the tricarboxylic acid cycle, allowing the incorporation of carbon, and thus, refilling oxaloacetate under carbon-limiting conditions. The targeting of ICL is complicated; however, GlcB, which accommodates the pantothenate tail of acetyl-CoA in the active site, is easier to target. A catalytic Mg2+ unit is located at the bottom of the cavity, and plays a very important role. Recently, the development of effective antituberculosis drugs based on phenyldiketo acids (PDKAs) has been reported. Interestingly, all the crystal structures of GlcB-inhibitor complexes exhibit close contact between the carboxylate of Asp633 and the face of the aromatic ring of the inhibitor. Remarkably, the replacement of the phenyl ring in PDKA by aliphatic moieties yields inactive inhibitors, suggesting that the aromatic moiety is crucial for inhibition. However, the aromatic ring of PDKA is not electron-deficient, and consequently, the anion-π interaction is expected to be very weak (dominated only by polarization effects). Herein, through a combination analysis of the recent X-ray structures of GlcB-PDKA complexes retrieved from the protein data bank (PDB) and computational ab initio studies (RI-MP2/def2-TZVP level of theory), we demonstrate the prominent role of the Mg2+ ion in the active site, which promotes long-range enhancement of the anion-π interaction. [ABSTRACT FROM AUTHOR]

Oilseed crops are an important natural resource because they can be used for food and renewable energy production. However, oilseed seedling establishment and vigor depend upon the capacity to overcome functional transition, a developmental stage characterized by the consumption of the remaining oil reserves, through β-oxidation and glyoxylate cycle, and the onset of autotrophic metabolism. The increased growth and the acclimation to full photosynthetic activity lead to production of reactive oxygen species and a reorganization of the cell antioxidant systems to achieve a new redox homeostasis. In the present study, catalase (CAT) was inhibited by 3-amino-1,2,4-triazole application during functional transition in sunflower and safflower seedlings to understand the effect of this antioxidant enzyme impairment on the mRNA expression of the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS), as well as the superoxide dismutase (SOD) activity and ascorbate peroxidase (APX) activity and expression. CAT inhibition led to significant seedling growth reduction and increases in HO content, SOD activity, and mRNA expression of CAT and APX in both species. However, APX activity was induced only in safflower plants. Additionally, ICL and MLS mRNA expressions were upregulated after 6 h of treatment when compared to the control values. These results indicate that under CAT impairment conditions, redox homeostasis at the functional transition phase was partially supported by the SOD and APX antioxidant systems to maintain the seedling photosynthetic establishment. [ABSTRACT FROM AUTHOR]

Stylosanthes (stylo) is a dominant leguminous forage in the tropics. Previous studies suggest that stylo has great potential for aluminium (Al) tolerance, but little is known about the underlying mechanism., A novel malic enzyme, Sg ME1, was identified from the Al-tolerant genotype TPRC2001-1 after 72 h Al exposure by two-dimensional electrophoresis, and the encoding gene was cloned and characterized via heterologous expression in yeast, Arabidopsis thaliana and bean ( Phaseolus vulgaris) hairy roots., Internal Al detoxification might be mainly responsible for the 72 h Al tolerance of TPRC2001-1, as indicated by 5.8-fold higher root malate concentrations and approximately two-fold higher Al concentrations in roots and root symplasts of TPRC2001-1 than those of the Al-sensitive genotype Fine-stem. An accompanying increase in malate secretion might also reduce a fraction of Al uptake in TPRC2001-1. Gene and protein expression of Sg ME1 was only enhanced in TPRC2001-1 after 72 h Al exposure. Overexpressing Sg ME1 enhanced malate synthesis and rescued yeast, A. thaliana and bean hairy roots from Al toxicity via increasing intracellular malate concentrations and/or accompanied malate exudation., These results provide strong evidence that superior Al tolerance of stylo is mainly conferred by Al-enhanced malate synthesis, functionally controlled by Sg ME1. [ABSTRACT FROM AUTHOR]

Malate synthase (Mls), a key enzyme in the glyoxylate cycle, is required for virulence in microbial pathogens. In this study, we identified the AoMls gene from the nematode-trapping fungus Arthobotrys oligospora. The gene contains 4 introns and encodes a polypeptide of 540 amino acids. To characterize the function of AoMls in A. oligospora, we disrupted it by homologous recombination, and the Δ AoMls mutants were confirmed by PCR and Southern blot analyses. The growth rate and colony morphology of the Δ AoMls mutants showed no obvious difference from the wild-type strains on potato dextrose agar (PDA) plate. However, the disruption of gene AoMls led to a significant reduction in conidiation, failure to utilize fatty acids and sodium acetate for growth, and its conidia were unable to germinate on minimal medium supplemented with sodium oleate. In addition, the trap formation was retarded in the Δ AoMls mutants, which only produced immature traps containing one or two rings. Moreover, the nematicidal activity of the Δ AoMls mutants was significantly decreased. Our results suggest that the gene AoMls plays an important role in conidiation, trap formation and pathogenicity of A. oligospora. [ABSTRACT FROM AUTHOR]