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Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme [F.sub.420], which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on [F.sub.420] biosynthesis nominated many actinobacterial proteins as candidate [F.sub.420]-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5'-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to [F.sub.420]-producing species. The LLM and PPOX families were observed in [F.sub.420]-producing species as well as species lacking [F.sub.420] but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism's ability to make [F.sub.420] allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify [F.sub.420]-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate [F.sub.420]-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of [F.sub.420] biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme [F.sub.420] for its redox reactions. This dependence and the cofactor's rarity may make [F.sub.420]-related proteins promising drug targets. doi: 10.1128/JB.00425-10

Propionyl-CoA is an inhibitor of both primary and secondary metabolism in Aspergillus species and a functional methylcitrate cycle is essential for the efficient removal of this potentially toxic metabolite. Although the genomes of most sequenced fungal species appear to contain genes coding for enzymes of the methylcitrate cycle, experimental confirmation of pathway activity in filamentous fungi has only been provided for Aspergillus nidulans and Aspergillus fumigatus. In this study we demonstrate that pathogenic Fusarium species also possess a functional methylcitrate cycle. Fusarium solani appears highly adapted to saprophytic growth as it utilized propionate with high efficiency, whereas Fusarium verticillioides grew poorly on this carbon source. In order to elucidate the mechanisms of propionyl-CoA detoxification, we first identified the genes coding for methylcitrate synthase from both species. Despite sharing 96% amino acid sequence identity, analysis of the two purified enzymes demonstrated that their biochemical properties differed in several respects. Both methylcitrate synthases exhibited low [K.sub.m] values for propionyl-CoA, but that of F. verticillioides displayed significantly higher citrate synthase activity and greater thermal stability. Activity determinations from cell-free extracts of F. solani revealed a strong methylcitrate synthase activity during growth on propionate and to a lesser extent on Casamino acids, whereas activity by F. verticillioides was highest on Casamino acids. Further phenotypic analysis confirmed that these biochemical differences were reflected in the different growth behaviour of the two species on propionyl-CoA-generating carbon sources. DOI 10.1099/mic.0.031781-0

DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A [[DELTA].sup.9] desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal [His.sub.6] or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ~10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

The coenzyme [B.sub.12] production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme [B.sub.12] gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQQP, sirA, hemACBL, and cobUSC, heroD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B12 itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme [B.sub.12] pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the [B.sub.12] biosynthesis genes. G + C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme [B.sub.12] biosynthesis and reuterin production, which might be implicated in Lb. reuteri's success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in [B.sub.12].

A study about an operon containing genes homologous to a formyl coenzyme A transferase gene (frc) and an oxalyl coenzyme A decarboxylase gene (oxc) that was in the genome of the probiotic bacterium Lactobacillus acidophilus is presented. Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of xalic acid at pH 3.5 compared with survival of the wild-type strain.

The ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5'-deoxycobinamide kinase enzyme (cobU in Salmonella enterica). The latter activity is required for cobinamide salvaging in bacteria. This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria. These studies demanded the functional characterization of two genes whose putative function had been annotated based solely on their homology to the bacterial genes encoding adenosylcobyric acid and adenosylcobinamide-phosphate synthases (cbiP and cbiB, respectively) of S. enterica. A cbiP mutant strain of the archaeon Halobacterium sp. strain NRC-1 was auxotrophic for adenosylcobyric acid, a known intermediate of the de novo cobamide biosynthesis pathway, but efficiently salvaged cobinamide from the environment, suggesting the existence of a salvaging pathway in this archaeon. A cbiB mutant strain of Halobacterium was auxotrophic for adenosylcobinamide-GDP, a known de novo intermediate, and did not salvage cobinamide. The results of the nutritional analyses of the cbiP and cbiB mutants suggested that the entry point for cobinamide salvaging is adenosylcobyric acid. The data are consistent with a salvaging pathway for cobinamide in which an amidohydrolase enzyme cleaves off the aminopropanol moiety of adenosylcobinamide to yield adenosylcobyric acid, which is converted by the adenosylcobinamide-phosphate synthase enzyme to adenosylcobinamide-phosphate, a known intermediate of the de novo biosynthetic pathway. The existence of an adenosylcobinamide amidohydrolase enzyme would explain the lack of an adenosylcobinamide kinase in archaea.

We found that Lactobacillus reuteri CRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin [B.sub.12]-free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propaniediol as a final product. The cell extract of L. reuteri corrected the coenzyme [B.sub.12] requirement of Lactobacillus delbrueckii subspi, lactis ATCC 7830 and allowed the growth of Salmonella enteriica serovar Typhimurium (metE cbiB) and Escherichia coil (metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes from L. reuteri allowed the identification of cob genes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced by L. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanociobalamin (vitamin [B.sub.12]).

Mycobacterium strains that grow on ethene and vinyl chloride (VC) are widely distributed in the environment and are potentially useful for biocatalysis and bioremediation. The catabolic pathway of alkene assimilation in mycobacteria is not well characterized. It is clear that the initial step is a monooxygenase-mediated epoxidation that produces epoxyethane from ethene and chlorooxirane from VC, but the enzymes involved in subsequent transformation of the epoxides have not been identified. We investigated epoxyethane metabolism in Mycobacterium strain JS60 and discovered a coenzyme M (CoM)-dependent enzyme activity in extracts from VC-and ethene-grown cells. PCR amplifications using primers targeted at epoxyalkane:CoM transferase (EaCoMT) genes yielded part of the JS60 EaCoMT gene, which was used to clone an 8.4-kb genomic DNA fragment. The complete EaCoMT gene (etnE) was recovered, along with genes (etnABCD) encoding a four-component monooxygenase and two genes possibly involved in acyl-CoA ester metabolism. Reverse transcription-PCR indicated that the etnE and etnA genes were cotranscribed and inducible by ethene and VC. Heterologous expression of the etnE gene in Mycobacterium smegmatis [mc.sup.2]155 using the pMV261 vector gave a recombinant strain capable of transforming epoxyethane, epoxypropane, and chlorooxirane. A metabolite identified by mass spectrometry as 2-hydroxyethyl-CoM was produced from epoxyethane. The results indicate that the EaCoMT and monooxygenase enzymes encoded by a single operon (etnEABCD) catalyze the initial reactions in both the VC and ethene assimilation pathways. CoM-mediated reactions appear to be more widespread in bacteria than was previously believed.

In the denitrifying member of the [beta]-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acylCoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoateCoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on [V.sub.max]/[K.sub.m], the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.

Analyses of the [F.sub.420]s present in Methanococcus jannaschii have shown that these cells contain a series of [gamma]-glutamyl-linked [F.sub.420]s capped with a single, terminal [alpha]-linked L-glutamate. The predominant form of [F.sub.420] was designated as [alpha]-[F.sub.420]-3 and represented 86% of the [F.sub.420]s in these cells. Analyses of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis showed that they contained only [gamma]-glutamyl-linked [F.sub.420]s.

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NA[D.sup.+]-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NA[D.sup.+] and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45[degrees]C. The kinetic parameters of the enzyme are highly influenced by temperatures. The [K.sub.m] for L-threonine was lowest at 20[degrees]C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NA[D.sup.+] binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.

Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4'-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.

Research has been conducted on protozoan parasites of the genus Plasmodium which cause malaria. The identification of the gene from Plasmodium falciparum which encodes malonyl-coenzyme A:acyl carrier proteins transacylase and the functional characterization of the enzyme are described.

SIR2 proteins have [NAD.sup.+]-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.

The amino acid [beta]-alanine is an intermediate in pantothenic acid (vitamin [B.sub.5]) and coenzyme A (CoA) biosynthesis. In contrast to bacteria, yeast derive the [beta]-alanine required for pantothenic acid production via polyamine metabolism, mediated by the four SPE genes and by the FAD-dependent amine oxidase encoded by FMS1. Because amine oxidases generally produce aldehyde derivatives of amine compounds, we propose that an additional aldehyde-dehydrogenase-mediated step is required to make [beta]-alanine from the precursor aldehyde, 3-aminopropanal. This study presents evidence that the closely related aldehyde dehydrogenase genes ALD2 and ALD3 are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to [beta]-alanine in vivo. While deletion of the nuclear gene encoding the unrelated mitochondrial Ald5p resulted in an enhanced requirement for pantothenic acid pathway metabolites, we found no evidence to indicate that the Ald5p functions directly in the conversion of 3-aminopropanal to [beta]-alanine. Thus, in Saccharomyces cerevisiae, ALD2 and ALD3 are specialized for [beta]-alanine biosynthesis and are consequently involved in the cellular biosynthesis of coenzyme A.

Research has been conducted on precursors of coenzymes-thiamin pyrophosphate and flavin mononucleotide/flavin adenine dinucleotide, riboflavin and thiamin. The study of riboflavin and thiamin gene regulation feedback has been carried out and the results demonstrate that this feedback relies on transcription attenuation mechanism.

Research has been conducted on Streptomyces coelicolor A3(2) acyl-coenzyme A synthetase. The physiological role of this coenzyme in S. coelicolor has been investigated via genetic and biochemical characterization of the putative acyl-coenzyme A synthetase with homology to the Escherichia coli protein and the details are reported.

Research has been conducted on the gene encoding acyl coenzyme A dehydrogenase of Escherichia coli fatty acid dergadation (fad) regulon. The experimental proof based on the transcriptional array analysis demonstrates that yafH and fadE are the same gene.

Research has been conducted on pantothenate, a cofactor synthesized in Salmonella enterica serovar Typhimurium. Mutations increasing pantothenate accumulation in the growth medium have been isolated and the results indicate that the causative mutations in these strains are the insertion of a CG base pair upstream of the panBCD operon transcription start site.

Research has been conducted on citrate lyase prosthetic group biosynthesis. The identification of the Klebsiella pneumoniae gene and its functions has been carried out and the results suggest that the gene is involved in citrate fermentation.

Research has been conducted on Mycobacterium bovis strain BCG mutants which could not make coenzyme F (sub)420. The transposon insertion into the M. bovis homologue of the M. tuberculosis gene has been carried out and the results indicate that this insertion creates mutants which cannot produce coenzyme F (sub)420 or the biosynthetic intermediate FO.

Research has been conducted on the serine cycle methylotrophic bacteria which converts acetyl coenzyme A to glyoxylate via a pathway. The additional genes involved in this pathway have been identified and the mutants defective in these genes have been investigated.

Research has been conducted on L-(+) isomer of 3-hydroxybutyrate of the wild-type Sinorhizobium meliloti. Results indicate that overexpression of the S. meliloti gene which encodes acetoacetyl coenzyme A synthetase confers L-(+)-3-hydroxybutyrate utilization ability.

Transposon mutagenesis demonstrates that both fbiA and fbiB genes are required for normal coenzyme F(sub)420-(sup)5,6 synthesis and together they constitute an operon. Data indicate that both the genes function between the intermediate FO and the coenzyme.

Research has been conducted on the transcriptional activator Cat8p of Kluyveromyces lactis (KlCat8p). Results indicate that KlCat8p is important for the induction of KlACS1 and KlACS2 genes and is required for the expression of KlJEN1 gene.

Reductions in the levels and activities of enzymes that utilize thiamine diphosphate (ThDP) as a cofactor are thought to be responsible for the tissue damage suffered during thiamine deficiency. Although loss of cofactor can account in part for loss of enzyme activity, thiamine and its phosphorylated derivatives may also regulate the expression of the genes encoding these proteins. To examine this possibility, steady-state mRNA levels for three ThDP-dependent enzymes were measured in human fibroblasts, lymphoblasts and neuroblastoma cells cultured under conditions of thiamine sufficiency and deficiency. In all three cell types, the mRNA levels of transketolase and the E1[Beta] subunit of pyruvate dehydrogenase complex were lower in thiamine-deficient cultures. In contrast, mRNA levels for a ThDP-binding subunit of [Alpha]-ketoglutarate dehydrogenase, the E1 subunit did not differ. These results indicate that thiamine or a thiamine metabolite regulates the expression in humans of some, but not all, genes encoding ThDP-utilizing enzymes. KEY WORDS: transketolase; [Alpha]-ketoglutarate dehydrogenase; pyruvate dehydrogenase complex; gene regulation; thiamine; humans

The hmgA gene for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase showed a high degree of sequence identity to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences indicated that the two archaeal genes are distant homologs of eukaryotic genes.

The adjacent 900-bp open reading frame (ORF3) of the mycocerosic acid synthase mas gene was expressed in Escherichia coli and Mycobacterium bovis to characterize its protein product. ORF3 protein that was characterized as a 61-kDa C-terminal fusion protein in Escherichia coli encoded an acyl coenzyme A synthase when expressed in Mycobacterium bovis BCG. Furthermore, the purified protein product from ORF3 catalyzed coenzyme A ester synthesis without affecting mycocerosic acid synthesis.

The effects of unesterified fatty acids within the cytosol of Escherichia coli on FadR activity were analyzed to determine the role of the transcription factor in the regulation of DNA binding by acyl coenzyme A. The synthesis of intracellular free(unesterified) fatty acids in the Escherichia coli cytoplasm efficiently induced the fad regulon. The effects of fatty acids on the fad regulon also indicated the role of acyl coenzyme A in mediating DNA binding by FadR by acting as a small molecule ligand.

The sequence and transcript of the genes encoding a coenzyme M methylase in Methanosarcina barkeri were investigated. This 480-kDa protein is composed of two subunits in equimolar concentrations which bind one corrinoid cofactor per alpha-beta dimer. The gene for the alpha polypeptide, mtsA, is upstream of that encoding the beta polypeptide, mtsB. The two genes are contiguous and overlap by several nucleotides. A 1.9-kb mRNA species which reacted with probes specific for either mtsA or mtsB was detected. Three possible methanogen consensus BoxA sequences and two sets of direct repeats were detected upstream of mtsA.

Twenty-six suppressor mutations that allowed growth of a delta-pdxH::omega null mutant in the absence of pyridoxal, were isolated in Escherichia coli. Each suppressor mapped to pdxJ and the eight suppressors sequenced contained the same glycine-to-serine change in the PdxJ polypeptide. This bypass suppression indicates that PdxJ may be involved in the formation of the pyridine ring of pyridoxine 5'-phosphate.

The benzoate-coenzyme A lipase gene badA was cloned and sequenced to study the molecular basis for the generation of benzoyl-coenzyme A by benzoate-coenzyme A ligase in Rhodopseudomonas palustris. R. palustris expresses another benzoate-activating enzyme in addition to BadA. Cyclohexane carboxylate-coenzyme A ligase is the third enzyme for activating benzoate. Findings show that R. palustris needs at least three enzymes to start the anaerobic benzoate degradation process.

The gene for human colipase was isolated, mapped and its tissue-specific expression studied to understand the regulation of this gene. A cDNA probe was used to locate the gene in a cosmid library. It was found to be located in chromosome 6. Furthermore, it was demonstrated to contain three exons in a 3,300 base BamHI restriction fragment. A TATA box, a GC box and a region composed of 28 bases similar to the pancreatic enhancer of rats were found in the 5' region of the gene. The region homologous to the rat pancreatic enhancer region binds a factor from nuclear extracts.

Acetyl-CoA carboxylase (ACC), the key enzyme in the fatty acid biosynthetic pathway, produces the donor of the two-carbon units that are polymerized into long chain fatty acids. The yeast FAS3 gene encoding ACC was isolated and characterized. Sequence analyses revealed the putative biotin carboxylase and transcarboxylase domains. Comparisons with chicken and rat ACCs showed an overall sequence identity of 34%, with the least conservation observed in the sequences surrounding the biotin binding site.

The baiB gene of Eubacterium sp. strain VPI 12708 which is part ofthe bile acid-inducible operon, was exprssed in E. coli and was found to encodea bile acid-coenzyme A (CoA) ligase. The polypeptide requires ATP, CoA and magnesium to optimize ligation activity. Sequencing of the polypeptide showedsimilarities with cyclic carboxylated compounds reacting with ATP or CoA fromother species. The enzyme catalyzes the initial step in the bile acid 7 alpha-dehydroxylation pathway.

A cipher of evolutionary design (CiED) is developed for identifying genetic perturbations, such as gene deletions and other network modifications, which has resulted in optimal phenotypes for the production of end products, such as recombinant natural products. Sequential overexpression of native Escherichia coli enzymes responsible for CoA biosynthesis are performed as the CiED algorithm predicted carbon flux has increased through the CoA biosynthetic pathway in concert with gene deletions, which has resulted in an optimal strain with improved flavanone production levels.

Research has been conducted on methane-oxidising archaea. The hypothesis that these bacteria have co-opted the methanogenic pathway has been tested via the isolation of methane-oxidising archaea genes by different approaches including PCR surveys of naturally occurring populations.

Research has been conducted on sulfate-reducing bacteria. The authors have investigated mercury methylation and acetyl-CoA activity in some of the bacterium's strains which do not utilize the acetyl-CoA pathway, and they report the results.

Research has been conducted on transmembrane bacterium Acinetobacter sp. strain ADP1. The authors have investigated the capture of hydroxycinnamate genes via endogenous vector integration, the regulatory control mechanis over these genes, and the structure of the inducer which triggeres hydroxycinnamate gene expression.

Research has been conducted on Aeromonas caviae R-specific enoyl-coenzyme A hydratase. The authors have investigated the feasibility of changing the acyl chain length substrate specificity of this hydratase via the use of site-directed mutagenesis.

Research has been conducted on coenzyme B (sub)12 -dependent glycerol and diol dehydratases. The isolation of these dehydrotases has been carried out via the construction and screening of complex libraries, and the genes encoding the targeted dehydratases have been purified.

Research has been conducted on Arthrobacter sp. strain SU 4-chlorobenzoate dehalogenation pathway wich converts 4-chlorobenzoate to 4-hydroxybenzoate. The fcbC thioesterase gene from the pathway operon has been subcloned and expressed in Escherichia coli, and its protein product has been characterized.

Research has been conducted on CobT enzyme of Salmonella typhimurium. The authors demonstrate that this enzyme has NAD (super)+ -dependent ADPribosyltransferase activity.

Research has been conducted on Eschrichia coli PhoP-PhoQ two-component system which regulates gene transcription in response to changes in pH and extracellular divalent cation concentration. The authors report that this system also can respond to acetate.

Research has been conducted on acyl coenzyme A carboxylase. The results of molecular characterization and purification of this carboxylase from Acidianus brierleyi are reported.

Research has been conducted on benzoyl coenzyme A biosynthesis pathway in Streptomyces maritimus. The study of this pathway carried out via target-directed mutations is presented.

Research has been conducted on 4-coumarate:coenzyme A ligase. The enzymatic properties of the ligase's homologue have been investigated, and the results are reported.