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Archives of Microbiology (v.180, #2)

Quinolinate dehydrogenase and 6-hydroxyquinolinate decarboxylase involved in the conversion of quinolinic acid to 6-hydroxypicolinic acid by Alcaligenes sp. strain UK21 by Akira Uchida; Mina Ogawa; Toyokazu Yoshida; Toru Nagasawa (pp. 81-87).

In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp. strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined. Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized. The enzyme catalyzed the incorporation of oxygen atoms of H₂O into the hydroxyl group. The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively. Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase. 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized. The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits. The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors. The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.

Keywords: Quinolinic acid; Hydroxylation; Quinolinate dehydrogenase; 6-Hydroxyquinolinate decarboxylase; Alcaligenes sp. strain UK21

A transporter of Escherichia coli specific for l- and d-methionine is the prototype for a new family within the ABC superfamily by Zhongge Zhang; Jérôme N. Feige; Abraham B. Chang; Iain J. Anderson; Vadim M. Brodianski; Alexei G. Vitreschak; Mikhail S. Gelfand; Milton H. Saier Jr (pp. 88-100).

An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds.

Keywords: Transport; Methionine; MetD; ATP-binding cassette; E. coli

Identification of a gene cluster encoding meilingmycin biosynthesis among multiple polyketide synthase contigs isolated from Streptomyces nanchangensis NS3226 by Yuhui Sun; Xiufen Zhou; Guoquan Tu; Zixin Deng (pp. 101-107).

A cluster encoding genes for the biosynthesis of meilingmycin, a macrolide antibiotic structurally similar to avermectin and milbemycin α11, was identified among seven uncharacterized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS3226 by hybridization with PCR products using primers derived from the sequences of aveE, aveF and a thioesterase domain of the avermectin biosynthetic gene cluster. Introduction of a 24.1-kb deletion by targeted gene replacement resulted in a loss of meilingmycin production, confirming that the gene cluster encodes biosynthesis of this important anthelminthic antibiotic compound. A sequenced 8.6-kb fragment had aveC and aveE homologues (meiC and meiE) linked together, as in the avermectin gene cluster, but the arrangement of aveF (meiF) and the thioesterase homologues differed. The results should pave the way to producing novel insecticidal compounds by generating hybrids between the two pathways.

Keywords: Antibiotic biosynthetic genes; Macrolide antibiotic; Polyketide synthase; Avermectin; Gene replacement in Streptomyces

The glutathione-mediated detoxification pathway in yeast: an analysis using the red pigment that accumulates in certain adenine biosynthetic mutants of yeasts reveals the involvement of novel genes by Kailash Gulshan Sharma; Rupinder Kaur; Anand K. Bachhawat (pp. 108-117).

The glutathione-mediated pathway for the detoxification of endogenously and exogenously derived toxic compounds was investigated using a pigment that accumulates in certain adenine biosynthetic mutants of yeasts. The ade1 / ade2 mutants of Saccharomyces cerevisiae, when grown on adenine-limiting medium, accumulate a characteristic red pigment (ade pigment) in their vacuoles. The precursors of the ade pigments are toxic intermediates that form conjugates with glutathione, followed by their transport inside the vacuole. In this study, this red pigment was used as a phenotypic screen to obtain insight regarding new genes involved in the three phases of this detoxification pathway: the activation phase (phase I), the conjugation phase (phase II), and the efflux phase (phase III). Components of the phase III (efflux) pathway which includes, in addition to the previously characterized Ycf1p and Bpt1p, another member of the 'Ycf1p family', Bat1p, as well as a vacuolar H⁺-ATPase-dependent transport were identified. In the investigation of phase II (conjugation), it was found that glutathione S-transferases, encoded by GTT1 and GTT2,do not appear to play a role in this process. By contrast, two other previously characterized genes, the oxidative stress transcription factor gene, SKN7 , and the yeast caesin protein kinase gene, YCK1, of S. cerevisiae do participate in this pathway.

Keywords: Glutathione; Detoxification; Adenine; Vacuole; Glutathione conjugate; Pump/

ISRm31, a new insertion sequence of the IS66 family in Sinorhizobium meliloti by Emanuele G. Biondi; Angelo Pietro Femia; Filippo Favilli; Marco Bazzicalupo (pp. 118-126).

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.

Keywords: Sinorhizobium meliloti ; Insertion sequence; ISRm31 ; nod genes; Inverse PCR

Complete denitrification in coculture of obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria from a hypersaline soda lake by Dimitry Y. Sorokin; Alexey N. Antipov; J. Gijs Kuenen (pp. 127-133).

Eight anaerobic enrichment cultures with thiosulfate as electron donor and nitrate as electron acceptor were inoculated with sediment samples from hypersaline alkaline lakes of Wadi Natrun (Egypt) at pH 10; however, only one of the cultures showed stable growth with complete nitrate reduction to dinitrogen gas. The thiosulfate-oxidizing culture subsequently selected after serial dilution developed in two phases. Initially, nitrate was mostly reduced to nitrite, with a coccoid morphotype prevailing in the culture. During the second stage, nitrite was reduced to dinitrogen gas, accompanied by mass development of thin motile rods. Both morphotypes were isolated in pure culture and identified as representatives of the genus Thioalkalivibrio, which includes obligately autotrophic sulfur-oxidizing haloalkaliphilic species. Nitrate-reducing strain ALEN 2 consisted of large nonmotile coccoid cells that accumulated intracellular sulfur. Its anaerobic growth with thiosulfate, sulfide, or polysulfide as electron donor and nitrate as electron acceptor resulted in the formation of nitrite as the major product. The second isolate, strain ALED, was able to grow anaerobically with thiosulfate as electron donor and nitrite or nitrous oxide (but not nitrate) as electron acceptor. Overall, the action of two different sulfur-oxidizing autotrophs resulted in the complete, thiosulfate-dependent denitrification of nitrate under haloalkaliphilic conditions. This process has not yet been demonstrated for any single species of chemolithoautotrophic sulfur-oxidizing haloalkaliphiles.

Keywords: Haloalkaliphilic; Sulfur-oxidizing bacteria; Soda lakes; Denitrification

Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway by Harry R. Harhangi; Anna S. Akhmanova; Roul Emmens; Chris van der Drift; Wim T. A. M. de Laat; Johannes P. van Dijken; Mike S. M. Jetten; Jack T. Pronk; Huub J. M. Op den Camp (pp. 134-141).

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min⁻¹mg protein^-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.

Keywords: Xylose isomerase; d-Xylulokinase; Phylogeny; Chytrid fungus; Piromyces

Bacterial degradation of arsenobetaine via dimethylarsinoylacetate by Richard O. Jenkins; Alisdair W. Ritchie; John S. Edmonds; Walter Goessler; Nathalie Molenat; Doris Kuehnelt; Christopher F. Harrington; Peter G. Sutton (pp. 142-150).

Microorganisms from Mytilus edulis (marine mussel) degraded arsenobetaine, with the formation of trimethylarsine oxide, dimethylarsinate and methylarsonate. Four bacterial isolates from these mixed-cultures were shown by HPLC/hydride generation-atomic fluorescence spectroscopy (HPLC/HG-AFS) analysis to degrade arsenobetaine to dimethylarsinate in pure culture; there was no evidence of trimethylarsine oxide formation. Two of the isolates ( Paenibacillus sp. strain 13943 and Pseudomonas sp. strain 13944) were shown by HPLC/inductively coupled plasma-mass spectrometry (HPLC/ICPMS) analysis to degrade arsenobetaine by initial cleavage of a methyl-arsenic bond to form dimethylarsinoylacetate, with subsequent cleavage of the carboxymethyl-arsenic bond to yield dimethylarsinate. Arsenobetaine biodegradation by pure cultures was biphasic, with dimethylarsinoylacetate accumulating in culture supernatants during the culture growth phase and its removal accompanying dimethylarsinate formation during a carbon-limited stationary phase. The Paenibacillus sp. also converted exogenously supplied dimethylarsinoylacetate to dimethylarsinate only under carbon-limited conditions. Lysed-cell extracts of the Paenibacillus sp. showed constitutive expression of enzyme(s) capable of arsenobetaine degradation through methyl-arsenic and carboxymethyl-arsenic bond cleavage. The work establishes the capability of particular bacteria to cleave both types of arsenic-carbon bonds of arsenobetaine and demonstrates that mixed-community functioning is not an obligate requirement for arsenobetaine biodegradation.

Keywords: Arsenobetaine; Dimethyarsinoylacetate; Dimethylarsinate; Bacteria; Degradation; Bioconversion

Purification and characterization of homodimeric methylmalonyl-CoA mutase from Sinorhizobium meliloti by Emi Miyamoto; Fumio Watanabe; Trevor C. Charles; Ryoichi Yamaji; Hiroshi Inui; Yoshihisa Nakano (pp. 151-154).

High activity (>60 munit/mg protein) of 5′-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000±5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0±2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH₂-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.

Keywords: Cobalamin; Methylmalonyl-CoA; Sinorhizobium meliloti ; Monovalent cation

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Archives of Microbiology (v.180, #2)

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Archives of Microbiology (v.180, #2)