Hiroya Yurimoto

Aerobic and Anaerobic Toluene Degradation by a Newly Isolated Denitrifying Bacterium, Thauera sp. Strain DNT-1

ABSTRACT A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.

Propane Monooxygenase and NAD+-Dependent Secondary Alcohol Dehydrogenase in Propane Metabolism by Gordonia sp. Strain TY-5

A new isolate, Gordonia sp. strain TY-5, is capable of growth on propane and n-alkanes with C(13) to C(22) carbon chains as the sole source of carbon. In whole-cell reactions, significant propane oxidation to 2-propanol was detected. A gene cluster designated prmABCD, which encodes the components of a putative dinuclear-iron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced. A mutant with prmB disrupted (prmB::Kan(r)) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane. These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium. Downstream of the prm genes, an open reading frame (adh1) encoding an NAD(+)-dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized. The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan(r)) indicate that Adh1 plays a major role in propane metabolism. Two additional NAD(+)-dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation. On the basis of these results, we conclude that Gordonia sp. strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol.

The Ribulose Monophosphate Pathway Substitutes for the Missing Pentose Phosphate Pathway in the Archaeon Thermococcus kodakaraensis

The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Delta hps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.

The physiological role of the ribulose monophosphate pathway in bacteria and archaea

3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes of the ribulose monophosphate pathway. This pathway, which was originally found in methylotrophic bacteria, is now recognized as a widespread prokaryotic pathway involved in formaldehyde fixation and detoxification. Recent progress, involving biochemical and genetic approaches in elucidating the physiological functions of HPS and PHI in methylotrophic as well as non-methylotrophic bacteria are described in this review. HPS and PHI orthologs are also found in a variety of archaeal strains. Some archaeal HPS orthologs are fused with other genes to form single ORF (e.g., the hps-phi gene of Pyrococcus spp. and the faeB-hpsB gene of Methanosarcina spp). These fused gene products exhibit functions corresponding to the individual enzyme activities, and are more efficient than equivalent systems made up of discrete enzymes. Recently, a novel metabolic function for HPS and PHI has been proposed in which these enzymes catalyze the reverse reaction for the biosynthesis of pentose phosphate in some archaeal strains. Thus the enzyme system plays a different role in bacteria and archaea by catalyzing the forward and reverse reactions respectively.

Anaerobic degradation of aromatic compounds by Magnetospirillum strains : Isolation and degradation genes

Four Magnetospirillum strains degrading toluene, phenol, benzoate, and other aromatic compounds under anaerobic conditions were isolated from denitrifying enrichment cultures. One of the isolates, toluene-degrading strain TS-6, contained genes that are homologous to those encoding benzylsuccinate synthase (Bss) and benzoyl-CoA reductase (Bcr), two key enzymes of anaerobic toluene and benzoate degradation respectively in known denitrifying bacteria. Transcription of the genes was confirmed. It was controlled by growth substrates and oxygen conditions, but bcr genes were unexpectedly expressed in aerobic cells grown on benzoate. It was confirmed that the genus Magnetospirillum represents the third genus of denitrifying bacteria capable of degrading aromatic compounds under anaerobic conditions, besides the genera Thauera and Azoarcus.

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

ABSTRACT Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid β-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.

Novel Acetone Metabolism in a Propane-Utilizing Bacterium, Gordonia sp. Strain TY-5

In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane --> 2-propanol --> acetone --> methyl acetate --> acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.

Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis.

Eukaryotic methylotrophs, which are able to obtain all the carbon and energy needed for growth from methanol, are restricted to a limited number of yeast species. When these yeasts are grown on methanol as the sole carbon and energy source, the enzymes involved in methanol metabolism are strongly induced, and the membrane-bound organelles, peroxisomes, which contain key enzymes of methanol metabolism, proliferate massively. These features have made methylotrophic yeasts attractive hosts for the production of heterologous proteins and useful model organisms for the study of peroxisome biogenesis and degradation. In this paper, we describe recent insights into the molecular basis of yeast methylotrophy.

Antioxidant System within Yeast Peroxisome BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERIZATION OF CbPmp20 IN THE METHYLOTROPHIC YEAST CANDIDA BOIDINII

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using thePMP20-deleted strain of C. boidinii(pmp20Δ strain). The His6-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activityin vitro toward alkyl hydroperoxides and H2O2. Catalytic activity and dimerization of His6-CbPmp20 depended on the only cysteine residue corresponding to Cys53. The pmp20Δ strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Δ growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Δ strain had a more severe growth defect than the cta1Δ strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, thecta1Δ strain accumulated H2O2, whereas the pmp20Δ strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface,e.g. lipid hydroperoxides, rather than to decompose H2O2. In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.

Methylovulum miyakonense gen. nov., sp. nov., a type I methanotroph isolated from forest soil.

A novel methanotroph, designated strain HT12(T), was isolated from forest soil in Japan. Cells of strain HT12(T) were Gram-reaction-negative, aerobic, non-motile, coccoid and formed pale-brown colonies. The strain grew only with methane and methanol as sole carbon and energy sources. Cells grew at 5-34 °C (optimum 24-32 °C). The strain possessed both particulate and soluble methane monooxygenases and assimilated formaldehyde using the ribulose monophosphate pathway. The major cellular fatty acids were C(16 : 0) (46.9 %) and C(14 : 0) (34.2 %), whereas unsaturated C(16) fatty acids, typical of type I methanotrophs, were absent. Comparative 16S rRNA gene sequence analysis showed that the most closely related strains were Methylosoma difficile LC 2(T) (93.1 % sequence similarity) and Methylobacter tundripaludum SV96(T) (92.6 % similarity). Phylogenetic analysis based on the pmoA gene indicated that strain HT12(T) formed a distinct lineage within the type I methanotrophs and analysis of the deduced pmoA amino acid sequence of strain HT12(T) showed that it had a 7 % divergence from that of its most closely related species. The DNA G+C content was 49.3 mol%. Based on this evidence, strain HT12(T) represents a novel species and genus of the family Methylococcaceae, for which the name Methylovulum miyakonense gen. nov., sp. nov. is proposed. The type strain of the type species is HT12(T) ( = NBRC 106162(T)  = DSM 23269(T)  = ATCC BAA-2070(T)).