Metabolism of 3-hydroxybenzoate and gentisate by strain <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP

The ability of strain Rhodococcus opacus 1CP to utilize 3-hydroxybenzoate (3-HBA) and gentisate in concentrations up to 600 and 700 mg/L, respectively, as sole carbon and energy sources in liquid mineral media was demonstrated. Using high-performance liquid chromatography (HPLC) and thin-layer chromatography, 2,5-dihydroxybenzoate (gentisate) was identified as the key intermediate of 3-hydroxybenzoate transformation. In the cell-free extracts of the strain grown on 3-HBA or gentisate, the activities of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, and maleylpyruvate isomerase were detected. During growth on 3-HBA, low activity of catechol 1,2-dioxygenase was detected. Based on the data obtained, the pathway of 3-HBA metabolism by strain R. opacus 1CP was proposed.     

The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum

Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC- and mshD- lost the ability to produce mycothiol, but mutant mshB- produced mycothiol as the wild type did. The phenotypes of mutants mshC- and mshD- were the same as the wild type when grown in LB or BHIS media, but mutants mshC- and mshD- were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 +/- 11.9 microM and 1520 +/- 57.4 micromol/min/mg, respectively (mycothiol concentration, 2.5 microM). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.     

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Evidence for isofunctional enzymes used in m-cresol and 2,5-xylenol degradation via the gentisate pathway in Pseudomonas alcaligenes.

Study of the reaction sequence by which Pseudomonas alcaligenes (P25X1) and derived mutants degrade m-cresol, 2,5-xylenol, and their catabolites has provided indirect evidence for the existence of two or more isofunctional enzymes at three different steps. Maleylpyruvate hydrolase activity appears to reside in two different proteins with different specificity ranges, one of which (MPH1) is expressed constitutively; the other (MPH11) is strictly inducible. Two gentisate 1,2-dioxygenase activities were found, one of which is constitutively expressed and possesses a broader specificity range than the other, which is inducible. From oxidation studies with intact cells, there appear to be two activities responsible for the 6-hydroxylation of 3-hydroxybenzoate, and again a broadly specific activity is present regardless of growth conditions; the other is inducible by 3-hydroxybenzoate. Three other enzyme activities are also detected in uninduced cells, viz., xylenol methylhydroxylase, benzylalcohol dehydrogenase, and benzaldehyde dehydrogenase. All apparently possess broad specificity. Fumarylpyruvate hydrolase was also detected but only in cells grown with m-cresol, 3-hydroxybenzoate, or gentisate. Mutants, derived either spontaneously or after treatment with mitomycin C, are described, certain of which have lost the ability to grow with m-cresol and 2,5-xylenol and some of which have also lost the ability to form the constitutive xylenol methylhydroxylase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase, 3-hydroxybenzoate 6-hydroxylase, and gentisate 1,2-dioxygenase. Such mutants, however, retain ability to synthesize inducibly a second 3-hydroxybenzoate 6-hydroxylase and gentisate 1,2-dioxygenase, as well as maleylpyruvate hydrolase (MPH11) and fumarylpyruvate hydrolase; MPH1 was still synthesized. These findings suggest the presence of a plasmid for 2,5-xylenol degradation which codes for synthesis of early degradative enzymes. Other enzymes, such as the second 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, maleylpyruvate hydrolase (MPH1 and MPH11), and fumarylpyruvate hydrolase, appear to be chromosomally encoded and, with the exception of MPH1, strictly inducible.     

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.     

Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens

An anaerobic phthalate isomer-degrading strain (JT^T) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JI^T, was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37°C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JT^T was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JI^T utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JI^T. Neither strain JT^T nor strain JI^T could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JT^T and JI^T were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in ‘Desulfotomaculum lineage I’. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JT^T and strain JI^T, respectively. The type strains are strains JT^T (= DSM 16121^T = JCM 11824^T = NBRC 100523^T) and JI^T (= JCM 12282^T = BAA-1053^T) for P. terephthalicum and P. isophthalicum, respectively.Nucleotide sequence accession number: The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequences of strains JT^T and JI^T are AB091323 and AB232785, respectively     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Deltancg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Pathways of 4-hydroxybenzoate degradation among species of Bacillus.

The pathways used by three bacterial strains of the genus Bacillus to degrade 4-hydroxybenzoate are delineated. When B. brevis strain PHB-2 is grown on 4-hydroxybenzoate, enzymes of the protocatechuate branch of the beta-ketoadipate pathway are induced. In contrast, B. circulans strain 3 contains high levels of the enzymes of the protocatechuate 2,3-dioxygenase pathway after growth on 4-hydroxybenzoate. B. laterosporus strain PHB-7a degrades 4-hydroxybenzoate by a novel reaction sequence. After growth on 4-hydroxybenzoate, strain PHB-7a contains high levels of gentisate oxygenase (EC 1.13.11.4) and maleylpyruvate hydrolase. Whole cells of strain PHB-7a (grown on 4-hydroxylbenzoate) accumulate 2,5-dihydroxybenzoate (gentisate) from 4-hydroxybenzoate when incubated in the presence of 1mM alpha,alpha'-dipyridyl. Thus, strain PHB-7a appears to convert 4-hydroxybenzoate to gentisate, which is further degraded by the glutathione-independent gentisic acid pathway. These pathway delineations provide evidence that Bacillus species are derived from a diverse evolutionary background.     

Evidence for two uptake systems in Rhizobium leguminosarum for hydroxy-aromatic compounds metabolized by the 3-oxoadipate pathway

Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii have separate uptake systems for 4-hydroxybenzoate and protocatechuate. The 4-hydroxybenzoate uptake system (pobP) is inhibited by a range of compounds with substitution at the 4-position on the aromatic ring whereas the uptake system for protocatechuate (pcaP) is markedly inhibited only by other dihydroxybenzoic acids. The rate of 4-hydroxybenzoate uptake is very low in Rhizobium leguminosarum and Rhizobium trifolii grown on protocatechuate but mutants defective in 4-hydroxybenzoate uptake transport protocatechuate at rates similar to the wild-type grown under similar conditions.     

Genetic analysis of benzoate metabolism in Aspergillus niger

Summary The benzoate metabolism of Aspergillus niger was studied as part of a design to clone the benzoate-4-hydroxylase gene of this fungus on the basis of complementation. Filtration enrichment techniques yielded mutants defective for different steps of benzoate degradation: bph (benzoate-4-hydroxylase), phh (4-hydroxybenzoate-3-hydroxylase) and prc (protocatechuate ring cleavage) mutants. In this way the degradation pathway for benzoate, involving the formation of 4-hydroxybenzoate and 3,4-dihydroxybenzoate has been confirmed. In addition a mutant sensitive to benzoate has been found. Complementation tests in somatic diploids showed that the bph mutants belonged to two complementation groups. The major group is probably defective in the structural gene (bphA). All phh mutants tested belonged to one complementation group. The prc mutants could be divided into several groups on the basis of their growth on different aromatic substrates and on the basis of the complementation test. The phh and both bph mutations are shown to be located on different chromosomes.Offprint requests to: C. J. Bos     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl^– and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.     

Arginine and proline genes ofAspergillus niger

Summary Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. Thearg genes belong to six different linkage groups and thepro genes to two. Onearg-mutant could be complemented by transformation with theA. nidulans arg B ⁺ gene, and thisA. niger gene thus appeared to be homologous to theA. nidulans arg B. We isolated anA. niger strain with theargB gene tightly linked with thenicA1 marker. This strain is very suitable as acceptor for transformation with anargB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using thenicA1 marker gene.     

Methanol and methylamine utilization result from mutational events in Thiosphaera pantotropha

With choline as carbon source Thiosphaera pantotropha GB17 grew with a doubling time (t_d) of 6 h. The cellular yield was 55.8 g dry cell weight per mol of choline, indicating that its methyl moieties were used for growth. However, T. pantotropha was unable to grow with methanol or with methylamine as carbon source. Mutants were isolated from liquid or from solid media able to grow with methanol (Mox⁺) as carbon or methylamine as nitrogen source (Mam⁺). The Mox⁺ mutant GB17M grew with a mean t_d of 11.7h and a growth yield of 8.9 g dry cell weight per mol of methanol. Diauxic growth of strain GB17M was observed with mixtures of pyruvate and methanol as substrates in batch culture. Methanol led to the formation of methanol dehydrogenase, formate dehydrogenase, ribulosebisphosphate carboxylase and of a soluble cytochrome c-551.5. Tn5-insertional mutants defective in the thiosulfate oxidizing enzyme system or in hydrogenase acquired the Mox⁺ phenotype. However, Tn5-insertional mutants defective in either a c-type cytochrome or the molybdenum cofactor did not mutate to the Mox⁺ phenotype, indicating common functions in thiosulfate and in methanol metabolism.     

Effect of molecular hydrogen on histidine utilization by Alcaligenes eutrophus

The effect of molecular hydrogen on heterotrophic metabolism of the facultative chemolithoautotrophic bacterium Alcaligenes eutrophus strain H 16 was representatively investigated on histidine utilization. The presence of hydrogen in a histidine or urocanate-containing medium had two effects (i) growth of the cells was inhibited, and (ii) formation of histidase was repressed. Both effects were relieved by supplying the cells with exogenous carbon dioxide. Studies on mutants defective in chemolithoautotrophic metabolism revealed that growth inhibition by hydrogen was exclusively mediated by the catalytic function of the soluble hydrogenase. Mutants containing only particulate hydrogenase activity did not exhibit growth inhibition. Repression of histidase formation, however, was mediated by the catalytic activity of the soluble as well as the particulate hydrogenase. Unexpectedly, mutants defective in autotrophic carbon dioxide fixation but unaffected in hydrogen oxidation showed an inhibition of growth by hydrogen but no repression of histidase synthesis. Mutants which formed histidase constitutively were still sensitive to repression in the presence of hydrogen. The results indicate that repression of enzyme synthesis by hydrogen is dependent on the function of both, the hydrogen-oxidizing and the carbon dioxide-fixing system. It is concluded that the hydrogen effect is a transient regulatory mechanism and only relevant for unbalanced conditions of growth.     

Anaerobic degradation ofm-cresol via methyl oxidation to 3-hydroxybenzoate by a denitrifying bacterium

The anaerobic degradation of m-cresol was studied in a denitrifying bacterium. In the initial studies, hypothetical intermediates of m-cresol degradation were tested in growth experiments and in adaptation studies with dense cell suspensions. Results suggested a degradation of m-cresol via 3-hydroxybenzoate. To verify this, the degradation of m-cresol was followed in concentrated cell suspensions in the presence of metabolic inhibitors. Fluoroacetate treatment resulted in the transient accumulation of substantial amounts of 3-hydroxybenzoate. In the presence of iodoacetamide, not only was 3-hydroxybenzoate transiently formed, but benzoate was also accumulated. These findings support a degradation of m-cresol via initial anaerobic methyl oxidation to 3-hydroxybenzoate, followed by reductive dehydroxylation to benzoate or benzoyl-CoA. Studies with extracts of m-cresol-grown cells showed the presence of several enzyme activities to be postulated for this pathway. No evidence was found for a carboxylation, hydroxylation of the aromatic ring, or direct ring reduction as the initial step in m-cresol metabolism. Received: 29 November 1994 / Accepted: 7 March 1995     

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit⁻, unable to grow in nitrate, and 28 Nit⁺, able to grow in nitrate). All the Nit⁻ strains lacked MoCo activity. Diploid complementation of Nit⁻, MoCo⁻ strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit⁺ strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c⁺) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c⁺ seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.