A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (< C4) and produces H2O2. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.

Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate.

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.

A two-component system involved in regulation of anaerobic toluene metabolism in Thauera aromatica.

The genes for a two-component regulatory system of the denitrifying toluene-degrading bacterium Thauera aromatica were identified immediately upstream of the genes for benzylsuccinate synthase (bssDCAB), the first enzyme involved in anaerobic toluene metabolism. The genes apparently encode the regulators of toluene catabolic enzymes and were therefore termed tdiSR (for toluene degradation including sensor and regulator). The tdiR gene product was overproduced in Escherichia coli and assayed for binding to a DNA fragment containing the 5' region of the bss operon. We observed specific DNA binding with cell extracts containing overproduced TdiR, but not with control extracts. The tdiSR genes are almost identical to two genes of Thauera strain T1, which have not been assigned a function so far. In addition, the derived gene products share similarity with regulators of toluene and styrene catabolic pathways in aerobic Pseudomonas species, and with the tutCB gene products of Thauera strain T1. The latter have previously been implicated in regulating anaerobic toluene metabolism. Our data suggest that toluene catabolism under aerobic and anaerobic conditions is regulated by similar, but distinct two-component systems.

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica.

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.

Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism.

Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (alpha), 90 (alpha'), 12 (beta) and 10 kDa (gamma) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the alpha- and alpha'-subunits were identical, suggesting a C-terminal degradation of half of the alpha-subunits to give the alpha'-subunit. The composition of native enzyme therefore appears to be alpha2beta2gamma2. A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the alpha'-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.