Attila Szentirmai

Microbial physiology of sidechain degradation of sterols

SummaryA large number of valuable starting materials for steroids synthesis (e.g. 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, 9α-hydroxy-4-androsten-17-one) have been produced by microbial transformation methods. This review helps to evaluate the microbial physiological interest of the widely used sterol sidechain degradation processes. Four inducible groups of the catabolic enzymes are involved in the sterol sidechain degradation pathway; the fatty acid β-oxidation system, the ω-oxidase reaction, a methyl-crotonyl-CoA carboxylation system and the propionyl-CoA carboylase system.Altogether nine catabolic enzymes are involved in the β-sitosterol sidechain degradation pathway. They work in 14 consecutive enzymatic steps. Summing up: three molecules of FADH2, three molecules of propionyl-SCoA, three of NADH and one molecule of acetic acid are formed, while the sidechain of one mole of sitosterol is removed selectively. The metabolism of the propionates and the acetate yield 18 molecules of NADH and 7 molecules of FADH2. Taking into consideration the whole process more than 80 molecules of ATP could be formed during the sitosterol sidechain degradation process.

GLUTATHIONE METABOLISM AND PROTECTION AGAINST OXIDATIVE STRESS CAUSED BY PEROXIDES IN PENICILLIUM CHRYSOGENUM

The filamentous fungus Penicillium chrysogenum showed remarkable resistance to the oxidative stress caused by high concentrations of either hydrogen peroxide (0.35-0.70 M) or tert-butyl hydroperoxide (tert-BOOH, 0.5-2.0 mM), which could be explained well with high levels of glutathione (GSH) peroxidase and catalase activities. The majority of exogenous H2O2 was likely removed by catalase from the cells while tert-BOOH was likely eliminated mainly by the GSH-dependent pathways. The GSH pool decreased considerably at high tert-BOOH concentrations, the glutathione disulphide (GSSG) pool increased at high H2O2 and tert-BOOH concentrations, meanwhile all the peroxide concentrations tested increased markedly the intracellular peroxide concentration. All the enzyme activities taking part in the glutathione metabolism (glutathione peroxidase, glutathione reductase, gamma-glutamyltranspeptidase and glutathione producing activities) except glutathione S-transferase increased significantly after exposing mycelia to both peroxides while the specific glucose-6-phosphate dehydrogenase and catalase activities remained unchanged. In the presence of 0.5 mM diamide both GSSG and GSH concentrations as well as the glutathione reductase and glutathione producing activities were elevated but no significant changes were found in the intracellular peroxide concentration or in any of the other enzyme activities examined.

Analysis of the oxidative stress response of Penicillium chrysogenum to menadione

The intracellular superoxide and glutathione disulphide concentrations increased in Penicillium chrysogeum treated with 50, 250 or 500 microM menadione (MQ). A significant increase in the intracellular peroxide concentration was also observed when mycelia were exposed to 250 or 500 microM MQ. The specific activity of Cu,Zn and Mn superoxide dismutases, glutathione reductase and glutathione S-transferase as well as the glutathione producing activity increased in the presence of MQ while glutathione peroxidase and gamma-glutamyltranspeptidase were only induced by high intracellular peroxide levels. The glucose-6-phosphate dehydrogenase and catalase activities did not respond to the oxidative stress caused by MQ.

Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens.

The sequences of the 16S rRNA gene of 40 strains of bacterial symbionts isolated from the nematodes Heterorhabditis spp. and seven bacterial symbionts of the nematodes Steinernema spp. which were isolated from different geographical areas, as well as the type strain of Xenorhabdus japonicus, were determined and compared to each other and to the sequences of several reference strains of members of the Enterobacteriaceae. The data confirmed the separate status of the two genera of symbionts of entomopathogenic rhabditid nematodes. The symbionts of Heterorhabditis spp. clustered with the type strain of Photorhabdus luminescens, while the symbionts of Steinernema spp. grouped with Xenorhabdus species. X. japonicus clustered with the other Xenorhabdus species. Phylogenetic analysis of 15 almost complete 16S ribosomal DNA (rDNA) sequences of the Heterorhabditis symbionts indicated that there were several subclusters. The properties correlated with these subclusters are not yet apparent, although there may be some geographical and ecological correlations. For example, among the nematode-symbiotic bacteria, the members of subclusters I and III are from southeastern and midwestern North America, respectively, while the members of subclusters II and IV are primarily from Europe and Australia, respectively. The nonsymbiotic strains of P. luminescens form a highly homologous subcluster by themselves. The results of DNA-DNA hybridization studies performed with a few selected strains of five of the 16S rDNA subclusters support the existence of several genospecies within P. luminescens.

Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae

Aims:  Broad‐spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes.

Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): A key to cellulase gene expression on lactose

The heterodisaccharide lactose (1,4-O-β-d-galactopyranosyl-d-glucose) induces cellulase formation in the ascomycete Hypocrea jecorina (= Trichoderma reesei). Lactose assimilation is slow, and the assimilation of its β-d-galactose moiety depends mainly on the operation of a recently described reductive pathway and depends less on the Leloir pathway, which accepts only α-d-galactose. We therefore reasoned whether galactomutarotase [aldose 1-epimerase (AEP)] activity might limit lactose assimilation and thus influence cellulase formation. We identified three putative AEP-encoding genes (aep1, aep2, aep3) in H. jecorina, of which two encoded intracellular protein (AEP1 and AEP2) and one encoded an extracellular protein (AEP3). Although all three were transcribed, only the aep3 transcript was detected on lactose. However, no mutarotase activity was detected in the mycelia, their cell walls, or the extracellular medium during growth on lactose. Therefore, the effect of galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal galactose mutarotase part of the Saccharomyces cerevisiae Gal10. These strains showed increased growth on lactose in a gene copy number-dependent manner, although their formation of extracellular β-galactosidase activity and transcription of the genes encoding the first steps in the Leloir and the reductive pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the β-anomer of d-galactose and reveal the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.

Regulation of formation of the intracellular β-gaiactosidase activity ofAspergillus nidulans

The regulation of formation of the single intracellular β-galactosidase activity ofAspergillus nidulans was investigated. β-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose orD-galactose.L-Arabinose, and — with lower efficacy —D-xylose also induced β-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in β-galactosidase activity. In contrast, in cultures growing onD-galactose, addition of glucose decreased the activity of β-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not ofD-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of β-galactosidase and partially interfered with the induction of β-galactosidase byD-galactose,L-arabinose, andD-xylose.D-Galactose phosphorylation was not necessary for β-galactosidase induction, since induction byD-galactose occurred in anA. nidulans mutant defective in galactose kinase, and by the non-metabolizableD-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced β-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible byD-galactose, whereas it was still inducible byL-arabinose. We conclude that biosynthesis of the intracellular β-galactosidase ofA. nidulans} is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced byD-galactose andL-arabinose in independent ways.

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum

Abstract Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed.

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

Aims:  The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram‐positive and Gram‐negative bacteria, and were tested against mastitis isolates from dairy cows.

Analysis of the relationship between growth, cephalosporin C production, and fragmentation in Acremonium chrysogenum

Mycelial fragmentation in submerged cultures of the cephalosporin C (CPC) producing fungus Acremonium chrysogenum was characterized by image analysis. In both fed-batch and chemostat cultures, the proportion of mycelial clumps seemed to be the most sensitive morphological indicator of fragmentation. In a fed-batch fermentation culture, this declined from roughly 60% at inoculation to less than 10% after 43 h. Subsequent additions of glucose resulted in a sharp increase back to near the initial value, an increase that reversed itself a few hours after glucose exhaustion. Meanwhile CPC production continued to decline steadily. On the other hand, the addition of soybean oil enhanced CPC production, but had no significant effect on the morphology. Although it may sometimes appear that morphology and productivity are related in batch or fed-batch cultures, this study suggests that this is because both respond simultaneously to more fundamental physiological changes, dependent on the availability of carbon. In circumstances, such as supplementary carbon source addition, the relationship is lost. Chemostat cultures supported this belief, as CPC-production rates were hardly affected by the specific growth rate, but the morphology showed significant differences, i.e., lower dilution rates resulted in a lower proportion of clumps and in smaller clumps.