AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production.     

The effect of thiourea on ureide metabolism in Neurospora crassa

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.     

Identification of glucose kinase‐dependent and ‐independent pathways for carbon control of primary metabolism,development and antibiotic production in Streptomyces coelicolor by quantitative proteomics

Members of the soil‐dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk‐independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the γ‐butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk‐independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium‐dependent antibiotic (CDA) in mannitol‐grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S. coelicolor on solid‐grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.     

Influence of cultivation conditions on production of lignocellulolytic enzymes by Ceriporiopsis subvermispora

The aim of this work was to make a survey describing factors that influence the production of extracellular enzymes by white-rot fungus Ceriporiopsis subvermispora responsible for the degradation of lignocellulolytic materials. These factors were: carbon sources (glucose, cellulose, hemicellulose, lignin, maltose and starch), nitrogen sources (ammonium sulphate, potassium nitrate, urea, albumin and peptone), pH, temperature and addition of three different concentrations of Cu²⁺ and Mn²⁺. The cellulase and xylanase activities were similar in medium with different carbon sources and the highest cellulase and xylanase activities were measured in medium with urea and potassium nitrate as nitrogen sources, respectively. The highest laccase activity was observed in medium with lignin and peptone as carbon and nitrogen sources. In other experiments, time course of production of lignocellulolytic enzymes by white-rot fungus C. subvermispora in medium with lignin or glucose as carbon sources was observed.     

Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin

The effect of ammonium ions on growth and tylosin biosynthesis in Streptomyces fradiae NRRL 2702 cultured on a chemically defined medium was studied. Mycelial growth and tylosin production were not affected when ammonium sulphate was added to idiophase cultures to a final concentration of 10 mm or 20 mm; however, when ammonium sulphate was added to tylosin cultures to a final concentration of 20 mm before the onset of antibiotic biosynthesis (trophophase), tylosin production was severely suppressed while mycelial growth was stimulated. The activities of propionyl-coenzyme A carboxylase (EC 6.4.1.3) and methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1), enzymes involved in the synthesis of tylonolide precursors, were depressed in high ammonium cultures. The activity of macrocin 3′-o-methyltransferase, which catalyses the methylation of macrocin to form tylosin, was also affected by high concentrations of ammonium ions added in the trophophase.     

Degradation and utilization of exogenous allantoin by intact soybean root

Allantoin is produced by soybean [ Glycine max (L.) Merr. cv. Harper] nodules during nitrogen fixation. Decomposed nodules, therefore, may release allantoin into the surrounding soil. If the released allantoin were to be taken up by the plant without degradation, it is possible that the exogenous allantoin might repress subsequent nodulation. Using a hydroponic growth system, degradation of exogenous allantoin by soybean root was studied. In the presence of intact soybean root exogenous allantoin was rapidly degraded, yielding ca 2 mmol of urea per mmol of allantoin. Hydrolysis of urea to ammonia proceeded very slowly. Instead, the urea seemed to be taken up by the intact soybean root. The enzyme(s) required for the production of urea from exogenous allantoin could not be detected in the aqueous rooting medium. Therefore, these enzymes seem to be attached to the exterior surface of the intact soybean root. This study shows that exogenous allantoin can be readily degraded and assimilated by the growing soybean plant.     

Nitrogen and carbon regulation of lignin peroxidase and enzymes of nitrogen metabolism inPhanerochaete chrysosporium

The regulation of nitrogen metabolism pathways was examined inPhanerochaete chrysosporium in relation to the repression of lignin peroxidase by nitrogen or carbon in this fungus. Under conditions of nitrogen derepression,P. chrysosporium synthesizes the amidohydrolases, formamidase (EC 3.5.1.9) and acetamidase (EC 3.5.1.4) and the enzymes of purine catabolism uricase (EC 1.7.3.3), allantoinase (EC 3.5.2.5), and allantoicase (EC 3.5.3.4). Formamidase is repressed to low levels in the presence of ammonium and there is no apparent control of this enzyme by carbon catabolite repression. Although formamide is a nitrogen source, it is not a carbon source forP. chrysosporium. Glutamate totally represses formamidase. Uricase, allantoinase, and allantoicase are also regulated by nitrogen repression but not carbon catabolite repression. Urease is synthesized at similar levels irrespective of the nitrogen or carbon conditions. The sensitivity of uricase, allantoinase, and allantoicase to nitrogen repression is less than that of formamidase. In contrast to formamidase, glutamate is not a more powerful repressor of uricase, allantoinase, and allantoicase compared with ammonium. No pathway-specific induction is required for the synthesis of formamidase, uricase, allantoinase, and allantoicase. Altogether these features indicate that nitrogen metabolism inP. chrysosporium is similar to that inAspergillus nidulans in its regulation, despite the absence of pathway-specific induction of the enzymes examined. These results are consistent with the existence of a regulatory gene mediating nitrogen catabolite repression similar to theA. nidulans areA gene inP. chrysosporium. Although glycerol acts as a nonrepressive carbon source for lignin peroxidase production (except when used at high concentrations), glutamate totally represses lignin peroxidase even in cultures with glycerol. This indicates that carbon regulation and nitrogen regulation of lignin peroxidase may not be separated inP. chrysosporium.     

Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.     

Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH₃ that is immediately protonated to form NH₄⁺. This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg²⁺ at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium.     

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.     

Detecting positive selection from genome scans of linkage disequilibrium

Background

During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.

Results

Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.

Conclusions

Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.     

Novel nitrogen sources for growth of Bacillus fastidiosus and their effect on the activity of NADP-dependent glutamate dehydrogenase

Abstract Although Bacillus fastidiosus assimilates ammonium formed internally during growth on urate, allantoin or allantoate via NADP-dependent glutamate dehydrogenase (NADP-GDH), growth on exogenous ammonium as nitrogen source has not been observed. Growth on ammonium, urea and ureidoglycolate, intermediates of the urate degradative pathway, was found to occur if the mineral growth medium containing glycerol as a carbon source was supplemented with both allantoin (0.5 mM) and brain heart infusion (BHI, 0.1%, w/v) or yeast extract. Neither allantoin nor BHI supported growth alone or in combination unless ammonium was present. NADP-GDH activity appeared to be regulated only by the extracellular concentration of allantoin or allantoate. Enzyme activity was not influenced by other nitrogen sources or the intracellular ammonium concentration.     

Effects of glutamate, glucose, phosphate, and alkali metal ions on cephamycin C production by Nocardia lactamdurans in defined media

A High cephamycin C producing strain of Nocardia lactam-durans was used to study cell growth and antibiotics production in defined media. Batch fermentations in shake flasks and stirred tanks showed that antibiotic production occurred during cell growth and the production rate rapidly decline as the growth slowed. Glutamate served as a primary substrate during this phase. Later, ammonia was utilized along with a remainder of the glucose. Rapid antibiotic production occurred in this phase. Increased glutamate promoted higher growth, a rise in ammonium ion concentration, and a marked reduction in antibiotic titers. An increase of the glucose concentration along with the glutamate concentration balanced to the medium; no ammonium ion rise occurred and a peak specific antibiotic titer comparable to the control medium was obtained. In a phosphate-limited medium, cell growth equivalent to the control medium and increased antibiotic titers were obtained. In these experiments, adjustment of Na(+) and K(+) ion concentration equal to that in the control medium was found to be important. Based on carbon and nitrogen balances, the activity of the key nitrogen metabolism enzymes, and the published literature, a two-stage model of regulation is suggested.     

Gluconic acid-producing Pseudomonas sp. prevent γ-actinorhodin biosynthesis by Streptomyces coelicolor A3(2)

Streptomyces are ubiquitous soil bacteria well known for their ability to produce a wide range of secondary metabolites including antibiotics. In their natural environments, they co-exist and interact with complex microbial communities and their natural products are assumed to play a major role in mediating these interactions. Reciprocally, their secondary metabolism can be influenced by the surrounding microbial communities. Little is known about these complex interactions and the underlying molecular mechanisms. During pairwise co-culture experiments, a fluorescent Pseudomonas, Pseudomonas fluorescens BBc6R8, was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 without altering the biosynthesis of the intracellular actinorhodin. A mutant of the BBc6R8 strain defective in the production of gluconic acid from glucose and consequently unable to acidify the culture medium did not show any effect on the γ-actinorhodin biosynthesis in contrast to the wild-type strain and the mutant complemented with the wild-type allele. In addition, when glucose was substituted by mannitol in the culture medium, P. fluorescens BBc6R8 was unable to acidify the medium and to prevent the biosynthesis of the antibiotic. All together, the results show that P. fluorescens BBc6R8 impairs the biosynthesis of the lactone form of actinorhodin in S. coelicolor by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. We propose some hypotheses on the ecological significance of such interaction.     

Ureide Catabolism of Soybeans : II. Pathway of Catabolism in Intact Leaf Tissue

Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit ¹⁴CO₂ release from [2,7-¹⁴C]allantoin (urea labeled), but inhibits urea dependent CO₂ release ≥99.9% under similar conditions. Furthermore, ¹⁴CO₂ and [¹⁴C] allantoate are the only detectable products of [2,7-¹⁴C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-¹⁴C]allantoin), its catabolism to [¹⁴C]allantoate, ¹⁴CO₂, [¹⁴C]glyoxylate, [¹⁴C]glycine, and [¹⁴C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH₄⁺ and CO₂ and that carbons 4 and 5 enter the photorespiratory pathway.     

N and C NMR determination of allantoin metabolism in developing soybean cotyledons

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state ¹³C and ¹⁵N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO₂. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO₂. The ¹³C-¹⁵N bonds of [5-¹³C;1-¹⁵N] and [2-¹³C;1,3-¹⁵N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH₄⁺ in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH₄⁺ produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.     

A remedy to oxygen limitation problem in antibiotic production: addition of perfluorocarbon

In this study, the effect of an oxygen carrier, perfluorocarbon, on actinorhodin fermentation by Streptomyces coelicolor A3(2) was investigated using a chemically defined medium in 2 and 20 l bioreactors. The inclusion of 50% (v/v) perfluorocarbon in the fermentation medium resulted in a five-fold increase in the maximum antibiotic concentration. The use of perfluorocarbon also caused remarkable increases in both glucose and oxygen consumption rates. Moreover, the increasing concentrations of perfluorocarbon improved the dissolved oxygen profile by raising the minimum dissolved oxygen concentration. It was found that observed increases in the antibiotic production were linearly related to the volumetric oxygen uptake rates. This result could perhaps be attributed to the enhancement of oxygen transfer in S. coelicolor cultures due to the higher oxygen solubilities of the fermentation medium through inclusion of perfluorodecalin.