Thymine metabolism in Pseudomonas aeruginosa strain 1: the presence of a salvage pathway

Exogenous thymine was found to be taken up very slowly by Pseudomonas aeruginosa in comparison to other pyrimidines, and most of it was catabolized by the cell. The existence of a functional, although inefficient, thymine salvage pathway was demonstrated and this pathway operated more effectively when de novo thymidine nucleotide biosynthesis was inhibited by trimethoprim or methotrexate. The mechanism of thymine salvage by P. aeruginosa appears to be different from that of Escherichia coli and Pseudomonas acidovorans as thymidine was not incorporated into the DNA. Like P. acidovorans, P. aeruginosa lacked thymidine phosphorylase activity. Unsuccessful attempts were made to isolate thymine auxotrophs.     

Enhancement of the Diversity of Polyoxins by a Thymine-7-Hydroxylase Homolog outside the Polyoxin Biosynthesis Gene Cluster

Polyoxins consist of 14 structurally variable components which differentiate at three branch sites of the carbon skeleton. Open reading frame (ORF) SAV_4805 of Streptomyces avermitilis, showing similarity to thymine-7-hydroxylase, was proved to enhance the diversity of polyoxins at the C-5 site of the 1-(5′-amino-5′-deoxy-β-d-allofuranuronosyl) pyrimidine moiety.The antifungal nucleoside antibiotic polyoxins synthesized naturally by Streptomyces cacaoi subsp. asoensis (S. cacaoi here) consist of a mixture of at least 14 different compounds called polyoxins A to N (Fig. ​(Fig.1)1) (6, 13, 25). Most of the polyoxins have a common nucleoside skeleton that is attached with variable side groups at three different places (polyoxins C, I, and N have a modified skeleton). Polyoxins are grouped into four different classes according to the identity of the side group R1 attached at C-5 of the 1-(5′-amino-5′-deoxy-β-d-allofuranuronosyl) pyrimidine moiety. Different classes of polyoxins differ markedly in their activity spectra against plant pathogenic fungi (1, 10, 20).Open in a separate windowFIG. 1.Chemical structure of polyoxin complex. Class is determined by R1. The × indicates a different skeleton that does not have this particular residue.It was deduced that the nucleoside moiety originated from the condensation of uridine with phosphoenolpyruvate (PEP) to generate octosyl acid as the intermediate (7, 12, 14). Then, a subsequent oxidative elimination of the two terminal carbons would create the nucleoside moiety (12). The detailed biosynthetic pathway of nucleoside moiety of polyoxins remains obscure. Our previous work showed that heterologous expression of the polyoxin biosynthetic gene cluster pol in Streptomyces lividans TK24 produces only thymine-derived polyoxin H (class III) (5). This prompted us to investigate the origin of a gene(s) related to the structural variation of polyoxins at the R1 site, which could be located outside the polyoxin biosynthetic gene cluster in the native producer.The pyrimidine rings of polyoxins correspond one to one to the intermediates of the thymidine salvage pathway, which was characterized only for some fungi (see Fig. S1 in the supplemental material) (22). In the prime pathway of nucleotide metabolism, dUMP could be converted to dTMP under the catalysis of thymidylate synthase (TS) with tetrahydrofolic acid as a methyl donor while dTMP could not be converted back to dUMP reversibly (4, 17). In the 1970s, Shaffer et al. separated two enzymes, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and isoorotate decarboxylase (IDCase), from fungal sources which showed potential ability to convert thymine to uracil (22). THase is a trifunctional oxygenase and catalyzes three enzymatic reactions: thymine to 5-hydroxymethyluracil, 5-hydroxymethyluracil to 5-formyluracil, and 5-formyluracil to uracil-5-carboxylic acid (also designated isoorotate) (18, 26). IDCase catalyzes the subsequent decarboxylation reaction, and uracil-5-carboxylic acid is converted to uracil in the end (21). This process is catalyzed by THase, and IDCase is the core step of the thymidine salvage pathway in fungi.BLAST analysis results with the amino acid sequence of THase from Rhodotorula glutinis and IDCase from Neurospora crassa as queries in the 25 sequenced Streptomyces strains (5 finished and 20 in process) of the genome database (from NCBI GenBank, accessed 24 July 2010) showed that the THase and IDCase gene homologs are distributed extensively in Streptomyces, such as open reading frame (ORF) SAV_4805 (sharing 27% identity and 45% similarity with the THase gene) from S. avermitilis strain MA4680 and SCO_6305 (sharing 25% identity and 44% similarity with the IDCase gene) from S. coelicolor strain A3(2) (2, 11, 23). The results indicated a potential thymidine salvage pathway in Streptomyces, and the salvage pathway provides alternative nucleoside monophosphates as precursors for polyoxin biosynthesis.     

Cloning and sequence analysis of thymidine kinase from the oral bacterium Streptococcus gordonii

Abstract Thymidine kinase is an important enzyme in the pyrimidine nucleotide salvage pathway and catalyzes the formation of thymidylate from thymidine using ATP as a phosphate donor. The gene encoding thymidine kinase of the oral bacterium Streptococcus gordonii was cloned and the nucleptide sequence determined. The inferred amino acid sequence of thymidine kinase (191 amino acids) exhibited 43% identity with type H thymidine kinase from Escherichia coli . The S. gordonii thymidine kinase expressed in Escherichia coli KY895 ( tdk⁻ ) was inhibited by thymidline triphosphate, a feature typical of type II thymidine kinases. Immediately 3' to the tdk gene, and possibly co-transcribed with it, was the gene encoding release factor 1 ( prfA ).     

Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans

Tetrathionate is one of the most important intermediates in dissimilatory sulfur oxidation and can itself be utilized as a sole energy source by some sulfur-oxidizing microorganisms. Tetrathionate hydrolase (4THase) plays a significant role in tetrathionate oxidation and should catalyze the initial step in the oxidative dissimilation when sulfur-oxidizing bacteria are grown on tetrathionate. 4THase activity was detected in tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells but not in iron-grown cells. A 4THase having a dimeric structure of identical 50kDa polypeptides was purified from tetrathionate-grown cells. The 4THase showed the maximum activity at pH 3.0 and high stability under acidic conditions. An open reading frame (ORF) encoding the N-terminal amino acid sequence of the purified 4THase was identified by a BLAST search using the database for the A. ferrooxidans ATCC 23270 genome. Heterologous expression of the gene in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. Antisera against the recombinant protein clearly recognized the purified native 4THase, indicating that the ORF encoded the 4THase.     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-beta-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-beta-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-beta-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.     

Transition metal-catalyzed nonoxidative decarboxylation reactions

Decarboxylases typically utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates. The recent characterization of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) has revealed that this enzyme adopts a TIM-barrel (beta/alpha)(8) fold and employs a mononuclear transition metal center to decarboxylate the substrate in an oxidant-independent fashion. Thus, ACMSD represents a type of decarboxylation reaction that has been so far uncharacterized in biological systems. Several close homologues of ACMSD were analyzed, including isoorotate decarboxylase (IDCase), 5-carboxyvanillic acid decarboxylase (5-CVD), gamma-resorcylate decarboxylase (gamma-RSD), and 4-oxalomesaconate hydratase (OMAH). These enzymes are involved in the catabolism of tryptophan and vanillate, the biodegradation of hydroxylbenzoates, and the thymidine salvage pathways in certain organisms. They possess the signature sequence motifs of the amidohydrolase superfamily and likely share the same structural and mechanistic characteristics as that of ACMSD. Analysis of the sequence conservation and evolutionary relationship of ACMSD-related proteins suggests an emerging ACMSD protein family that includes ACMSD and ACMSD-like decarboxylases and hydratases with diverse substrate specificities, many of which are poorly understood in regard to their functions and mechanisms. Progress in the biochemical and structural characterization of ACMSD not only sheds light on the active site of this protein family but also promises the elucidation of the detailed catalytic mechanism of these novel transition metal-dependent nonoxidative decarboxylation reactions.     

Comparative nucleotide sequence analysis of growth-rate-regulated gnd alleles from natural isolates of Escherichia coli and from Salmonella typhimurium LT-2.

A comparative study of gnd genes from Escherichia coli strains isolated from natural populations and laboratory strains and from Salmonella typhimurium was undertaken. In the accompanying paper (G. J. Barcak and R. E. Wolf, Jr., J. Bacteriol. 170:365-371, 1988), we showed that the growth-rate-dependent regulation of gnd expression was conserved among four natural E. coli isolates and E. coli B/r in a manner qualitatively similar to that of the gene from E. coli K-12. Here, we report the DNA sequence of the 5' regulatory region and the first 125 codons of the structural gene for the five E. coli gnd genes and the gnd gene from S. typhimurium LT-2. The sequences differed from one another by 5% on the average. All sequences defined putative secondary structures of the mRNA leader, which were previously proposed to be important in the regulation of the K-12 gene. In addition, a sequence between codons 69 and 74, which is highly complementary to the ribosome-binding site of the mRNA, was conserved in all the genes. The sequence data are discussed with respect to potential regulatory consequences.     

Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene

The chemical monomer p-hydroxystyrene (pHS) is used for producing a number of important industrial polymers from petroleum-based feedstocks. In an alternative approach, the microbial production of pHS can be envisioned by linking together a number of different metabolic pathways, of which those based on using glucose for carbon and energy are currently the most economical. The biological process conserves petroleum when glucose is converted to the aromatic amino acid L-tyrosine, which is deaminated by a tyrosine/phenylalanine ammonia-lyase (PAL/TAL) enzyme to yield p-hydroxycinnamic acid (pHCA). Subsequent decarboxylation of pHCA gives rise to pHS. Bacteria able to efficiently decarboxylate pHCA to pHS using a pHCA decarboxylase (PDC) include Bacillus subtilis, Pseudomonas fluorescens and Lactobacillus plantarum. Both B. subtilis and L. plantarum possess high levels of pHCA-inducible decarboxylase activity and were chosen for further studies. The genes encoding PDC in these organisms were cloned and the pHCA decarboxylase expressed in Escherichia coli strains co-transformed with a plasmid encoding a bifunctional PAL/TAL enzyme from the yeast Rhodotorula glutinis. Production of pHS from glucose was ten-fold greater for the expressed L. plantarum pdc gene (0.11mM), compared to that obtained when the B. subtilis PDC gene (padC) was used. An E. coli strain (WWQ51.1) expressing both tyrosine ammonia-lyase(PAL) and pHCA decarboxylase (pdc), when grown in a 14L fermentor and under phosphate limited conditions, produced 0.4g/L of pHS from glucose. We, therefore, demonstrate pHS production from an inexpensive carbohydrate feedstock by fermentation using a novel metabolic pathway comprising genes from E. coli, L. plantarum and R. glutinis.     

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.     

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.     

Gene cloning, expression, and functional characterization of an ornithine decarboxylase protein from Serratia liquefaciens IFI65

Putrescine has a negative effect on health and is also used as an indicator of quality on meat products. We investigated the genes involved in putrescine production by Serratia liquefaciens IFI65 isolated from a spoiled Spanish dry-cured ham. We report here the genetic organization of its ornithine decarboxylase encoding region. The 5506-bp DNA region showed the presence of three complete and two partial open reading frames. Putative functions have been assigned to several gene products by sequence comparison with proteins included in the databases. The second gene putatively coded for an ornithine decarboxylase. The functionality of this decarboxylase has been experimentally demonstrated by complementation to an E. coli defective mutant. Based on sequence comparisons of some enterobacterial ornithine decarboxylase regions, we have elaborated a hypothetical pathway for the acquisition of putrescine biosynthetic genes in some Enterobacteriaceae strains.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

Characterization of a UV endonuclease gene from the fission yeast Schizosaccharomyces pombe and its bacterial homolog.

From the fission yeast Schizosaccharomyces pombe, a cDNA fragment was isolated, which confers UV resistance on repair deficient Escherichia coli host cells. The cloned cDNA encodes a protein of 68,815 Da, which has a 36.6% identity of amino acid sequence with the previously identified 74 kDa UV endonuclease of the filamentous fungus Neurospora crassa. Analysis of several truncated gene constructs shows that only the C-terminal two thirds region, which has 54% identity of amino acid sequence with the C-terminal region of the Neurospora homolog, is necessary for complementing activity of UV-sensitivity in the E. coli host cells. Purified recombinant protein from E. coli host cells incises both UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts at the sites immediately 5' to the DNA damage in the same fashion as the Neurospora protein. Furthermore, a bacterial homologous sequence was isolated from Bacillus subtilis and shows a similar complementing activity of UV sensitivity in E. coli host cells, indicating a wide distribution of this alternative excision repair mechanism in life.     

Regulation of thymidine metabolism in Neurospora crassa.

The utilization of thymidine by Neurospora crassa is initiated by the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and the consequent formation of thymine and ribose. Thymine must then be oxidatively demethylated by the thymine 7-hydroxylase and uracil-5-carboxylic acid decarboxylase reactions. This article shows that the 2'-hydroxylase reaction can be regulated differently than the oxidative demethylation process and suggests that the 2'-hydroxylase has, in addition to the role of salvaging the pyrimidine ring, the role of providing ribose not only for the utilization of the demethylated pyrimidine but also for other metabolic processes. One way that this difference in regulation was observed was with the uc-1 mutation developed by Williams and Mitchell. The present communication shows that this mutation increases the activities of the 7-hydroxylase and the decarboxylase but has no comparable effect on the 2'-hydroxylase. Qualitatively similar effects on these enzymes were bought about by growth of wild-type Neurospora in media lacking ammonium ion, such as the Westergaard-Mitchell medium. The 2'-hydroxylase and 7-hydroxylase are also differently affected by the carbon dioxide content of the atmosphere above the growing culture and the growth temperature. Studies with inhibitors indicated that the carbon dioxide effect is dependent on protein synthesis.     

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: an alternate cycloisomerase motif.

3-Carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.