pyrE Gene Research Articles

Regulation of the Bacillus subtilis Pyrimidine Biosynthetic Operon by Transcriptional Attenuation: Control of Gene Expression by an mRNA-Binding Protein

The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA. Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure. This model is supported by extensive molecular genetic analysis. PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases. Purified B. subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences. The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds. PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B. subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor. PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized.

Sequence and phylogenetic analysis of the Rhizobium leguminosarum biovar trifolii pyrE gene, overproduction, purification and characterization of orotate phosphoribosyltransferase

The pyrE gene of Rhizobium leguminosarum biovar trifolii ( Rl) was subcloned and its sequence is presented. The nucleotide sequence analysis suggests that this gene is not regulated by transcriptional attenuation as seen for the pyrE and pyrB genes of Escherichia coli ( Ec) and Salmonella typhimurium. The Rl pyrE gene was subcloned into Ec AT2538 pyrE60 where the Rl pyrE gene product, orotate phosphoribosyltransferase (OPRTase), was overproduced. Using Ec AT2538 pyrE60 overproducing Rl OPRTase, the enzyme was purified to homogeneity utilizing ammonium sulfate fractionation and affinity chromatography with an orotate monophosphate agarose matrix. The electrophoretically pure OPRTase was characterized and found to be a 24.7±0.3-kDa protein with a K m of 27.6 μmol l −1. The deduced amino acid sequence for OPRTase was compared with OPRTases from other organisms and found to be most similar to that of Bacillus subtilis ( Bs). The Rl OPRTase exhibits 37% identity and 46% similarity to the Bs OPRTase.

A new Thermus-Escherichia coli shuttle integration vector system.

We established a Thermus thermophilus strain in which the pyrE gene (coding for orotate phosphoribosyltransferase of the pyrimidine biosynthetic pathway) was totally deleted. We also constructed an integration vector, which consisted of the Escherichia coli plasmid vector pBluescript and a 2.1-kb segment of the T. thermophilus leu operon sequence, for the integration of a foreign gene into a chromosome of the thermophile. pyrE and leuB genes were used as probes to test the integration vector. The integration vector pINV, bearing the pyrE gene, transformed the delta pyrE strain at a frequency of 6 x 10(-5) through a single crossover event. The leuB gene could also be used as another marker of the integration vector system. The vector could be integrated at the expected site. By digesting the chromosomal DNA of the T. thermophilus transformants with a unique restriction enzyme, the vector could be recovered into E. coli after the recircularization in vitro. The kanamycin nucleotidyltransferase gene could be successfully expressed in the thermophile by using pINV.

Utilization of the Aspergillus nidulans pyrG gene as a selectable marker for transformation and electroporation of Neurospora crassa

We report the complementation of the Neurospora crassa pyr-4 mutation with the Aspergillus nidulans pyrG gene. The use of the pyrG gene as a selectable marker for N. crassa transformation offers several advantages: a non toxic nutritional selection, the ability to put single-copy transformants through a sexual cross without the occurrence of RIP (Repeat-Induced Point mutation) by alleles of the pyr-4 gene, and a relatively-high electroporation efficiency. These traits were exploited for targeted gene replacement in N. crassa. In addition, the high electroporation efficiency of pyrG achieved using supercoiled circular DNA renders it an ideal marker for random insertional mutagenesis studies in N. crassa.

Structure and expression of a pyrimidine gene cluster from the extreme thermophile Thermus strain ZO5.

On a 4.7-kbp HindIII clone of Thermus strain ZO5 DNA, complementing an aspartate carbamoyltransferase mutation in Escherichia coli, we identified a cluster of four potential open reading frames corresponding to genes pyrR, and pyrB, an unidentified open reading frame named bbc, and gene pyrC. The transcription initiation site was mapped at about 115 nucleotides upstream of the pyrR translation start codon. The cognate Thermus pyr promoter also functions in heterologous expression of Thermus pyr genes in E. coli. In Thermus strain ZO5, pyrB and pyrC gene expression is repressed three- to fourfold by uracil and increased twofold by arginine. Based on the occurrence of several transcription signals in the Thermus pyr promoter region and strong amino acid sequence identities (about 60%) between Thermus PyrR and the PyrR attenuation proteins of two Bacillus sp., we propose a regulatory mechanism involving transcriptional attenuation to control pyr gene expression in Thermus. In contrast to pyr attenuation in Bacillus spp., however, control of the Thermus pyr gene cluster would not involve an antiterminator structure but would involve a translating ribosome for preventing formation of the terminator RNA hairpin. The deduced amino acid sequence of Thermus strain ZO5 aspartate carbamoyltransferase (ATCase; encoded by pyrB) exhibits the highest similarities (about 50% identical amino acids) with ATCases from Pseudomonas sp. For Thermus strain ZO5 dihydroorotase (DHOase; encoded by pyrC), the highest similarity scores (about 40% identity) were obtained with DHOases from B. caldolyticus and Bacillus subtilis. The enzyme properties of ATCase expressed from truncated versions of the Thermus pyr gene cluster in E. coli suggest that Thermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase complex.

Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans.

The effect of altering the conditions of transformation on the efficiency of gene targeting in filamentous fungi was studied using Aspergillus nidulans as a model organism. The niaD and amdS genes of A. nidulans, which are both involved in nitrogen source utilisation, were selected as target loci. Homologous recombination of transforming DNA at these loci resulted in niaD and amdS mutants with an impaired ability to utilise nitrate or acetamide as the sole nitrogen source, respectively. Vectors were constructed that contained the Neurospora crassa pyr4 gene as a selectable marker and an internal segment of the amdS (0.6-1.27 kb) or niaD (0.9-2.15 kb) genes. The parameters investigated for their effect on gene targeting included (a) length of homologous DNA in the disruption cassette, (b) conformation of the transforming vector (circular or linear), (c) transcriptional status (on/off) of the targeted gene, (d) concentration of DNA in the transformation mix and (e) temperature of incubation of the transformation reaction and of protoplast regeneration on selective media. Parameters shown to have an effect on the targeting frequency at the niaD locus were tested at the amdS locus. The level of gene targeting when circular DNA was used was found to correlate with the size of the homologous segment at both loci. Similarly the level of targeting was shown to increase when vectors were linearised within the region of homology. The level of targeting was unaltered at the niaD locus when transcription was induced at different stages in the transformation procedure. Likewise, targeting was unaffected by altering the amount of DNA in the reaction mix over the concentration range tested. The regeneration temperature did have an effect on targeting, with enhanced targeting observed at 25 degrees compared with 37 degrees C. However, the most dramatic effect was the difference between targeting efficiency at different genetic loci, with targeting of niaD being at least five fold more efficient than amdS under all conditions tested.

Structure and organisation of the pyrimidine biosynthesis pathway genes in Lactobacillus plantarum: a PCR strategy for sequencing without cloning

This report describes the sequence and structural organisation of the pyrimidine biosynthesis pathway genes of Lactobacillus plantarum CCM 1904. It also describes an in vitro technique based on PCR for sequencing without cloning. This new technique was developed because it was impossible to clone certain parts of the L. plantarum genomic DNA in the Escherichia coli host. L. plantarum pyr genes are organised as a 9.8-kb operon with the following order: pyrR, pyrB, pyrC, pyrAA, pyrAB, pyrD, pyrF and pyrE. There are two major differences from the pyrimidine operons of Bacillus subtilis (Quinn et al., J. Bacteriol. 266 (1991) 9113–9127; Turner et al., J. Bacteriol. 176 (1994) 3708–3722) and Bacillus caldolyticus (Ghim et al., Microbiology 140 (1994) 479–491): the absence of pyrP encoding for uracil permease, and the absence of an open reading frame named orf2, whose function is unknown. Two mutually exclusive stem-loop structures were predicted at the 5′-end of L. plantarum pyr mRNA; this operon could be regulated by transcriptional attenuation under the control of PyrR. Complementation of E. coli pyrD, pyrF and pyrE mutants was obtained with a L. plantarum genomic DNA library. Alignment of the L. plantarum Pyr proteins with other known procaryotic Pyr proteins indicates that they display highly conserved regions in Gram-positive and Gram-negative bacteria.

Evidence that the Bacillus subtilis pyrimidine regulatory protein PyrR acts by binding to pyr mRNA at three sites in vivo.

The Bacillus subtilis pyr operon is regulated by a transcriptional attenuation mechanism that requires the PyrR regulatory protein. Multicopy plasmids that could be transcribed to yield segments of RNA from the attenuation regions of the pyr operon induced derepression of chromosomal pyr genes, whereas plasmids that could not yield pyr RNA did not. We conclude that pyr RNA acts by titrating the PyrR protein and preventing it from regulating pyr attenuation.

Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus.

We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus. A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced. The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile.

Orotate Phosphoribosyltransferase from Thermus thermophilus: Overexpression in Escherichia coli, Purification and Characterizatiton

Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified. The pyrE gene of T. thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content. Since this gene expressed itself inefficiently in E. coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to major codons for E. coli. Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli. This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity. Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide. The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP. The activation energy of this enzyme reaction was 20.3 kJ/mol. The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions. The purified OPRTase was stable for 20 min at 85 degrees C.

Cloning, sequence and expression of the gene coding for rhamnogalacturonase of Aspergillus aculeatus; a novel pectinolytic enzyme.

Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da. The protein contains a potential signal peptidase cleavage site behind Gly-18 and three potential sites for N-glycosylation. Limited homology with A. niger polygalacturonase amino acid sequences is found. A genomic clone of rhgA was isolated from a recombinant phage lambda genomic library. Comparison of the genomic and cDNA sequences revealed that the coding region of the gene is interrupted by three introns. Furthermore, amino acid sequences of four different peptides, derived from purified A. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence of rhgA. A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either the A. niger pyrA gene or the A. aculeatus pyrA gene as selection marker. For expression of rhamnogalacturonase in A. awamori the A. awamori pyrA gene was used as selection marker. Degradation patterns of modified hairy regions, determined by HPLC, show the recombinant rhamnogalacturonase to be active, and the enzyme was found to have a positive effect in the apple hot-mash liquefaction process.

Isolation of uracil auxotrophic mutants of Trichoderma harzianum and their transformation with heterologous vectors

Nine uracil auxotrophic mutants were isolated from a mycoparasitic strain of Trichoderma harzianum. One of these could be transformed with the pKIM7 cosmid, which contained the pyr-4 gene of Neurospora crassa. Another uracil-requiring mutant was transformed with heterologous vectors containing the orotate pyrophosphoribosyl transferase genes of Podospora anserina and Trichoderma reesei. The transformation frequencies varied between 150 and 1000 μg −1 plasmid DNA. Hybridization analysis of the transformants revealed distinct locations of integrated vectors in the genomes, but in some transformants freely replicating plasmids could also be detected.

Isolation of uracil auxotrophic mutants ofTrichoderma harzianumand their transformation with heterologous vectors

Nine uracil auxotrophic mutants were isolated from a mycoparasitic strain of Trichoderma harzianum. One of these could be transformed with the pKIM7 cosmid, which contained the pyr-4 gene of Neurospora crassa. Another uracil-requiring mutant was transformed with heterologous vectors containing the orotate pyrophosphoribosyl transferase genes of Podospora anserina and Trichoderma reesei. The transformation frequencies varied between 150 and 1000 μg−1 plasmid DNA. Hybridization analysis of the transformants revealed distinct locations of integrated vectors in the genomes, but in some transformants freely replicating plasmids could also be detected.

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5'-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli beta-glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.

A mutualistic fungal symbiont of perennial ryegrass contains two different pyr4 genes, both expressing orotidine-5′-monophosphate decarboxylase

A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5′-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lpl), which grows as an endophyte in perennial ryegrass ( Lolium perenne). Three positive clones, λMC11, λMC12 and λMC14, were isolated. Two of these clones, λMC12 and λMC14, were overlapping clones from the same locus, while λMCll was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other Pyrenomycete fungi. The pyr4 gene from λMC12 and λMC14 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5′ non-coding sequences were the least conserved with 88.5% identity and the 3′ non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lpl demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lpl showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307–1317] that the two pyr4 in Lpl arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5′ non-coding regions of pyr4, from possible ancestors to Lpl supports this hypothesis. Comparisons of these sequences to the 5′ non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lpl.

Sequence of the pyr4 gene encoding orotidine-5′-phosphate decarboxylase from the biocontrol fungus Trichoderma harzianum

The pyr4 gene, encoding orotidine-5′-phosphate decarboxylase (OMP decarboxylase) from the biocontrol fungus Trichoderma harzianum, has been isolated by hybridization, using a polymerase chain reaction (PCR)-derived fragment as a probe. The PCR primers corresponded to conserved regions of OMP decarboxylase-encoding genes from other filamentous fungi. A 2490-bp genomic fragment, which complemented a pyr4-auxotrophic T. reesei mutant, was sequenced. The gene showed high homology to pyr4 genes from other pyrenomycetes.

Molecular characterization of pyrimidine biosynthesis genes from the thermophile Bacillus caldolyticus.

The genes encoding the six pyrimidine biosynthesis enzymes from the thermophile Bacillus caldolyticus were characterized by cloning and complementation in Escherichia coli, and by nucleotide sequence analysis. Nine cistrons are clustered within an 11 kb region of the chromosome, the gene order being: orf1-pyrB-pyrC-pyrAa-pyrAb-orf2-p yrD-pyrF-pyrE. This organization of the cluster is very similar to that of the pyr operon of Bacillus subtilis. Different parts of the B. caldolyticus cluster were cloned in two orientations in the expression shuttle vector pHPS9. Complementation studies in B. subtilis established that expression of the pyr genes was dependent on the vector-borne promoter, suggesting that they are part of an operon, and that the native promoter of the operon had not been cloned. The deduced amino acid sequence of the individual cistrons showed 49 to 78% identity with the corresponding B. subtilis cistrons. Measurements of the aspartate transcarbamylase (pyrB), orotidine monophosphate decarboxylase (pyrF) and orotate phosphoribosyltransferase (pyrE) levels in cells grown under different conditions indicated that expression of the operon is repressed 7-9-fold by addition of uracil to the growth medium. Based on the nucleotide sequence in the intercistronic region between orf1 and pyrB a regulatory mechanism involving transcriptional termination and antitermination is proposed to control expression of the operon.

Molecular cloning, expression and structure of the endo-1,5-alpha-L-arabinase gene of Aspergillus niger.

Secretion of endo-1,5-alpha-L-arabinase A (ABN A) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing L-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA+ RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-transformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized.

Use of Aspergillus overproducing mutants, cured for intergrated plasmid, to overproduce heterologous proteins

Aspergillus niger var. awamori was previously transformed with a vector designed to express a fused glucoamylase-prochymosin gene and bearing the Neurospora crassa pyr4 gene as a selectable marker. Mutant strains that overproduced the glucoamylase-prochymosin fusion protein were derived from one of the transformants. Despite the fact that the expression vector was integrated into the genome of these strains it was possible to obtain strains from which the vector sequences had been removed. This was performed by selection against the pyr4 gene present on the expression vector using 5-fluoroorotic acid. The cured strains were retransformed in order to investigate production of heterologous proteins using other expression vectors. In addition to the glucoamylase-prochymosin fusion protein, the mutant Aspergillus strains also overproduced Rhizomucor miehei aspartic proteinase but not preprochymosin produced as a non-fusion protein. The ability to select for loss of integrated plasmid from Aspergillus transformants may prove to be important for a variety of applications.

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels

The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.