RESUMO
The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subject to control by the purR gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the purA and purR genes. A consensus sequence for the binding of PurR, named the PurBox, has been suggested (M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900-3906, 1998). To determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the B. subtilis genome sequence and found several candidate PurBoxes. By the use of transcriptional lacZ fusions, five selected genes or operons (glyA, yumD, yebB, xpt-pbuX, and yqhZ-folD), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR. Using a machine-learning algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two PurBoxes in their upstream control regions. The two boxes are divergently oriented, forming a palindromic sequence with the inverted repeats separated by 16 or 17 nucleotides. A computerized search revealed one additional PurR-regulated gene, ytiP. The significance of the tandem PurBox motifs was demonstrated in vivo by deletion analysis and site-directed mutagenesis of the two PurBox sequences located upstream of glyA. All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism. Functional analysis showed that yebB encodes the previously characterized hypoxanthine-guanine permease PbuG and that ytiP encodes another guanine-hypoxanthine permease and is now named pbuO. yumD encodes a GMP reductase and is now named guaC.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Regiões Operadoras Genéticas , Regulon , Proteínas Repressoras/fisiologia , Bacillus subtilis/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos , Biologia Computacional , Sequência Consenso , Modelos Químicos , Dados de Sequência Molecular , Purinas/metabolismoRESUMO
The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285 degrees on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Purinas/metabolismo , Regulon , Transativadores/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Mapeamento Cromossômico , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Nitrogênio/metabolismo , Transativadores/genéticaRESUMO
Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias , Proteínas de Escherichia coli , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Regulação Alostérica/efeitos dos fármacos , Bacillus subtilis/genética , Sequência de Bases , Sítios de Ligação , Cromatografia de Afinidade , DNA/genética , DNA/metabolismo , Pegada de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Cinética , Peso Molecular , Regiões Operadoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribosemonofosfatos/metabolismo , Ribosemonofosfatos/farmacologia , TermodinâmicaRESUMO
In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.
Assuntos
Bacillus subtilis/metabolismo , Nucleosídeos/metabolismo , Óperon/genética , Fosfotransferases/genética , Purina-Núcleosídeo Fosforilase/genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Sequência de Bases , Carbono/metabolismo , Códon de Iniciação/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Dados de Sequência Molecular , Fosfotransferases/biossíntese , Nucleosídeos de Purina/metabolismo , Purina-Núcleosídeo Fosforilase/biossíntese , Esporos Bacterianos/fisiologia , Transcrição Gênica/genéticaRESUMO
The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions. It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Proteínas de Membrana Transportadoras , Regiões Operadoras Genéticas , Óperon , Proteínas Repressoras/genética , Aldeído Liases/genética , Bacillus subtilis/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Expressão Gênica , Genes Bacterianos , Óperon Lac , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Regiões Operadoras Genéticas/efeitos dos fármacos , Pentosiltransferases/genética , Reação em Cadeia da Polimerase , Pirimidina Fosforilases , Ribosemonofosfatos/metabolismo , Ribosemonofosfatos/farmacologia , Deleção de SequênciaRESUMO
The xpt and pbuX genes from Bacillus subtilis were cloned, and their nucleotide sequences were determined. The xpt gene encodes a specific xanthine phosphoribosyltransferase, and the pbuX gene encodes a xanthine-specific purine permease. The genes have overlapping coding regions, and Northern (RNA) blot analysis indicated an operon organization. The translation of the second gene, pbuX, was strongly dependent on the translation of the first gene, xpt. Expression of the operon was repressed by purines, and the effector molecules appear to be hypoxanthine and guanine. When hypoxanthine and guanine were added together, a 160-fold repression was observed. The regulation of expression was at the level of transcription, and we propose that a transcription termination-antitermination control mechanism similar to the one suggested for the regulation of the purine biosynthesis operon exists. The expression of the xpt-pbuX operon was reduced when hypoxanthine served as the sole nitrogen source. Under these conditions, the level of the hypoxanthine- and xanthine-degrading enzyme, xanthine dehydrogenase, was induced more than 80-fold. The xanthine dehydrogenase level was completely derepressed in a glnA (glutamine synthetase) genetic background. Although the regulation of the expression of the xpt-pbuX operon was found to be affected by the nitrogen source, it was normal in a glnA mutant strain. This result suggests the existence of different signalling pathways for repression of the transcription of the xpt-pbuX operon and the induction of xanthine dehydrogenase.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica/fisiologia , Óperon/genética , Xantinas/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sequência de Bases , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Glutamato-Amônia Ligase/metabolismo , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Nitrogênio/farmacologia , Conformação de Ácido Nucleico , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Biossíntese de Proteínas/genética , Purinas/metabolismo , Purinas/farmacologia , RNA Bacteriano/análise , RNA Bacteriano/química , RNA Mensageiro/análise , RNA Mensageiro/química , Proteínas Recombinantes de Fusão , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética , XantinaRESUMO
The isolation of mutants defective in adenine metabolism in Bacillus subtilis has provided a tool that has made it possible to investigate the role of adenine deaminase in adenine metabolism in growing cells. Adenine deaminase is the only enzyme that can deaminate adenine compounds in B. subtilis, a reaction which is important for adenine utilization as a purine and also as a nitrogen source. The uptake of adenine is strictly coupled to its further metabolism. Salvaging of adenine is inhibited by the stringent response to amino acid starvation, while the deamination of adenine is not. The level of adenine deaminase was reduced when exogenous guanosine served as the purine source and when glutamine served as the nitrogen source. The enzyme level was essentially the same whether ammonia or purines served as the nitrogen source. Reduced levels were seen on poor carbon sources. The ade gene was cloned, and the nucleotide sequence and mRNA analyses revealed a single-gene operon encoding a 65-kDa protein. By transductional crosses, we have located the ade gene to 130 degrees on the chromosomal map.
Assuntos
Adenina/metabolismo , Aminoidrolases/fisiologia , Bacillus subtilis/metabolismo , Genes Bacterianos , Nitrogênio/metabolismo , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Aminoidrolases/genética , Bacillus subtilis/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Dados de Sequência MolecularRESUMO
The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).
Assuntos
Bacillus subtilis/genética , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana Transportadoras , Óperon/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Clonagem Molecular , Desoxiadenosinas/farmacologia , Repressão Enzimática/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Dados de Sequência Molecular , Mutagênese Insercional , Pentosiltransferases/biossíntese , Pentosiltransferases/genética , Regiões Promotoras Genéticas/genética , Pirimidina Fosforilases , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Repressoras/fisiologia , Mapeamento por Restrição , Análise de Sequência de DNA , Succinatos/farmacologia , Ácido Succínico , Regiões Terminadoras Genéticas/genética , Timidina/farmacologia , Transcrição Gênica/genéticaRESUMO
The purT gene from Bacillus subtilis encoding the formate-dependent glycinamide ribonucleotide transformylase T was cloned by functional complementation of an Escherichia coli purN purT double mutant. The nucleotide sequence revealed an open reading frame of 384 amino acids. The purT amino acid sequence showed similarity to the enzyme phosphoribosylaminoimidazole carboxylase encoded by the purK gene but not to the N10-formyltetrahydrofolate-dependent glycinamide ribonucleotide transformylase N enzyme encoded by the purN gene. The glycinamide ribonucleotide transformylase T level was repressed in cells grown in rich medium compared to minimal-medium-grown cells. However, when the culture entered the stationary-growth phase the enzyme level increased in rich medium and decreased in minimal medium. By comparing the deduced amino acid sequence of the B. subtilis purT gene product with translated nucleotide sequences in various databanks, evidence for the existence of putative purT genes in the Gram-negative bacteria Pasteurella haemolytica and Pseudomonas aeruginosa was obtained.
Assuntos
Aciltransferases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Carboxiliases , Proteínas de Escherichia coli , Genes Bacterianos , Hidroximetil e Formil Transferases , Aciltransferases/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Teste de Complementação Genética , Mannheimia haemolytica/genética , Dados de Sequência Molecular , Fosforribosilglicinamido Formiltransferase , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Purinas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms. Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant. The genes were sequenced, and the putative amino acid sequences were deduced. One gene showed a high level of homology to UPRTases from other organisms, whereas the other gene with a low level of homology to other UPRTases turned out to be the pyrR gene--the repressor of the pyr operon. The role of these genes in uracil metabolism was established by an analysis of the phenotypes of upp and pyrR mutants.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias , Genes Bacterianos/genética , Pentosiltransferases/genética , Proteínas Repressoras/genética , Uracila/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
We have found that Bacillus subtilis possesses a second 5'-phosphoribosyl-1-glycinamide (GAR) transformylase catalysing the first one-carbon transfer reaction in the purine biosynthetic pathway. Inactivation of the purN gene encoding the N10-formyl tetrahydrofolate-dependent enzyme did not result in purine auxotrophy. However, growth of a purN strain was stimulated when either purine or formate was added to the growth medium. In cell-free extracts GAR could be formylated, provided formate was added to the assay mixture. From the purN strain, purine-requiring mutants were isolated. One of these mutant strains was defective in the formate-dependent formylation of GAR in vitro. The gene containing this second mutation was designated purT, and was mapped to approximately 20 degrees on the genetic map between the cysA and aroI markers.
Assuntos
Aciltransferases/metabolismo , Bacillus subtilis/enzimologia , Genes Bacterianos , Hidroximetil e Formil Transferases , Aciltransferases/genética , Bacillus subtilis/genética , Clonagem Molecular , Formiatos/farmacologia , Mutação , Fosforribosilglicinamido Formiltransferase , Transformação GenéticaRESUMO
Random genomic Bacillus subtilis lacZ fusions were screened in order to identify the possible existence of regulons responding to the stimuli generated by partial purine starvation. A leaky pur mutation (purL8) was isolated and used to generate the partial purine starvation conditions in the host strain used for screening. On the basis of their induction during partial purine starvation, seven genomic lacZ fusions were isolated. None of the fusions map in loci previously reported to contain purine-regulated genes. One fusion maps very close to the citB locus and may very well be a citB fusion. The fusions were divided into two types on the basis of their response to complete starvation for either ATP or GTP or both components at the same time. Except for one, type 2 fusions were induced by specific starvation for ATP and by simultaneous starvation for ATP and GTP, but not by specific GTP starvation in a gua strain or by GTP starvation induced by the addition of decoyinine. Type 1 fusions were equally well induced by all three kinds of purine starvation including GTP starvation induced by decoyinine. Further subdivisions of the fusions were obtained on the basis of their responses to the spo0A gene product. A total of five fusions showed that spo0A affected expression. One class was unable to induce lacZ expression in the absence of the spo0A gene product, whereas the other class had increased lacZ expression during partial purine starvation in a spo0A background.
Assuntos
Bacillus subtilis/genética , DNA Recombinante , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Óperon Lac , Purinas/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Mapeamento Cromossômico , Guanosina Trifosfato/metabolismo , Mutagênese Insercional , Fenótipo , Regulon/genética , Fatores de Transcrição/genéticaRESUMO
The genes encoding the enzymes of IMP biosynthesis in Bacillus subtilis constitute the pur operon, whereas the genes encoding GMP biosynthetic enzymes, guaA (GMP synthetase) and guaB (IMP dehydrogenase), and the purA gene encoding adenylosuccinate (sAMP) synthetase all occur as single units. The purB gene encodes an enzyme involved in both IMP and AMP biosynthesis and is located in the pur operon. The levels of purine biosynthetic enzymes (except for GMP synthetase) were repressed in cells grown in the presence of purine compounds. Transcription of the pur operon is regulated negatively by adenine and guanine compounds. Our results suggest that ATP and guanine (or hypoxanthine) act as low molecular mass repressors. The level of IMP dehydrogenase was repressed by guanosine, but not in the presence of adenine, and was negatively correlated with the GTP/ATP pools ratio. The level of sAMP synthetase was repressed by adenine and increased by guanosine, and was positively correlated with the GTP/ATP pools ratio. It appears that the mode of regulating purine biosynthetic enzyme levels coincides with the cellular need for the individual enzymes.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Nucleotídeos de Purina/farmacologia , Purinas/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Retroalimentação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Óperon , Fosforribosil Pirofosfato/metabolismoRESUMO
The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophosphate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (pur A), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.
Assuntos
Bacillus subtilis/genética , Purinas/biossíntese , Adenina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Mapeamento Cromossômico , Clonagem Molecular , Genes Bacterianos , Ligação Genética , Guanina/metabolismo , Hipoxantinas/metabolismo , Mutação , Transdução GenéticaRESUMO
Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.