putA Gene Research Articles

Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putA was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the HpaI site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.

Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region.

The two genes required for proline utilization (put) in Salmonella typhimurium form a divergent operon. Extensive genetic evidence suggests that transcription of the put operon is autoregulated by the putA gene product, a membrane-associated dehydrogenase. In order to understand the mechanism of regulation, we characterized plasmid clones of the put operon. A 7.5 kb clone contains both of the put structural genes and regulatory sites. This clone only expressed two unique proteins corresponding to the putA and putP gene products. By comparing the physical and genetic maps of the put operon, the position of the put regulatory region was defined and the DNA sequence of this region was determined. Analysis of the DNA sequence indicated several potential regulatory sites for the put genes. Based on genetic and physical mapping studies, the most likely regulatory sites are two convergent promoters approximately 30 bp apart. A 27 bp palindrome located between the two promoters may be the operator for autoregulation by the PutA protein. The putA translational start site is 40 bp downstream of its putative mRNA start site. The putP promoter and its translational start site are separated by a 400 bp untranslated region.

Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product.

The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo. The transcription initiation and termination sites of the PUT1 mRNA were determined. By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied. PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2. The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells. Oxygen regulation is abolished when cells are respiratory deficient. PUT1 expression in a [rho-] strain grown either aerobically or anaerobically is as high as that seen in a [rho+] strain grown aerobically. Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.

Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product.

The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo. The transcription initiation and termination sites of the PUT1 mRNA were determined. By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied. PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2. The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells. Oxygen regulation is abolished when cells are respiratory deficient. PUT1 expression in a [rho-] strain grown either aerobically or anaerobically is as high as that seen in a [rho+] strain grown aerobically. Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.

Nucleotide sequence of putC, the regulatory region for the put regulon of Escherichia coli K12.

The nucleotide sequence of the putC region, from which divergent transcription of the putP and putA genes starts, was determined. The promoter region for the putA gene was restricted to the location between the HindIII site and the NcoI site or at the NcoI site by using putA-lacZ fusion plasmids and the transcriptional start for the putA gene was identified in the region between the HindIII site and the NcoI site by Sl mapping. This region also contains a potential CAP binding site, a ribosome binding site, and a sequence that is highly homologous to argTr. Five potential promoters (putPp1-putPp5) for the putP gene, which were separate from the promoter region for the putA gene, were indicated by S1 mapping analysis of the putP gene transcripts. We concluded that the putC region is 419 bp long and contains two independent sets of promoters, regulating the expression of putP and putA genes in opposite directions. In addition, this region was found to contain an open reading frame (orf) capable of encoding a polypeptide of 111 amino acids in overlapping fashion. But studies using an orf-lacZ fusion gene showed that this open reading frame was not expressed.

Evidence for positive regulation of the proline utilization pathway in Saccharomyces cerevisiae.

A mutation has been identified that prevents Saccharomyces cerevisiae cells from growing on proline as the sole source of nitrogen, causes noninducible expression of the PUT1 and PUT2 genes, and is completely recessive. In the put3-75 mutant, the basal level of expression (ammonia as nitrogen source) of PUT1-lacZ and PUT2-lacZ gene fusions as measured by beta-galactosidase activity is reduced 4- and 7-fold, respectively, compared with the wild-type strain. Normal regulation is not restored when the cells are grown on arginine as the sole nitrogen source and put3-75 cells remain sensitive to the proline analog, L-azetidine-2-carboxylic acid, indicating that the block is not at the level of transport of the inducer, proline. In a cross between the put3-75 strain and the semidominant, constitutive mutation PUT3c-68, only parental ditype tetrads were found, indicating allelism of the two mutations. Further support for allelism derives from the comparison of enzyme levels in heteroallelic and heterozygous diploid strains. The constitutive allele appears to be fully dominant to the noninducible allele but only partially dominant to the wild type, suggesting an interaction between the wild-type and PUT3c-68 gene products. The PUT3 gene maps on chromosome XI, about 5.7 cM from the centromere. The phenotypes of alleles of the PUT3 gene, either recessive and noninducible (the put3-75 phenotype) or semidominant and constitutive (the PUT3c-68 phenotype), and their pleiotropy suggest that the PUT3 gene product is a positive activator of the proline utilization pathway.

Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12.

The nucleotide sequence of the putP gene coding for the proline carrier in Escherichia coli has been determined and the amino acid sequence of the proline carrier deduced from it. The proline carrier is predicted to consist of 502 amino acids, resulting in a molecular weight of 54,343. The predicted protein is very hydrophobic (70% nonpolar amino acids), and its hydropathy profile suggests that it is composed of 12 hydrophobic segments with a mean length of 24.4 residues/segment. If these segments are assumed to be alpha-helical, the mean length of each domain corresponds to the thickness of the hydrophobic core of the membrane. Potential promoter, catabolite gene activator protein (CAP) binding sites and several palindromic sequences, which might be regulatory regions by the putA gene product, were also found in the 5' flanking region of the postulated putP gene. A typical rho-independent transcription termination signal was found after the terminator codon of the putP gene.

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.

Amino-Terminal Fragments of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Direct β-Galactosidase to the Mitochondrial Matrix in Saccharomyces cerevisiae

delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids. The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains. The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions. The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids. The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains. The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions. The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12.

Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product). Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.

Primary structure of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in Saccharomyces cerevisiae.

The PUT2 gene, believed to encode delta 1-pyrroline-5-carboxylate dehydrogenase, has been completely sequenced. The DNA contains an open reading frame of 1,725 base pairs encoding a protein of 575 amino acids. Transcript mapping with both S1 nuclease and primer extension methods revealed numerous initiation sites of RNA synthesis 50 to 80 base pairs downstream from several TATA boxes. The deduced amino acid sequence of delta 1-pyrroline-5-carboxylate dehydrogenase contains a highly basic amino terminus that may serve as the signal sequence that targets this protein to the mitochondrion.

Primary structure of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in Saccharomyces cerevisiae.

The PUT2 gene, believed to encode delta 1-pyrroline-5-carboxylate dehydrogenase, has been completely sequenced. The DNA contains an open reading frame of 1,725 base pairs encoding a protein of 575 amino acids. Transcript mapping with both S1 nuclease and primer extension methods revealed numerous initiation sites of RNA synthesis 50 to 80 base pairs downstream from several TATA boxes. The deduced amino acid sequence of delta 1-pyrroline-5-carboxylate dehydrogenase contains a highly basic amino terminus that may serve as the signal sequence that targets this protein to the mitochondrion.

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT2 gene.

The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (delta 1-pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae. Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the put2 gene into which the S. cerevisiae HIS3+ gene had been inserted. The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level. The PUT2-specific mRNA was approximately 2 kilobases in length and was used in S1 nuclease protection experiments to locate the gene to a 3-kilobase HindIII fragment. When delta 1-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains. Effects due to increased copy number or position were also seen. The cloned gene on a 2 mu-containing vector was used to map the PUT2 gene to chromosome VIII.

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT2 gene.

The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (delta 1-pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae. Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the put2 gene into which the S. cerevisiae HIS3+ gene had been inserted. The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level. The PUT2-specific mRNA was approximately 2 kilobases in length and was used in S1 nuclease protection experiments to locate the gene to a 3-kilobase HindIII fragment. When delta 1-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains. Effects due to increased copy number or position were also seen. The cloned gene on a 2 mu-containing vector was used to map the PUT2 gene to chromosome VIII.

Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap, lac) operon fusions.

The genes for proline utilization were fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mu d(Ap lac). Stable deletion derivatives of these fusions were selected and used to study the transcriptional regulation of the put genes. Analysis of these fusions showed that the putA gene product, a bifunctional oxidase-dehydrogenase, also serves to negatively control transcription of the putA and putP genes. Transcription of the put genes is repressed only in putA+ strains; this repression is lifted when exogenous proline is supplied. Transcription of the put genes is stimulated by cyclic AMP in putA+ and putA strains. Maximal induction of the put genes in putA+ strains requires oxygen or an alternative electron acceptor. This oxygen effect is mediated by the putA protein since putA mutants show maximal transcription even without an electron acceptor. The orientation of the Mu d(Ap lac) insertions was determined by formation of Hfr's via the lac homology on F'ts114 lac+. The direction of chromosome mobilization by these Mu d(Ap lac)-directed Hfr's demonstrated that the putP and putA genes are divergently transcribed from a central regulatory region lying between them.

Purification of the putA gene product. A bifunctional membrane-bound protein from Salmonella typhimurium responsible for the two-step oxidation of proline to glutamate.

In this paper we report the purification of a protein which is able to catalyze both the proline oxidase and the pyrroline-5-carboxylic acid dehydrogenase activities necessary for the oxidation of proline to glutamic acid. The purification involves the preparation of a crude membrane pellet, detergent solubilization, ammonium sulfate fractionation, and DEAE-chromatography. We are able to obtain an essentially pure preparation (greater than 95% pure) after only a 52-fold purification, demonstrating that the protein is a major protein in cells fully induced for proline utilization. Both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-purity throughout our purification. Velocity sedimentation of the purified protein demonstrates that both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-sediment. Early in the purification procedure we are able to detect two species of protein which have both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities. Our procedure purifies only the larger molecular weight species. The purified protein is a dimer composed of identical 132,000-dalton subunits. Analysis of mutants defective for proline utilization demonstrate that the bifunctional enzyme is the putA gene product.

Enzymatic properties of the purified putA protein from Salmonella typhimurium.

In the previous paper (Menzel, R., and Roth, J. (1981) J. Biol. Chem. 256, 9755-9761) we have described the purification of a protein, the putA gene product, which has both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities. In this paper we demonstrate that these enzyme activities are distinct with respect to a number of characteristics. The oxidase activity proceeds by a ping-pong mechanism involving the reduction of an enzyme-bound flavin. The dehydrogenase activity utilizes an ordered reaction mechanism.

Regulation of the genes for proline utilization in Salmonella typhimurium: Autogenous repression by the putA gene product

Two genes are involved in the degradation of proline to glutamic acid. The putP gene encodes a proline permease; the putA gene encodes a bifunctional degradative enzyme. Although the two genes are transcribed independently, both genes are induced by exogenous proline and are subject to catabolite repression. The putA gene product appears to function also as an autogenous repressor protein acting on both the putA and putP transcription units. Regulatory mutations map throughout the putA gene; these are correctable by an episomal putA + gene, which acts in trans to restore repression.