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1.  Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of th proA step. 
Journal of Bacteriology  1996;178(15):4412-4419.
Two chromosomal loci containing the Corynebacterium glutamicum ATCC 17965 proB and proC genes were isolated by complementation of Escherichia coli proB and proC auxotrophic mutants. Together with a proA gene described earlier, these new genes describe the major C. glutamicum proline biosynthetic pathway. The proB and proA genes, closely linked in most bacteria, are in C. glutamicum separated by a 304-amino-acid open reading frame (unk) whose predicted sequence resembles that of the 2-hydroxy acid dehydrogenases. C. glutamicum mutants that carry null alleles of proB, proA, and proC were constructed or isolated from mutagenized cultures. Single proC mutants are auxotrophic for proline and secrete delta1-pyrroline-5-carboxylate, which are the expected phenotypes of bacterial proC mutants. However, the phenotypes or proB and proA mutants are unexpected. A proB mutant has a pleiotropic phenotype, being both proline auxotrophic and affected in cell morphology. Null proA alleles still grow slowly under proline starvation, which suggests that a proA-independent bypass of this metabolic step exists in C. glutamicum. Since proA mutants are complemented by a plasmid that contains the wild-type asd gene of C. glutamicum, the asd gene may play a role in this bypass.
PMCID: PMC178206  PMID: 8755867
2.  Genetic analysis of the proBA genes of Salmonella typhimurium: physical and genetic analyses of the cloned proB+ A+ genes of Escherichia coli and of a mutant allele that confers proline overproduction and enhanced osmotolerance. 
Journal of Bacteriology  1983;156(3):1249-1262.
Because of the fact that proline overproduction relieves the inhibitory effects of high external osmotic strength in a number of procaryotes, we wished to clone a mutant allele, pro-74, that confers proline overproduction and enhanced osmotolerance on Salmonella typhimurium and Escherichia coli. Therefore, the pro-74 allele, originally located on an E. coli episome F'128, was cloned into pBR322. In a parallel experiment, the wild type proB+ A+ genes of E. coli were also cloned from F'128 into pBR322. Both the pro-74 and the proB+ A+ alleles were obtained on a 10.4-kilobase-pair fragment that also contained the unrelated phoE gene. Strains carrying either the wild-type proB+ A+ or the pro-74 alleles on pBR322 grew more slowly, both in minimal medium and media of elevated osmotic strength, than strains carrying the same alleles on the low-copy plasmid, F'128, indicating that some gene in the cloned region is deleterious in high copy. We constructed Tn5 insertion mutations in the proB and the proA genes of E. coli, carried on F'128 in S. typhimurium. Using P22 transduction in S. typhimurium, we transferred these proB and proA::Tn5 insertions from F'128 into the cloned proBA genes on pBR322. From the restriction maps of the plasmids thus generated, we determined the approximate locations of the proB and the proA genes. We also performed complementation tests of S. typhimurium and E. coli proB and proA mutants by using the F'128 proB and proA::Tn5 insertions. These tests revealed that the proBA genes of S. typhimurium form an operon, whose direction of transcription is from proB to proA. They also indicated that in S. typhimurium, as in E. coli, the proB+ gene encodes gamma-glutamyl kinase, and the proA+ gene encodes gamma-glutamyl phosphate reductase. Complementation tests also indicated that the pro-74 mutation is either in the proB structural gene, or its promoter-operator.
PMCID: PMC217975  PMID: 6315682
3.  Suppression of Proline Requirement of proA and proAB Deletion Mutants in Salmonella typhimurium by Mutation to Arginine Requirement 
Journal of Bacteriology  1969;98(2):593-598.
Eleven variants able to grow without proline (provided arginine was absent) were obtained by spontaneous mutation from Salmonella typhimurium LT7 proA and proAB deletion mutants. Suppression resulted from mutation at argG, which specifies Nα-acetylornithine δ-transaminase. In the absence of exogenous arginine, deficiency of this enzyme would cause derepression of the arginine pathway and accumulation of N-acetylglutamic γ-semialdehyde. N-acetylglutamic γ-semialdehyde, if deacetylated, would produce glutamic γ-semialdehyde, the proline precursor whose synthesis from glutamate is blocked in proA and proAB mutants. All of the mutants grew only slowly (some very slowly) if not supplied with arginine. Sonic-treated preparations of eight mutants had no measurable acetylornithine δ-transaminase activity, but those of the three mutants least dependent on arginine had 0.11, 0.28, and 1.48 of wild-type activity; presumably, their enzymes have low specific activity, at least in vivo. Phage P22 cotransduced argG and strA. Genetic analysis showed that the minor degree of arginine dependence of the mutant with greater than wild-type in vitro enzyme activity was a characteristic of its argG allele, not the result of modification of the argG phenotype by mutation elsewhere.
PMCID: PMC284859  PMID: 4891261
4.  Arginine Catabolism in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Involves the Urea Cycle and Arginase Pathway 
Journal of Bacteriology  2000;182(4):1008-1015.
Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of l-[14C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of l-[14C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [14C]ornithine preceded that of [14C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with l-[14C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Δ1pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [14C]citrulline from [14C]arginine; (ii) a proC (Δ1pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [14C]proline, [14C]glutamate, and [14C]glutamine from [14C]arginine or [14C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [14C]glutamate from l-[14C]arginine, l-[14C]ornithine, or l-[14C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Δ1pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [14C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-l-arginyl-poly(l-aspartic acid)], a reserve material unique to cyanobacteria.
PMCID: PMC94377  PMID: 10648527
5.  Specific Action of 4-Nitropyridine 1-Oxide on Escherichia coli K-12 Pro+ Strains Leading to the Isolation of Proline-Requiring Mutants: Isolation and Characterization of Pro− Mutants 
A specific action of 4-nitropyridine 1-oxide on Escherichia coli K-12 Pro+ strains leading to highly efficient, selective isolation of Pro− mutants is described. Incubation of Pro+ cells with a sublethal concentration of 4-nitropyridine 1-oxide in Penassay broth gave Pro− mutants, which lacked either the biosynthetic pathway of proline from glutamic acid to glutamyl γ-phosphate (proB−) or the pathway from glutamyl γ-phosphate to glutamic γ-semialdehyde (proA−) or both. Pro− mutants, which have the metabolic block between Δ1 pyrroline-5-carboxylate (the cyclized dehydration product of glutamic γ-semialdehyde) and proline (proC−) were not found among survivors. Treatment of Pro+ cells with N-methyl-N′-nitro-N-nitrosoguanidine led to isolation of all three types of Pro− mutants, suggesting that the action of 4-nitropyridine 1-oxide on Pro+ cells is apparently distinct from the action of N-methyl-N′-nitro-N-nitrosoguanidine. F-duction and interrupted mating experiments led to determination of the correlation between proline loci and the biosynthetic pathway of proline from glutamic acid.
PMCID: PMC429743  PMID: 791096
6.  Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis 
The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.
PMCID: PMC4272716  PMID: 25344233
7.  Regulation of cytoplasmic proline levels in Salmonella typhimurium: effect of osmotic stress on synthesis, degradation, and cellular retention of proline. 
Journal of Bacteriology  1988;170(5):2374-2378.
I investigated the effects of osmotic stress on the synthesis and catabolism of proline in Salmonella typhimurium by measuring the intracellular and extracellular proline levels in various strains. In the wild-type strain, exposure to 0.8 M NaCl did not cause a significant change in the intracellular proline level; however, it brought about a 6.5-fold increase in the intracellular glutamate pool size. These results indicate that gamma-glutamyl kinase is inhibited by proline in wild-type cells in media of normal or elevated osmolarity. I also tested whether proline is subject to turnover in cells wild type with respect to the enzymes of the proline degradation pathway. In strains that were wild type for proline biosynthesis, the loss of the proline catabolic enzymes, due to putA mutations, did not result in a statistically significant increase in the intracellular proline levels. Therefore, in the wild-type strain, proline turnover does not seem to be important for control of the intracellular proline levels. However, in a proline-overproducing mutant, a putA lesion caused a threefold increase in the intracellular proline level and a 6.5-fold increase in the extracellular proline level, indicating that proline is subject to turnover in the overproducing mutant. The proline-overproducing mutants excreted large quantities of the proline into the culture medium; osmotic stress altered the partitioning of proline such that the ratio of intracellular to extracellular levels of proline increased with increased osmotic stress. The increased cellular retention of proline in media of high osmolarity is probably due to the functioning of the ProP and ProU proline transport systems, which are stimulated under conditions of osmotic stress.
PMCID: PMC211132  PMID: 3283109
8.  Identification and Disruption of the proBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection 
Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.
PMCID: PMC92909  PMID: 11375165
9.  Isolation and Characterization of Proline Peptidase Mutants of Salmonella typhimurium 
Journal of Bacteriology  1974;120(1):364-371.
The proline requirement of Salmonella typhimurium strain proB25 can be satisfied by either of the peptides Leu-Pro or Gly-Pro-Ala. A mutant derivative of strain proB25 isolated by penicillin selection in medium containing Leu-Pro as proline source fails to use either Leu-Pro or Gly-Pro-Ala as a source of proline. This strain is a double mutant that lacks two aminoacyl-proline-specific peptidases. One of these enzymes (peptidase Q) catalyzes the rapid hydrolysis of Leu-Pro but does not hydrolyze Gly-Pro-Ala or poly-l-proline. Mutations at a site (pepQ) near metE lead to loss of this activity. The other peptidase (peptidase P) catalyzes the hydrolysis of Gly-Pro-Ala and poly-l-proline but is only weakly active with Leu-Pro as substrate. This enzyme is similar to aminopeptidase P previously described in Escherichia coli (16). Mutations at a locus (pepP) near serA lead to loss of this enzyme.
PMCID: PMC245771  PMID: 4607625
10.  Salinity-Dependent Switching of Osmolyte Strategies in a Moderately Halophilic Bacterium: Glutamate Induces Proline Biosynthesis in Halobacillus halophilus▿  
Journal of Bacteriology  2007;189(19):6968-6975.
The moderately halophilic bacterium Halobacillus halophilus copes with the salinity in its environment by the production of compatible solutes. At intermediate salinities of around 1 M NaCl, cells produce glutamate and glutamine in a chloride-dependent manner (S. H. Saum, J. F. Sydow, P. Palm, F. Pfeiffer, D. Oesterhelt, and V. Müller, J. Bacteriol. 188:6808-6815, 2006). Here, we report that H. halophilus switches its osmolyte strategy and produces proline as the dominant solute at higher salinities (2 to 3 M NaCl). The proline biosynthesis genes proH, proJ, and proA were identified. They form a transcriptional unit and encode the pyrroline-5-carboxylate reductase, the glutamate-5-kinase, and the glutamate-5-semialdehyde dehydrogenase, respectively, catalyzing proline biosynthesis from glutamate. Expression of the genes was clearly salinity dependent and reached a maximum at 2.5 M NaCl, indicating that the pro operon is involved in salinity-induced proline biosynthesis. To address the role of anions in the process of pro gene activation and proline biosynthesis, we used a cell suspension system. Chloride salts lead to the highest accumulation of proline. Interestingly, chloride could be substituted to a large extent by glutamate salts. This unexpected finding was further analyzed on the transcriptional level. The cellular mRNA levels of all three pro genes were increased up to 90-fold in the presence of glutamate. A titration revealed that a minimal concentration of 0.2 M glutamate already stimulated pro gene expression. These data demonstrate that the solute glutamate is involved in the switch of osmolyte strategy from glutamate to proline as the dominant compatible solute during the transition from moderate to high salinity.
PMCID: PMC2045198  PMID: 17660292
11.  Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs. 
Journal of Bacteriology  1979;138(3):816-822.
Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.
PMCID: PMC218109  PMID: 378940
12.  Evolution of a New Gene Substituting for the leuD Gene of Salmonella typhimurium: Origin and Nature of supQ and newD Mutations 
Journal of Bacteriology  1974;120(3):1176-1185.
The second specific enzyme in the biosynthesis of leucine, α-isopropylmalate isomerase, is coded for by two genes, leuC and leuD. Leucine auxotrophs carrying mutations in the leuD gene (including deletions of the entire leuD gene) revert to leucine prototrophy by secondary mutations at the locus supQ, which is located in the proline region of the chromosome. The mechanism of the supQ function is explained by the following model. The supQ gene and an additional gene, newD, code for two different subunits of a multimeric enzyme, whose normal function is yet to be determined. The newD gene protein is able, without genetic alterations, to form an active complex with the leuC protein, thus replacing the nonfunctional or missing leuD protein and restoring leucine prototrophy. The newD protein has, however, a higher affinity for the supQ protein than for the leuC protein; therefore, mutations in the supQ gene are needed to make sufficient amounts of the newD protein available. The following gene order has been established: gpt-proB-proA-ataA-supQ-newD. Different supQ mutations have been identified, i.e., insertion in the supQ gene, point mutations, and deletions of various extent. Some deletions remove the P22 phage attachment site ataA. Other supQ deletions are simultaneously Pro−, because they extend into the proA or proA and proB genes; some extend even further, i.e., into the gpt gene (guanine phosphoribosyl transferase). Mutations in the newD gene caused renewed leucine auxotrophy in leuD supQ mutant strains. One newD mutation causes a temperature-sensitive Leu+ phenotype. Alternate models for the supQ-newD interactions are discussed.
PMCID: PMC245897  PMID: 4612005
13.  Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum. 
We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.
PMCID: PMC167790  PMID: 8572700
14.  Effect of the Proline Analogue Baikiain on Proline Metabolism in Salmonella typhimurium 
Journal of Bacteriology  1972;112(3):1134-1141.
A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Δ1-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 × 10−4m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.
PMCID: PMC251541  PMID: 4565530
15.  Proline Catabolism by Pseudomonas putida: Cloning, Characterization, and Expression of the put Genes in the Presence of Root Exudates 
Journal of Bacteriology  2000;182(1):91-99.
Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5–Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to ′lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.
PMCID: PMC94244  PMID: 10613867
16.  Construction of a Proline-Producing Mutant of the Extremely Thermophilic Eubacterium Thermus thermophilus HB27 
Applied and Environmental Microbiology  1998;64(11):4328-4332.
Growth of Thermus thermophilus HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the γ-glutamyl kinase (the product of the proB gene) was inhibited by feedback inhibition in T. thermophilus. DHP-resistant mutants were reported previously for Escherichia coli (A. M. Dandekar and S. L. Uratsu, J. Bacteriol. 170:5943–5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693–699, 1992), and their mutated sites in the proB gene were identified. Comparison of the amino acid sequence of T. thermophilus γ-glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the proB gene as reported for the proline-producing mutant of S. marcescens. The mutant, TH401, excreted about 1 mg of l-proline per liter at 70°C after 12 h of incubation. It was also suggested that T. thermophilus had a proline degradation and transport pathway since it was able to grow in minimal medium containing l-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize l-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of l-proline per liter in minimal medium at 70°C after 12 h of incubation.
PMCID: PMC106647  PMID: 9797285
17.  Gene Dosage Effect of l-Proline Biosynthetic Enzymes on l-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae 
Applied and Environmental Microbiology  2003;69(11):6527-6532.
We have previously reported that l-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with l-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding γ-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant γ-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three l-proline biosynthetic enzymes, including Δ1-pyrroline-5-carboxylate reductase, which converts Δ1-pyrroline-5-carboxylate into l-proline, on l-proline accumulation and freeze tolerance in a non-l-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on l-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant γ-glutamyl kinase and the wild-type γ-glutamyl phosphate reductase was effective for l-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Δ1-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of l-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular l-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.
PMCID: PMC262311  PMID: 14602584
18.  Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein. 
Journal of Bacteriology  1995;177(22):6432-6439.
Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.
PMCID: PMC177492  PMID: 7592417
19.  Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus. 
Applied and Environmental Microbiology  1987;53(12):2733-2738.
Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.
PMCID: PMC204189  PMID: 2829719
20.  CcpA Mediates Proline Auxotrophy and Is Required for Staphylococcus aureus Pathogenesis▿  
Journal of Bacteriology  2010;192(15):3883-3892.
Human clinical isolates of Staphylococcus aureus, for example, strains Newman and N315, cannot grow in the absence of proline, albeit their sequenced genomes harbor genes for two redundant proline synthesis pathways. We show here that under selective pressure, S. aureus Newman generates proline-prototrophic variants at a frequency of 3 × 10−6, introducing frameshift and missense mutations in ccpA or IS1811 insertions in ptsH, two regulatory genes that carry out carbon catabolite repression (CCR) in staphylococci and other Gram-positive bacteria. S. aureus Newman variants with mutations in rocF (arginase), rocD (ornithine aminotransferase), and proC (Δ1-pyrroline 5-carboxylate [P5C] reductase) are unable to generate proline-prototrophic variants, whereas a variant with a mutation in ocd (ornithine cyclodeaminase) is unaffected. Transposon insertion in ccpA also restored proline prototrophy. CcpA was shown to repress transcription of rocF and rocD, encoding the first two enzymes, but not of proC, encoding the third and final enzyme in the P5C reductase pathway. CcpA bound to the upstream regions of rocF and rocD but not to that of proC. CcpA's binding to the upstream regions was greatly enhanced by phosphorylated HPr. The CCR-mediated proline auxotrophy was lifted when nonpreferred carbohydrates were used as the sole carbon source. The ccpA mutant displayed reduced staphylococcal load and replication in a murine model of staphylococcal abscess formation, indicating that carbon catabolite repression presents an important pathogenesis strategy of S. aureus infections.
PMCID: PMC2916365  PMID: 20525824
21.  Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity▿‡ 
Journal of Bacteriology  2011;193(19):5335-5346.
Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded γ-glutamyl kinase, the proA-encoded γ-glutamyl phosphate reductase, and the proH-encoded Δ1-pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.
PMCID: PMC3187420  PMID: 21784929
22.  Mutations in the Listerial proB Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection 
Applied and Environmental Microbiology  2001;67(10):4560-4565.
The observed sensitivity of Listeria monocytogenes to the toxic proline analogue l-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of γ-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (ΔproBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZr) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.
PMCID: PMC93203  PMID: 11571156
23.  Regulation of Proline Degradation in Salmonella typhimurium 
Journal of Bacteriology  1970;103(1):144-152.
The pathway for proline degradation in Salmonella typhimurium appears to be identical to that found in Escherichia coli and Bacillus subtilis. Δ1-Pyrroline-5-carboxylic acid (P5C) is an intermediate in the pathway; its formation consumes molecular oxygen. Assays were devised for proline oxidase and the nicotinamide adenine dinucleotide phosphate-specific P5C dehydrogenase activities. Both proline-degrading enzymes, proline oxidase and P5C dehydrogenase, are induced by proline and are subject to catabolite repression. Three types of mutants were isolated in which both enzymes are affected: constitutive mutants, mutants with reduced levels of enzyme activity, and mutants unable to produce either enzyme. Most of the mutants isolated for their lack of P5C dehydrogenase activity have a reduced level of proline oxidase activity. All the mutations are cotransducible. A genetic map of some of the mutations is presented. The actual effector of the pathway appears to be proline.
PMCID: PMC248050  PMID: 4912518
24.  Subcellular compartmentation in control of converging pathways for proline and arginine metabolism in Saccharomyces cerevisiae. 
Journal of Bacteriology  1981;145(3):1359-1364.
Enzymes of proline biosynthesis and proline degradation which act on the same compound, delta 1-pyrroline-5-carboxylate, are physically separated in yeast cells. The enzyme responsible for the final step in proline biosynthesis, pyrroline-5-carboxylate reductase, converts pyrroline-5-carboxylate to proline and is located in the cytoplasm. The last enzyme in the proline degradative pathway, pyrroline-5-carboxylate dehydrogenase, converts pyrroline-5-carboxylate to glutamate and is found in the particulate fraction of the cell, presumably in the mitochondrion. By subcellular compartmentation, yeast cells avoid futile cycling between proline and pyrroline-5-carboxylate.
PMCID: PMC217140  PMID: 7009582
25.  Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli 
Journal of Bacteriology  2014;197(3):431-440.
The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.
PMCID: PMC4285992  PMID: 25384482

Results 1-25 (390086)