Genetics of L-proline utilization in Escherichia coli.

L-Azetidine-2-carboxylate (AC) and 3,4-dehydro-D,L-proline (DHP) are toxic L-proline analogs that can be used to select bacterial mutants defective for L-proline transport. Mutants resistant to AC and DHP are defective for proline transport alone (putP mutants), and mutants resistant to AC but not to DHP are defective both in putP and in the closely linked proline dehydrogenase gene putA. Proline dehydrogenase oxidizes DHP but not AC, probably detoxifying the former compound. These observations were exploited in preparing an otherwise isogenic set of Escherichia coli K-12 strains with well-defined defects in the putP and putA genes. The results of this study suggest that the genetic and biochemical characteristics of proline utilization in E. coli K-12 are closely analogous to those of Salmonella typhimurium.     

Two proline porters in Escherichia coli K-12

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.     

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein.

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.     

Identification of a trans-dominant mutation affecting proline dehydrogenase in Escherichia coli

L-Proline dehydrogenase catalyzes the oxidation of L-proline to delta 1-pyrroline-5-carboxylate, a reaction that is an important step in the utilization of proline as a carbon or nitrogen source by bacteria. A mutant of Escherichia coli K-12 lacking L-leucyl-tRNA:protein transferase had been found previously to contain about five times as much proline dehydrogenase activity as its parent strain. This difference has now been shown to be due to the presence in the parent strain of a previously unrecognized mutation. This mutation, which has been designated put-4977, specifically affects proline dehydrogenase rather than proline uptake. Although proline dehydrogenase remains inducible by L-proline in strains carrying the mutation, there is a premature cessation of differential synthesis during induction that results in a lower specific activity. The mutation shows about 50% P1-mediated cotransduction with pyrC and is therefore located at about 22 min on the E. coli chromosome. Merodiploids containing a normal F' factor still exhibit decreased enzyme activity, indicating that the put-4977 mutation is trans-dominant. The mutation cannot be detected in present stocks of the transferase-deficient mutant, suggesting that this mutant is a revertant for put-4977.     

鼠李糖乳杆菌D-(+)-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达

利用PCR的方法从鼠李糖乳杆菌基因组DNA中扩增到D-(+)-乳酸脱氢酶基因(ldhD),并连接到载体pSE380上,构建表达质粒pSE-ldhD,将重组质粒pSE-ldhD转化大肠杆菌BL21(DE3),重组菌株经IPTG诱导表达,SDS-PAGE电泳分析表明ldhD在大肠杆菌中实现了表达,表达产物的分子量约为37kD。同时采用紫外分光光度法测定D-乳酸脱氢酶的酶活,测得重组菌株的D-乳酸脱氢酶活力为5.4U/mL,最适反应温度为35℃,最适pH为5.6。     

Possible involvement of a L-delta 1-pyrroline-5-carboxylate (P5C) reductase in the synthesis of proline in Desulfovibrio desulfuricans Norway

A L-delta 1-pyrroline-5-carboxylate reductase activity has been detected in crude extracts of Desulfovibrio desulfuricans Norway. This P5C reductase activity is also found when a 2.5 kb D. desulfuricans DNA fragment is introduced into an Escherichia coli proC mutant. Although it restores growth of the proC mutant, the ProDd enzyme might be detrimental to the E. coli host since the plasmid carrying the cognate proDd gene is segregated at high rate by the cells but is stabilized by small deletions which lead to a loss of the P5C reductase activity.     

D-Alanine dehydrogenase. Its role in the utilisation of alanine isomers as growth substrates by Pseudomonas aeruginosa PA01.

Pseudomonas aeruginosa PA01 was found to utilise both the D- and L-isomers of alpha-alanine and also beta-alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation of D-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation of L-alanine required its conversion to the directly oxidisable D-form by a soluble racemase; (iii) utilisation of beta-alanine, like L-alanine, involves both the racemase and D-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity of which is still speculative; (iv) P. aeruginosa, like Escherichia coli, appears to take up D-alanine and L-alanine by means of two specific permeases.     

E. coli histidine triad nucleotide binding protein 1 (ecHinT) is a catalytic regulator of D-alanine dehydrogenase (DadA) activity in vivo

Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.     

Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline

A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized. The proline 4-hydroxylase activity was l600-fold higher than that in Dactylosporangium sp. RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.     

Biotransformation of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase

The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding "P6C reductase" that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%.     

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation.

1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.     

Multicopy proC in Streptomyces coelicolor A3(2) elicits a transient production of prodiginines, while proC deletion does not yield a proline auxotroph

The last step of proline biosynthesis is typically catalysed by the enzyme Δ(1)-pyrroline-5-carboxylate reductase, encoded by the proC gene. Complete genome sequencing of Streptomyces coelicolor, a soil-dwelling Gram-positive bacterium that uses proline as a precursor for synthesis of prodiginine, revealed a single copy of this gene. Unexpectedly, disruption of this proC homologue (Sco3337) in S. coelicolor M145 yielded a prototrophic strain, yet the reductase activity of Sco3337 was confirmed by complementation of an Escherichia coli proC mutant. Multicopy proC within different genetic contexts elicited a transient production of prodiginines, which showed differential production kinetics of the two most common forms of this natural product produced by S. coelicolor, i.e. streptorubin B (cyclic) and undecylprodigiosin (linear). The metabolic and evolutionary implications of these observations are discussed.     

Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli.

Proline utilization by Escherichia coli and Salmonella typhimurium requires expression of genes putP (encoding a proline transporter) and putA. Genetic data indicate that the PutA protein is both put repressor and a respiratory chain-linked dehydrogenase. We report a redesigned purification procedure as well as the physical characteristics and biological activities of the PutA protein purified from E. coli. The purified protein was homogeneous as determined by electrophoresis performed under denaturing and nondenaturing conditions. Its N-terminal sequence corresponded to that predicted by the DNA sequence. We showed copurification of proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities. Purified PutA protein bound put DNA in vitro in an electrophoretic band-shift assay and it could be reconstituted to inverted membrane vesicles, yielding proline dehydrogenase activity. The Stokes radius and Svedberg coefficient of the protein were determined to be 7.1 nm and 9.9 S, respectively. These hydrodynamic data revealed that the protein in our preparation was dimeric with a molecular mass of 293 kDa and that it had an irregular shape indicated by the friction factor (f/f0) of 1.6.     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates

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.     

Proline-hyperproducing strains of Serratia marcescens: enhancement of proline analog-mediated growth inhibition by increasing osmotic stress

Proline-producing strains of Serratia marcescens were more osmotolerant than wild-type strains. Growth inhibition by proline analogs was significantly enhanced by increasing the osmotic stress of the medium. Mutants resistant to azetidine-2-carboxylate were derived from a proline-producing strain, SP126, under a high osmotic condition. One of the mutants, strain SP187, produced 56 mg of L-proline per ml of medium containing sucrose and urea. This amount was ca. 3 times larger than that produced by strain SP126. The intracellular glutamate content which decreased in strain SP126 was restored in strain SP187. The glutamate dehydrogenase level of strain SP187 was 5 times higher than that of strain SP126.     

Proline transport carrier-defective mutants of Escherichia coli K-12: properties and mapping.

A series of mutants of Escherichia coli K-12 requiring a high concentration of L-proline for growth were isolated from a proline auxotroph strain, JE2133. Genetic studies of the mutants, PT19, PT21, and PT22, showed that all the mutations (proT) were point mutations, and these were mapped at 82 min on the E. coli genetic map. Intact cells and cytoplasmic membrane vesicles of these mutants were specifically defective in L-proline transport activity. Strain PT21 had no detectable activity of the L-proline transport carrier at all, and strains PT19 and PT22 had only 1/35 and 1/70, respectively, of the transport activity of the parental strain. The mutants were also shown to have a defect in proline-binding function of the carrier by measuring specific binding of proline to sonically disrupted membranes. These results indicate that the gene proT determines the function of proline carrier in the cytoplasmic membrane.     

Solubilization and functional reconstitution of the proline transport system of Escherichia coli

The membrane carrier for L-proline (product of the putP gene) of Escherichia coli K12 was solubilized and functionally reconstituted with E. coli phospholipid by the cholate dilution method. The counterflow activity of the reconstituted system was studied by preloading the proteoliposomes with either L-proline or the proline analogues: L-azetidine-2-carboxylate or 3,4-dehydro-L-proline. The dilution of such preloaded proteoliposomes into a buffer containing [3H]proline resulted in the accumulation of this amino acid against a considerable concentration gradient. A second driving force for proline accumulation was an electrochemical potential difference for Na+ across the membrane. More than a 10-fold accumulation was seen with a sodium electrochemical gradient while no accumulation was found with proton motive force alone. The optimal pH for the L-proline carrier activities for both counterflow and sodium gradient-driven uptake was between pH 6.0 and 7.0. The stoichiometry of the co-transport system was approximately one Na+ for one proline. The effect of different phospholipids on the proline transport activity of the reconstituted carrier was also studied. Both phosphatidylethanolamine and phosphatidylglycerol stimulate the carrier activity while phosphatidylcholine and cardiolipin were almost inactive.     

Proline-hyperproducing strains of Serratia marcescens: enhancement of proline analog-mediated growth inhibition by increasing osmotic stress.

Proline-producing strains of Serratia marcescens were more osmotolerant than wild-type strains. Growth inhibition by proline analogs was significantly enhanced by increasing the osmotic stress of the medium. Mutants resistant to azetidine-2-carboxylate were derived from a proline-producing strain, SP126, under a high osmotic condition. One of the mutants, strain SP187, produced 56 mg of L-proline per ml of medium containing sucrose and urea. This amount was ca. 3 times larger than that produced by strain SP126. The intracellular glutamate content which decreased in strain SP126 was restored in strain SP187. The glutamate dehydrogenase level of strain SP187 was 5 times higher than that of strain SP126.