Proline Auxotroph Research Articles

MppA, a Periplasmic Binding Protein Essential for Import of the Bacterial Cell Wall Peptide l -Alanyl-γ- d -Glutamyl- meso -Diaminopimelate

Mutants of a diaminopimelic acid (Dap)-requiring strain of Escherichia coli were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide, L-alanyl-gamma-D-glutamyl-mesodiaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type mppA gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the opp operon. An E. coli strain whose entire opp operon was deleted but which carried the mppA locus was unable to grow on murein tripeptide unless it was provided with oppBCDF genes in trans. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.

A cryptic proline permease in Salmonella typhimurium.

Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy done of the proY+ gene or both a single copy of the proY+ gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.

Proline is biosynthesized from arginine in Staphylococcus aureus.

Staphylococcus aureus NCTC 8325 exhibited a long lag phase (11 h) when inoculated into defined medium lacking proline, that could be shortened by increasing the concentration of arginine in the medium, or by supplying ornithine. Radioactivity from L-[14C]arginine, but not L-[14C]glutamate was incorporated into a spot with the chromatographic mobility of [14C]proline in the pool metabolites fraction. Selection for transposon Tn917-lacZ mutants impaired in arginine catabolism yielded four proline auxotrophs. Enzyme assays and precursor feeding experiments suggested that the major pathway for proline biosynthesis in S. aureus was from arginine via ornithine and delta'-pyrroline 5-carboxylate, rather than from glutamate. Strain 8325 Pro+, a proline prototrophic variant obtained by cultivation of 8325 in the absence of proline, accumulated L-[14C]arginine from the medium at about eight times the rate of strain 8325, suggesting its response to proline starvation was to increase arginine uptake.

Corrected gene assignments of Escherichia coli pro- mutations

We have reevaluated the gene assignments of the proline mutant alleles of some known Pro- Escherichia coli strains. Of nine proline auxotrophs included in the study, five presented phenotypes inconsistent with their previously assigned genotypes. We discuss the possible sources and the consequences of these assignment errors.

Molecular characterization of the proline-1 (pro-1) locus of Neurospora crassa, which encodes delta 1-pyrroline-5-carboxylate reductase.

delta 1-pyrroline-5-carboxylate reductase (P5CR; [L-proline: NAD(P+) 5-oxidoreductase]; EC 1.5.1.2) catalyzes the final step in proline biosynthesis. We have shown that the proline-1 (pro-1) locus of Neurospora crassa encodes P5CR. The pro-1 gene was localized to a 3.2 kb region by complementation of (restoration of proline-independent growth to) a proline auxotroph carrying a recessive mutation at the pro-1 locus. The nucleotide sequence of this 3.2 kb region contains an open reading frame with coding capacity of 311 amino acids. The deduced polypeptide shows significant similarity to P5CR amino acid sequences. Similarity of N. crassa P5CR is greatest to that of the yeast, Saccharomyces cerevisiae, but is also strong to P5CR sequences from archaea, eubacteria, plants, and humans. In N. crassa, amino acid imbalance, including deficiency or excess of a single amino acid, such as histidine, induces expression of many amino acid biosynthetic genes that are under cross-pathway control, a general regulatory system analogous to general amino acid control in Saccharomyces. Although P5CR catalyzes the only committed step in proline biosynthesis, pro-1 expression was unaltered by histidine starvation and independent of CPC1, a positively acting transcription factor that mediates cross-pathway control in N. crassa.

Directed evolution of novel biosynthetic pathways: Overexpression of leucine aminopeptidase allows an Escherichia coli proline auxotroph to grow faster on prolinamide

Selection of plasmids which confer a rapid growth phenotype on prolinamide from a library maintained in an Escherichia coli proline auxotroph reveals aminopeptidase I to be responsible for the growth; the enzyme has been overproduced, purified to homogeneity, characterised and crystallised in a form suitable for structure determination.

Directed evolution of novel biosynthetic pathways: Construction of a randomly mutated gene library in an Escherichia coli proline auxotroph and selection for growth on potential proline precursors

By introducing a plasmid library into a highly mutagenising Escherichia coli proline auxotroph the evolutionary scenario of gene doubling and mutagenesis has been mimicked allowing for evolved growth on certain potential proline precursors.

Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis.

Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a mothbean (Vigna aconitifolia) cDNA expression library. This novel strategy, "trans-complementation," allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta-OAT). This enzyme transaminates ornithine to glutamic-gamma-semialdehyde (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant. The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as well as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases. The V. aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa. The native enzyme expressed in E. coli appears to be a monomer with Km of 2 mM for ornithine and 0.75 mM for alpha-ketoglutarate. Levels of mRNA in V. aconitifolia for delta 1-pyrroline-5-carboxylate synthetase (P5CS) and delta-OAT, the two key enzymes for proline synthesis, were monitored under different physiological conditions. Salt stress and nitrogen starvation induced P5CS mRNA levels and depressed OAT mRNA levels. Conversely, OAT mRNA level was elevated in plants supplied with excess nitrogen while the P5CS mRNA level was reduced. These data suggest that the glutamate pathway is the primary route for proline synthesis in plants during conditions of osmotic stress and nitrogen limitation whereas the ornithine pathway assumes prominence under high nitrogen input.

Directed evolution of novel biosynthetic pathways: growth of an escherichia coli proline auxotroph on Δ 1-pyrroline-2-carboxylic acid.

A directed approach to the evolution of novel biosynthetic pathways is exemplified by the selection of plasmids which enhance the growth of an Escherichia coli proline auxotroph on an unnatural precursor, Δ 1-pyrroline-2-carboxylic acid 2.

Reduction of Δ 1-pyrroline-2-carboxylic acid to proline by an escherichia coli proline auxotroph.

A new synthesis of Δ 1-pyrroline-2-carboxylic acid 2 and various isotopomers is described; the conversion of 2 to proline by Escherichia coli is shown to proceed by a direct reduction.

Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae.

Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. We cloned a human pyrroline-5-carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100. Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy. The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy. The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable. The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein. The derived amino acid sequence is 32% identical with that of S. cerevisiae. By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17.

A Bacillus subtilis dipeptide transport system expressed early during sporulation

Two previously identified Bacillus subtilis DNA segments, dciA and dciB, whose transcripts accumulate very rapidly after induction of sporulation, were found in the same 6.2 kb transcription unit, now known as the dciA operon. Analysis of the sequence of the dciA operon showed that its putative products are homologous to bacterial peptide transport systems. The product of the fifth gene, DciAE, is similar to peptide-binding proteins from Escherichia coli and Salmonella typhimurium (DppA and OppA) and B. subtilis (OppA). A null mutation in dciAE abolished the ability of a proline auxotroph to grow in a medium containing the dipeptide Pro-Gly as sole proline source, suggesting that the dciA operon encodes a dipeptide transport system.

Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein

We describe the isolation and analysis of an Escherichia coli gene, dppA, and its role in dipeptide transport. dppA maps near min 79 and encodes a protein (DppA) that has regions of amino acid similarity with a peptide-binding protein from Salmonella typhimurium (OppA). Like OppA, DppA is found in the periplasmic space and thus is most likely a dipeptide-binding protein. Insertional inactivation of dppA results in the inability of a proline auxotroph to utilize Pro-Gly as a proline source. dppA-dependent Pro-Gly utilization does not require any of the three major proline transport systems, demonstrating that DppA is not simply a dipeptidase. An in vivo competition assay was used to show that DppA is probably involved in the transport of dipeptides other than Pro-Gly. Transcription of dppA is repressed by the presence of casamino acids, suggesting that the cell alters its dipeptide transport capabilities in response to an environmental signal.

Biochemical and genetic studies with arginine and proline auxotrophs of Neisseria gonorrhoeae

The prevalence of specific arginine biosynthesis gene defects was studied for 319 arginine-requiring clinical isolates of Neisseria gonorrhoeae by using the ability of the strains to utilize intermediates of arginine biosynthesis. Only 11% of the uracil-requiring strains defective in the carbamylation of ornithine to yield citrulline had a defective carbamoylphosphate synthetase gene (carAB). Strains defective in carAB were of auxotype CUH. The other strains (89%) having a dual requirement for citrulline and uracil, which were mostly of auxotype PCU, were defective in the ornithine transcarbamoylase gene (argF). Over 90% of the strains were defective either in argJ (174 strains) or in argF (126 strains). Three argininosuccinate-requiring strains (i.e., defective in argG) of auxotype PAU were identified. Some of the arginine auxotrophs of N. gonorrhoeae defective in carAB, argJ, argF, or argG were complemented by genetic transformation with DNA from recombinant bacteriophages carrying characterized gonococcal arginine biosynthesis genes. Gene defects in proA (five strains) and in proB (six strains) were identified by gonococcal transformation assays with recombinant bacteriophages or plasmids carrying proline biosynthesis genes from N. gonorrhoeae. None of the 11 proline-requiring strains tested was defective in proC.

Fermentative production of L-glutamic-.GAMMA.-semialdehyde and its chemical conversion into L-tryptophan.

Proline auxotrophs, which were induced from an i -prolific-producing strain of Brevibacterium flavum by indirect suppression mutation, accumulated L-glutamic-γ-semialdehyde (GSA) in the fermentative broth. The addition of sulfite salts to the culture medium, when bacteria were exponentially growing, improved the accumulation of GSA from 2.3 to a maximum of 13.2 mg/ml. The direct use of GSA in the fermentative broth for the chemical synthesis of L-tryptophan was studied. The yield of L-tryptophan was 48% on the basis of GSA in the reaction mixture.

Proline in the osmoregulation of Brevibacterium lactofermentum.

Osmoregulation in Brevibacterium lactofermentum was studied. Proline was accumulated up to approximately 35mg/g dry cell weight in the cells of a wild strain of the bacterium grown under osmotic stress. The osmotic tolerance of a proline auxotroph mutant obtained from the bacterium was lower than that in the wild strain. The activity of pyrroline-5-carboxylate reductase, one of the enzymes in the proline biosynthetic pathway, increased about 3-fold when the cells of B. lactofermentum were grown under osmotic stress. These data indicated that proline is important in osmoregulation in the bacterium.

Two membrane-bound proteins associated with alanine resistance and increased A-system amino acid transport in mutants of CHO-K1.

Growth of CHO-K1, a proline auxotroph, is inhibited by amino acids that prevent proline transport. From a hydroxyurea-treated, alanine-resistant, constitutive mutant, alar4, we isolated, in a stepwise fashion, mutants, resistant to higher concentrations of alanine, that have increased velocity of amino acid transport through the A system. Two such mutants, alar4-H2.1 and alar4-H3.9, isolated as resistant to 50 mM and 125 mM alanine, respectively, showed increases in Vmax of proline transport through the A system that are directly proportional to their resistance to alanine. Alar4-H3.9, as compared to alar4 and CHO-K1, has six and 29 times the Vmax of proline transport through the A system and two and five times the velocity of transport through the combined ASC and P systems, respectively, and no change in system L. No double-minute or homologous staining regions were detectable in alar4-H3.9. A-system activity of alar4-H2.1 and alar4-H3.9, when grown under nonselective conditions, was stable for 20 generations and then declined. The phenotype of alar4-H3.9 is codominant with that of alar4 and partially recessive to that of CHO-K1. Membrane vesicles prepared from alar4-H3.9 show increases mainly in A-system transport. In sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of A-system active membrane vesicles and endoplasmic reticulum, two bands of molecular weight of approximately 62-66 kd and 29 kd are present in higher concentrations in alar4-H3.9 than in CHO-K1. These results are compatible with the hypothesis that the phenotype of alar4-H3.9 is the result of gene amplification of an A-system transporter structural gene and that the two bands may represent this transporter.

Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae.

The PRO1, PRO2, and PRO3 genes were isolated by functional complementation of pro1, pro2, and pro3 (proline-requiring) strains of Saccharomyces cerevisiae. Independent clones with overlapping inserts were isolated from S. cerevisiae genomic libraries in YEp24 (2 microns) and YCp50 (CEN) plasmids. The identity of each gene was determined by gene disruption, and Southern hybridization and genetic analyses confirmed that the bona fide genes had been cloned. Plasmids containing each gene were introduced into known bacterial proline auxotrophs, and the ability to restore proline prototrophy was assessed. Interspecies complementation demonstrated that the S. cerevisiae PRO1 gene encoded gamma-glutamyl kinase, PRO2 encoded gamma-glutamyl phosphate reductase, and PRO3 encoded delta 1-pyrroline-5-carboxylate reductase. The presence of the PRO3 gene on a high-copy-number plasmid in S. cerevisiae caused a 20-fold overproduction of delta 1-pyrroline-5-carboxylate reductase. The PRO2 gene mapped on chromosome XV tightly linked to cdc66, and the PRO3 gene was located on the right arm of chromosome V between HIS1 and the centromere.

Naturally occurring auxotrophs of Campylobacter jejuni and Campylobacter coli.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.

1] Chinese hamster ovary cells

Publisher Summary This chapter describes Chinese hamster ovary (CHO) cells that use for genetic analysis in tissue culture. These cells have been used for the isolation of mutants affecting intermediary metabolism; DNA, RNA, and protein synthesis; membrane functions; and several more complex forms of cell behavior such as cell growth and endocytosis. A recent compilation of CHO mutants lists more than 80 classes of mutants that are isolated using this cell line. The methodologies described in this chapter are developed for work with the CHO-S sublines but most of the methods, with the exception of suspension culture, may be used for CHO-K1 cell lines. CHO cells are proline auxotrophs, unlike most other cultured cell lines, and require medium containing this nutrient, such as Ham's F12 or a-modified Eagle's medium (a-MEM) without ribonucleoside or deoxyribonucleosides, both of which are commercially available. These rich media must in addition contain other limiting nutrients for CHO cells, as they support more rapid growth of CHO lines than is possible in MEM alone supplemented with proline.