Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

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.     

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

Summary 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.Abbreviations pCMB p-chloromercuribenzoate - DM Davis and Mingioli - Ap ampicillin - NTG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate - Str streptomycin - Tet tetracycline - Ac l-azetidine-2-carboxylic acid - DHP 3, 4-dehydro-d,l-proline - MTT 3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - Kan kanamycin - Spc spectinomycin     

The effect of amino acids on the production of ochratoxin A in chemically defined media

The specific ability ofl-glutamic acid and ofl-proline to induce the formation of ochratoxin by a strain of the fungusAspergillus ochraceus in chemically defined media has been further confirmed. This induction was inhibited by 22 other amino acids as well as by analogues and antagonists of glutamic acid and proline. The inhibition could be reversed by increasing the concentrations of glutamic acid or proline and by adding lactic acid in low concentrations.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Lincomycin,rational selection of high producing strain and improved fermentation by amino acids supplementation

Based on the report that the introduction of the biosynthetic precursor of lincomycin, propylproline, could increase the production of lincomycin (Bruce et al. in US Patent 3,753,859, 1973), a mutant strain pro10–20, with resistance of feedback suppression of proline (an analog of propylproline) was thus selected and lincomycin production increased by 10%. The addition of three amino acids (l-proline, l-tyrosine, l-alanine) which are the precursors of propylproline to the fermentation medium was found to enhance the accumulation of l-dopa through different pathways and was favorable to lincomycin biosynthesis. The production of lincomycin was increased by 23, 10, 13%, respectively, with the addition of 0.05 g L⁻¹ l-proline at 60 h, 0.005 g L⁻¹ l-tyrosine and 0.1 g L⁻¹ l-alanine directly in the medium.     

Integration of the mutated biotin biosynthetic genes to the chromosome of a d-biotin-producing strain of Serratia marcescens

To construct a genetically stable strain of Serratia marcescens for d-biotin production, we integrated the mutated biotin-biosynthetic (bio) genes into the chromosome of a d-biotin-producing strain of S. marcescens Sr41. Temperature-sensitive suicide vectors consisting of the Tn5 element were used as the integration tool. Extra bio genes carried between the Tn5 termini were integrated into the chromosome of strain ETA23, a d-biotin-producing mutant. Resultant integrants produced 120 mg of d-biotin per l of a medium containing sucrose and urea whereas ETA23 produced d-biotin at 55 mg/l in the same medium. The integrated bio genes were stably maintained on the chromosome of ETA23 for 100 generations.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Construction of a threonine-hyperproducing strain of Serratia marcescens by amplifying the phosphoenolpyruvate carboxylase gene

Summary The phosphoenolpyruvate carboxylase gene (ppc) of Escherichia coli K-12 was cloned on the multi-copy plasmid pLG339. Plasmid pST101, which carried a 4.3-kb SalI fragment, was introduced into Serratia marcescens T-1165, which carried the seven regulatory mutations for three aspartokinases and two homoserine dehydrogenases. Strain T-1165[pST101] produced phosphoenolpyruvate carboxylase at a rate 26 times higher than the control strain T-1165[pLG339]. While T-1165[pST101] produced 63 mg/ml l-threonine in a medium containing sucrose and urea, whereas T-1165 only produced 52 mg/ml.     

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.     

Benzoylarginine peptidase and iminopeptidase profiles ofTreponema denticola strains isolated from the human periodontal pocket

Seven clinical isolates and the ATCC strain 35405 ofTreponema denticola, obtained from human periodontal pockets, were studied for peptidase activity with several chromogenic compounds as substrates. The cell sonicates of all strains hydrolyzed phenylazobenzyloxycarbonyl-l-prolyl-l-leucyl-glycyl-l-prolyl-d-arginine (a collagenase substrate), azocasein, and the 2-naphthylamines ofl-proline,l-hydroxyproline,l-pyrrolidine, and benzoyl-l-arginine, but the rates of hydrolysis varied considerably from strain to strain. Fast protein liquid chromatography on gel and anion exchange columns revealed further biochemical differences between the strains. The ATCC strain consistently produced several proline iminopeptidases, whereas four of the clinical isolates yielded high and three yielded low iminopeptidase activity. The ATCC strain and six clinical isolates displayed high benzoylarginine peptidase activity. The use ofN-l-prolyl-2-naphthylamine as substrate revealed more differences between the strains than other substrates. The substrate specificity of the enzymes discovered suggests that they may be important for the nutrition of the organism or in the protection of the organism against chemical defense factors present in the gingival pocket.     

Mutational Engineering of the Cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis> for Resistance to Growth-Inhibitory Action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N₂ fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li⁺-R and Na⁺-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O₂-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol m⁻³ proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline. Received: 10 July 2002 / Accepted: 13 August 2002     

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 × 10⁷  viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole^–) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole^– mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim^– (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 ×g supernatant, but produced wild-type levels of penicillin. Received: 16 April 1997 / Accepted: 26 July 1997     

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production

Microbial production of trans-4-hydroxy-l -proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l -proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l⁻¹ Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l -proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb_(L)-DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l⁻¹, 87.4% and 1.38 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.     

l-proline as a nitrogen source increases the susceptibility ofSaccharomyces cerevisiae S288c to fluconazole

Fluconazole inhibition ofSaccharomyces cerevisiae S288c growth was evaluated in media containing ammonia,l-proline orl-leucine as a nitrogen source. Growth inhibition by fluconazole was maximum whenl-proline was used as a nitrogen source, while rhodamine 6G accumulation and fluconazole resistance were the highest when ammonia was the sole nitrogen source.     

Construction of a Proline-Producing Mutant of the Extremely Thermophilic Eubacterium Thermus thermophilus HB27

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.Thermus thermophilus, a gram-negative aerobic eubacterium, is one of the most widely studied species of extremely thermophilic microorganisms. We have been working on the molecular genetics and molecular reproduction of T. thermophilus HB27. We have already cloned and sequenced three proline biosynthetic genes, proB, proA, and proC, and reported that the proB and proA genes exist in tandem (7, 9).We have also constructed physical maps of the HB27 chromosome and of a large plasmid, pTT27, and determined the locations of all proline biosynthetic genes on the chromosomal DNA (20, 21). We have already succeeded in overproducing carotenoids in T. thermophilus HB27 (6), but at present there is no report about extracellular production of amino acids in extreme thermophiles. We have elucidated the consensus sequences for strong promoters of T. thermophilus (11) and developed a thermostable antibiotic resistance gene (12). It is also easy to disrupt or mutate genes on chromosomal DNA in T. thermophilus HB27 (8). Among the extreme thermophiles, a host-vector system has been established only in T. thermophilus. Generally, the reaction rate of thermostable enzymes which are produced from T. thermophilus is higher than those of enzymes from mesophiles. In a fermentation process such as amino acid production, T. thermophilus may contribute to the improvement of amino acid productivity since fermentation at a high temperature eliminates the problems of contamination and cooling procedures. So, we decided to attempt excretion of proline at a high temperature with T. thermophilus mutants.l-Proline is synthesized from glutamate by the sequential reaction of γ-glutamyl kinase, γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase in bacteria (1). Genes proB and proA, which encode γ-glutamyl kinase and γ-glutamyl phosphate reductase, respectively, were found to comprise an operon in T. thermophilus (9), Escherichia coli (5), and Serratia marcescens (13). In E. coli and S. marcescens, γ-glutamyl kinase is subject to feedback control by l-proline (3, 13), but γ-glutamyl phosphate reductase and pyrroline-5-carboxylate reductase are not inhibited by proline (3, 15). Meanwhile, E. coli and S. marcescens rapidly degrade proline by proline dehydrogenase (proline oxidase), encoded by the putA gene (3, 14, 22). So far, it has been reported that E. coli mutants resistant to proline analogs, dl-3,4-dehydroproline and l-azetidine-2-carboxylic acid, excreted l-proline into the medium. But the amount of l-proline excreted was too small for practical use because of the existence of the proline degradation pathway (2). For S. marcescens, Sugiura et al. (18, 19) have constructed a proline-overproducing strain, SP126, as a double mutant resistant to 3,4-dehydroproline and thiazolidine-4-carboxylate and derived from a proline dehydrogenase-deficient mutant (18). Strain SP126 produced about 20 mg of l-proline per ml in the fermentation medium (18).In T. thermophilus, the control system in proline biosynthesis has not been elucidated. However, we thought that the feedback control of proline biosynthesis in T. thermophilus should be similar to that of E. coli and S. marcescens, since the amino acid sequences of proline biosynthetic enzymes in T. thermophilus show a high similarity to sequences of those of E. coli and S. marcescens (7, 9). E. coli and S. marcescens mutants resistant to 3,4-dehydroproline have already been determined to be proB mutants (4, 14). The comparison of the amino acid sequences of γ-glutamyl kinases in E. coli, S. marcescens, and T. thermophilus showed that these mutations occurred in the positions conserved among the three microorganisms (Fig. ​(Fig.1).1). We thought that it was possible to construct a 3,4-dehydroproline-resistant mutant of T. thermophilus by introducing the same mutations into the proB gene found in the mutants of E. coli and S. marcescens. We determined the strategy for construction of a proline-producing strain of T. thermophilus by following two steps: first, construction of a 3,4-dehydroproline-resistant mutant by introduction of mutations into the proB gene, and second, isolation of a mutant which cannot utilize proline for its growth by mutagenizing the dehydroproline-resistant mutant. Open in a separate windowFIG. 1Comparison of the amino acid sequences of γ-glutamyl kinases in E. coli, S. marcescens, and T. thermophilus. The amino acid substitutions found in E. coli (4) and S. marcescens (14) are shown by arrows. Asterisks show the amino acid residues conserved in the three microorganisms.     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Na+ (Li+)-proline cotransport inEscherichia coli

Summary Na⁺ and Li⁺ were found to stimulate the transport ofl-proline by cells ofEscherichia coli induced for proline utilization. The gene product of the put P gene is involved in the expression of this transport activity since the put P⁺ strains CSH 4 and WG 148 show activity and the put P^– strain RM 2 fails to show this cation coupled transport. The addition of proline was found to stimulate the uptake of Li⁺ and of Na⁺. Attempts to demonstrate proline stimulated H⁺ uptake were unsuccessful. It is concluded that the proline carrier (coded by the put P gene) is responsible for Na⁺ (or Li⁺)-proline cotransport.     

Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity

Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl--alanyl-l-proline (PhAc-Ala-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The K _m of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.