Methionine-containing Medium Research Articles

Mechanism analysis of methionine inhibiting growth of Bifidobacterium bifidum

Bifidobacterium bifidum rarely utilises yeast extract, which causes significant challenges in its industrial production. This study characterised the utilisation of amino acids by B. bifidum to improve the viability of the strain. In this study, the proliferation effect of B. bifidum in different nitrogen sources was determined, and the amino acid contents of different nitrogen sources were analysed, which indicated that hydrophobic amino acids may inhibit B. bifidum growth. Among different hydrophobic amino acids, methionine was identified as the strongest growth inhibitor. Furthermore, the intracellular pH and NH4+ contents of B. bifidum CCFM16 were higher in methionine-free medium. Among the NH3-producing enzymes related to amino acid metabolism, the metC gene was significantly transcribed in methionine-free medium. In addition, tcyP, metB, metC and metE genes encoding cysteine transporter proteins and the conversion pathway of cysteine to methionine were transcribed 2.03-fold, 7.03-fold, 3.05-fold and 2.92-fold, respectively, in methionine-free medium compared to methionine-containing medium. Compared with methionine-containing medium, the activity of tricarboxylic acid cycle rate-limited enzyme isocitrate dehydrogenase in methionine-free medium also increased from 198.89 to 410.56 nmol/min/mg protein, which probably enhanced the carbon flux and promoted growth. Meanwhile, the activity of pyrroline-5-carboxylate reductase, an enzyme that synthesises proline, increased from 14.76 to 35.28 nmol/min/mg protein, which promoted proline synthesis in response to osmotic stress. Cyclopropane fatty acids were also significantly higher in methionine-free medium, which may promote growth by decreasing cell membrane permeability to protons and enhancing cell membrane fluidity. This study provides a theoretical basis for high-density culture of B. bifidum.

Oncogenes and Methionine Addiction of Cancer: Role of c-MYC.

Methionine addiction is a general and fundamental hallmark of cancer cells, termed the Hoffman effect. Previously Vanhamme and Szpirer showed that methionine addiction could be induced by transfection of the activated HRAS1 gene to a normal cell line. In the present study, we investigated the role of the c-MYC oncogene in methionine addiction of cancer, by comparison of c-Myc expression and malignancy of methionine-addicted osteosarcoma cells and rare methionine-independent revertants, derived from the methionine-addicted cells. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were derived from methionine-addicted parental 143B osteosarcoma cells (143B-P), by continuous culture in medium depleted of methionine by recombinant methioninase. To compare in vitro malignancy of methionine-addicted parental cells and methionine-independent revertant cells, the following experiments were performed: for 143B-P and 143B-R cells, cell proliferation capacity was measured with a cell-counting assay, and colony-formation capacity was determined on plastic and in soft agar, all in methionine-containing Dulbecco's Modified Eagle's Medium (DMEM). Tumor growth was measured in orthotopic xenograft nude-mouse models, to compare in vivo malignancy of 143B-P and 143B-R cells. c-MYC expression was examined with western immunoblotting and compared in 143B-P and 143B-R cells. 143B-R cells had reduced cell proliferation capacity, compared to 143B-P cells, in methionine-containing medium (p=0.003). 143B-R cells had reduced colony formation capacity on plastic (p=0.003) and in soft agar, compared to 143B-P cells in methionine-containing medium. 143B-R cells had reduced tumor growth in orthotopic xenograft nude-mouse models, compared to 143B-P cells, (p=0.002). These results demonstrate that 143B-R methionine-independent revertant cells lost malignancy. Expression of c-MYC was reduced in 143B-R methionine-independent revertant osteosarcoma cells, compared to 143B-P cells, (p=0.0007). The present study demonstrated that c-MYC expression is linked to malignancy and methionine addiction of cancer cells. The present study on c-MYC, and the previous study on HRAS1, suggest that oncogenes may play a role in methionine addiction, which is a hallmark of all cancers, as well as in malignancy.

A putative phospholipase C is involved in Pichia fermentans dimorphic transition

BackgroundPichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi. MethodsPichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition. ResultsFive genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18. ConclusionPhospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition. General significancePhospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.

Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

p21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors that includes p21, p27, and p57. Recent studies have suggested that Cdk2 activity may promote p21 degradation through a pathway similar to that for p27, although the mechanism by which this occurs has not been clarified. In the current report, co-expression with cyclin E and Cdk2 stabilized p21 in a manner that required the CDK-binding site of p21 and a cyclin-binding site (cy1) located in the p21 N terminus. Strikingly, however, a kinase-dead Cdk2 mutant stabilized p21 to a greater extent than did wild-type Cdk2, consistent with the notion that Cdk2 activity can destabilize p21. The ability of wild-type Cdk2 to destabilize p21 required a potential Cdk2 phosphorylation site in p21 at serine 130 and an intact cyclin-binding motif (cy2) in the p21 C terminus. Finally, p21 was phosphorylated by Cdk2 at Ser-130 in vitro, and this ability of Cdk2 to phosphorylate p21 was dependent, in large part, on the presence of cy2. These results support a model in which active Cdk2 destabilizes p21 via the cy2 cyclin-binding motif and p21 phosphorylation.

Expression of Xanthomonas oryzae pv. oryzae hrp Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae.

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo.

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway.

Regulated turnover of a cell surface-associated pool of newly synthesized apolipoprotein E in HepG2 cells

Newly synthesized apolipoprotein E (apoE) is incompletely secreted and partially degraded by hepatocytes. To evaluate current models concerning apoE's role in remnant lipoprotein clearance by the liver, we performed a detailed and quantitative pulse-chase analysis of apoE secretion, subcellular distribution, and proteolytic degradation by the human hepatoma cell line HepG2. Only about 35% of newly synthesized apoE were found to be secreted to the culture medium. As determined by a protease-protection assay, a substantial amount of newly synthesized apoE was transported to the cell surface, constituting more than half of the cellular apoE under steady-state conditions. A subpopulation representing almost 40% of newly secreted apoE was rapidly rebound to the cell surface, indicating a dynamic equilibrium between cell surface and secreted apoE. These pools of newly synthesized apoE were subject to proteolytic turnover that occurred in lysosomes, presumably after re-endocytosis. We found that the proteolytic turnover of cell surface and secreted apoE was regulated by the availability of apoE ligands, being almost completely suppressed by the presence of lipoprotein-containing human serum or isolated lipoproteins, namely LDL and HDL. The characteristics of regulated degradation of cell surface apoE shed new light on potential physiological functions of this pool of apoE. Our results provide evidence that hepatocytes are capable of forming a large pool of cell surface-associated apoE, thereby supporting the previously proposed secretion-recapture model for apoE.

The Escherichia coli K-12 metJ193 allele contains a point mutation which alters the hydrophobic pocket responsible for in vitro binding of S-adenosylmethionine: effects on cell growth and induction of met regulon expression

The metJ193 allele encodes one of two identified temperature-sensitive Escherichia coli K-12 met repressors. The nucleotide sequence of the metJ193 allele was determined. The point mutation was a T to A transversion at base 170 of the metJ193 open reading frame and resulted in the substitution of leucine by glutamine at the 56th amino acid residue of the MetJ193 protein. The mutational lesion altered the hydrophobic pocket responsible for in vitro binding of the corepressor S-adenosylmethionine by wild-type MetJ. MetJ193 protein formed at the permissive temperature (28 degrees C) allowed slow derepression of met regulon expression when cultures were shifted to the nonpermissive temperature (34 degrees C). When 28 degrees C cultures of strains bearing two metJ193 alleles were transferred from methionine-containing medium to minimal medium, derepression of met regulon expression did not occur quickly enough to avoid a lag in growth due to the methionine deprivation. The inability of the MetJ193 protein to easily accomplish transition between apo- and active-repressor conformations was also demonstrated by using a maxicell system to study expression of a plasmid-borne copy of the E. coli metF transcription unit. These results confirm the importance of the leucine 56 residue for the structure and function in vivo of the wild-type MetJ protein.

Differential synthesis of crystallins in the developing rat eye lens

The patterns of protein synthesis in rat lenses ranging in age from newborn to 4 months were compared. After incubation of lenses in [ 35S]methionine-containing medium it was possible to identify the de novo synthesized crystallins by two-dimensional gel electrophoresis and fluorography, in combination with peptide mapping and immunoblotting. It was found that the relative synthesis of αA and βA3 stays fairly constant in rat lenses of all investigated ages. The relative synthesis of βB2 and γs shows a pronounced increase with age in these post-natal lenses. A differential decrease can be observed in the relative synthesis of the other six γ-crystallins (γA-γF). There appears to be a good correlation between the changes in relative synthesis of the various crystallins and previously reported alterations in mRNA levels, although certain mRNAs exhibit marked differences in translational efficiency.

Transferrin is an autocrine growth factor secreted by Reuber H-35 cells in serum-free culture.

We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the absence and presence of [35S]methionine. A 1:4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell numbers per assay dish from 1.59 +/- 0.12 X 10(5) to 3.35 +/- 0.34 X 10(5) after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P less than 0.05) less growth (2.14 +/- 0.41 X 10(5) cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [35S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate that 29.1 +/- 1.2 ng of transferrin was secreted per 10(6) cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P less than 0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35 cells into serum-free culture is due to transferrin.

Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development.

The role in early development of proteins secreted by oviduct epithelial cells has been investigated. Secreted proteins devoid of serum contamination have been produced by the surgical removal and immediate incubation of oviduct cells in [35S]methionine-containing medium. After electrophoretic separation, secreted polypeptides could be divided into those that were secreted uniformly throughout the oestrous cycle and a second class that showed a cyclical pattern of secretion. The first class of proteins represented a small proportion of total output whilst the predominant second class was composed mainly of polypeptides of Mr 92 and 46 x 10(3), respectively. Both of these polypeptide species, referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), are detected only during the first 4 to 5 days after oestrus when the embryos are located in the oviduct. Oviduct cells collected at oestrus and maintained thereafter in culture secrete the same pattern of proteins and follow the same time course as their counterparts in vivo. The interaction between the oviduct proteins and the developing embryo was studied firstly by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination studies showed that two polypeptides of Mr 92 and 46 x 10(3), respectively, were bound to the zona pellucida of eggs removed from the oviduct but were absent from eggs that had not had contact with the oviduct epithelium. That these newly acquired proteins represent SOP 92 and 46 is suggested by their electrophoretic mobility and their ability to bind to the zona of follicular eggs when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. The collection of unlabelled secreted proteins enabled us to produce a monoclonal antibody, which was used in the second series of experiments on oviduct-embryo interactions. The results confirmed that SOP 92 binds to the zona pellucida and moreover showed that this protein crosses the zona and becomes associated with the individual blastomeres of the developing embryo. These findings provide evidence that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by its epithelium.

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe 3+, EDTA and oxygen.

Biology of human megakaryocyte factor V

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S- methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.

Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect.

A metabolic defect that is prevalent in human cancer cell lines was exploited to selectively kill these cells without killing cocultured normal human fibroblasts. Methionine dependence, a metabolic defect seen only in cancer cells or immortalized cell lines in vitro, precludes the cells from growing in media in which methionine is replaced by its immediate precursor, homocysteine, a condition that allows the growth of all normal cell strains tested. The methionine-dependent cells become reversibly blocked in late S-G2 (i.e., late-S and G2 phases) under the above condition, a block that was exploited for selective chemotherapy against these cells. In cultures that were initiated with equal amounts of cancer cells and human diploid fibroblasts, substitution of homocysteine and doxorubicin for methionine in the culture medium followed by methionine repletion with vincristine was totally effective at selectively eliminating a methionine-dependent human sarcoma and 3 methionine-dependent human carcinomas. The above protocol was nearly totally effective against a partially methionine-independent revertant of the sarcoma. The chemotherapeutic procedure used was not lethal to normal cells growing alongside the tumor cells and was ineffective when conducted totally in methionine-containing medium. The optimal procedure was 10(-10) M doxorubicin in methionine-free, homocysteine-containing medium for 10 days followed by 2 x 10(-7) M vincristine in methionine-containing, homocysteine-free medium for 1 day, in turn followed by drug-free methionine-containing, homocysteine-free medium. These results demonstrate the potential for treatment of solid tumors with chemotherapy based on metabolic differences between normal and tumor cells.

Ethylene formation by cultures of Escherichia coli.

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH4Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe3+ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH4Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.

Neoplastic conversion of rat liver epithelial cells in culture by ethionine and S-adenosylethionine.

Rat liver epithelial cells were grown in the presence of the hepatocarcinogen, DL-ethionine, for 12 weeks. The cells were then cultured for an additional 32 weeks in carcinogen-free medium. At approximately monthly intervals, the cells were tested for transformation by assaying their ability to grow in soft agar and to produce tumors when injected s.c. into irradiated syngeneic rats. After a total of 34 weeks in culture, cells treated with 7.5 mM DL-ethionine were transformed according to both criteria. The experiment was repeated using medium in which methionine was replaced by its metabolic precursor, homocysteine. The cells were treated for 12 weeks with L-ethionine and its metabolite, S-adenosyl-L-ethionine (L-AdoEt), then cultured in their absence in methionine-containing medium for an additional 24 weeks. After a total of 20 weeks in culture, cells treated with 0.375 mM L-ethionine and cells treated with 0.2 mM L-AdoEt produced tumors following their injection into animals. Positive growth in soft agar was observed 5 and 11 weeks later, respectively. A total of 31 weeks in culture was required before cells treated with 0.2 mM L-ethionine became tumorigenic. Furthermore, tumors resulting from the injection into animals of cells treated with 0.2 mM L-ethionine arose 8-10 weeks later than did the corresponding tumors arising from the cells treated with 0.2 mM L-AdoEt. Thus, the results indicate that AdoEt may be a proximal carcinogenic metabolite of ethionine.

Influence of cysteine upon the glutathione status of isolated rat hepatocytes

Contrary to a previous report [J. Viña, R. Hems and H. A. Krebs, Biochem. J. 170, 627 (1978)], we have observed that incubation of isolated rat hepatocytes in a medium containing high concentrations of cysteine did not deplete intracellular glutathione (GSH), but instead caused a net increase of GSH. Hepatocyte-dependent accumulation of the mixed disulfide of cysteine and GSH (CYSSG) in incubation media within a 2-hr period was increased several-fold in medium containing cysteine or cystine compared to methionine-containing medium. Hepatocytes (10 6 cells/ml) placed in medium containing normal levels of cysteine, cystine, or methionine established about a 1:1 ratio of extracellular GSH to GSSG at a concentration of about 6μM each. These results indicate a possible intracellular-extracellular relationship for glutathione and an intracellular response to the extracellular status of glutathione and cyst(e)ine.

An increased requirement for methionine by transformed rat liver epithelial cells in vitro

The growth of two normal and four transformed rat liver epithelial cell lines in a methionine-containing medium and a methionine-deficient medium supplemented with homocysteine was examined. The growth rates of the normal cells on the homocysteine-supplemented medium were approximately one-half the growth rates shown by the same cells in the methionine-containing medium. In contrast, three of the four transformed cell lines studied showed virtually no growth on the homocysteine-supplemented medium, although they grew quite rapidly on the methionine-containing medium. The fourth, transformed by N-methyl- N-nitrosourea, was able to grow on the homocysteine-supplemented medium at about one-third the rate as on the methionine-containing medium. Thus, transformed rat liver epithelial cells resemble other malignant cells in their reduced capacity to grow on homocysteine in the absence of methionine.

New method for study of peptide transport in bacteria.

The transport system for glycylmethionine in Escherichia coli B and Salmonella typhimurium LT2 was examined by a new approach which may be applied to other types of exogenous materials. Physiological auxotrophs were prepared by growing wild strains in a methionine-containing medium to repress the methionine biosynthetic enzymes. Immediate protein synthesis was shown to take place in such physiological auxotrophs only in the presence of either exogenous methionine or a methionine peptide, e.g., glycylmethionine. Protein synthesis was dependent on glycylmethionine taken up by the cell and was indicated by assaying for the inducible enzyme lysine decarboxylase at 5- to 15-min intervals. Uptake was studied by using low concentrations of glycylmethionine, therefore making uptake by permease the limiting step in incorporation of methionine into protein, and by addition of competitor peptides to media containing saturating concentrations of glycylmethionine. Lysine decarboxylase activity in S. typhimurium LT2 was about 80 times that present in E. coli B. Glycylmethionine transport had a K(m) of the order of 1 muM in S. typhimurium. Structural specificities observed for peptide transport by other workers were confirmed for E. coli B. Competitive inhibition of glycylmethionine uptake by dipeptides was observed in E. coli.

New Method for Study of Peptide Transport in Bacteria1

The transport system for glycylmethionine in Escherichia coli B and Salmonella typhimurium LT2 was examined by a new approach which may be applied to other types of exogenous materials. Physiological auxotrophs were prepared by growing wild strains in a methionine-containing medium to repress the methionine biosynthetic enzymes. Immediate protein synthesis was shown to take place in such physiological auxotrophs only in the presence of either exogenous methionine or a methionine peptide, e.g., glycylmethionine. Protein synthesis was dependent on glycylmethionine taken up by the cell and was indicated by assaying for the inducible enzyme lysine decarboxylase at 5- to 15-min intervals. Uptake was studied by using low concentrations of glycylmethionine, therefore making uptake by permease the limiting step in incorporation of methionine into protein, and by addition of competitor peptides to media containing saturating concentrations of glycylmethionine. Lysine decarboxylase activity in S. typhimurium LT2 was about 80 times that present in E. coli B. Glycylmethionine transport had a K(m) of the order of 1 muM in S. typhimurium. Structural specificities observed for peptide transport by other workers were confirmed for E. coli B. Competitive inhibition of glycylmethionine uptake by dipeptides was observed in E. coli.