Transport of Methylamine by Pseudomonas sp. MA

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JOURNAL OF BACTRIOLOGY, June 198, p. 786-79 21-9193/8/6-786/5$2./ Vol. 142, No. 3 Transport of Methylamine by Pseudomonas sp. MA DWARD BLLION,* M. YOUSUFUDDIN ALI KHAN, AND MICHAL J.

1 JOURNAL OF BACTRIOLOGY, June 198, p /8/6-786/5$2./ Vol. 142, No. 3 Transport of Methylamine by Pseudomonas sp. MA DWARD BLLION,* M. YOUSUFUDDIN ALI KHAN, AND MICHAL J. ROMANO Department of Chemistry, The University of Texas at Arlington, Arlington, Texas 7619 Pseudomonas sp. MA grows on methylamine as a sole source of carbon, nitrogen, and energy. The transport of methylamine into the organism was investigated. It was found that this organism possesses an inducible transport system for methylamine having the following physical parameters: ph optimum, 7.2; temperature optimum, 3 to 35C; Km, 1 to 3 mm; Vmax, 9 to 12 nmol/min per mg (dry weight) of cells. Methylamine uptake was curtailed by azide, cyanide, and carbonyl cyanide-m-chlorophenylhydrazone; osmotic shock treatment reduced the uptake by 5%. The uptake was not effectively inhibited by ammonium ion, amino acids, or amides, but was competitively inhibited by short-chain alkylamines. Cells grown on succinate-ammonium chloride did not possess the transport system, but it could be induced in such cells by methylamine in 2 h. Cells grown with methylamine as a sole nitrogen, but not carbon, source transported methylamine at a reduced rate. Many microorganisms capable of growth on C, compounds such as methane, methanol, or methylamine are known (for a review, see reference 4). Although much of the biochemistry and physiology of such methyltrophs is now known in a substantial degree of detail, very little is known about the transport of C, compounds into these cells. However, the transport of methylamine by other types of microorganisms has been studied, usually with methylamine as a probe to study the ammonia transport systems of the organisms. Thus, Roon et al. (11) showed that methylamine was transported by Saccharomyces cerevisiae. The process was energy-dependent but was not regulated to any great degree by the nature of the nitrogenous growth source. This is in contrast to work with Aspergillus nidulans (1), where the transport system was derepressed by growth on amino acids but was repressed by growth in the presence of a high ammonium ion concentration or on urea, and with Penicillium chrysogenum (5), where the transport system developed during nitrogen starvation. Stevenson and Silver studied methylamine uptake by scherichia coli (13) and found that it is also energy dependent. This organism exhibits two systems, with ph optima of 7 and 9, respectively. All of the above transport systems were strongly inhibited by ammonium ions. We report here the results of experiments on the transport of methylamine by the organism Pseudomonas sp. MA. This organism grows on methylamine as a sole source of carbon, nitrogen, and energy, and its pathway for CQ metabolism is fairly well established. We wanted to deternine whether a unique transport system 786 for methylamine existed in this organism and to examine the characteristics of the system. Methylamine is converted in this organism to ammonia and N-methylglutamate (12), which is then oxidized to glutamate and formaldehyde (6). Formaldehyde is further metabolized by the isocitrate lyase-positive variant of the serine pathway (1, 3). MATRIALS AND MTHODS The growth and maintenance of the organism have been described previously (2, 12). Cells were harvested by centrifugation at a cell density corresponding to an optical density at 54 nm of.8 to.9 (1-mm cuvettes in a Bausch & Lomb Spectronic 2 spectrometer). They were then washed three times with cold.2 M phosphate buffer, ph 7., and used immediately. Transport assay. Fresh cells were suspended to produce a cell density of.2 to.3, as described above, in the growth medium minus methylamine and incubated in a shaking water bath at 32C. [14C]methylamine (Amersham Corp.) was then added to samples of this suspension to produce final volumes of 5 ml. The final specific radioactivity of methylamine was in the range of 6 x 14 to 3 x 15 dpm/,umol. At appropriate intervals, usually 5 min,.5-mil samples were withdrawn and filtered through 25-mm polycarbonate membrane filters (Bio-Rad Laboratories) having a pore size of.45,um. The filtered cells were then washed at once with 1 ml of water at 2 C. The washed filters plus cells were then placed in scintillation vials, covered with 5 ml of scintillation fluid, and counted. Methylamine uptake was linear for at least 3 min. The dry weights of cells used in the transport assay were determined from a standard curve relating the dry weight of cells per milliliter to an optical density at 54 nm (1-mm cuvettes). Uptake rates are expressed as nanomoles of methylamine per minute per milligram (dry weight) of cells.

VOL. 142, 198 Radioactive counting. The samples were counted by the liquid scintillation method with a Beckman LS23 instrument. The scintillation fluid contained 3.

2 VOL. 142, 198 Radioactive counting. The samples were counted by the liquid scintillation method with a Beckman LS23 instrument. The scintillation fluid contained 3.5 g of 2,5-diphenyloxazole, 25 mg of 1,4-bis-(5-phenyloxazole)benzene, 5 g of naphthalene, and p-dioxane to a total volume of 5 ml. Chemicals. [14C]methylamine was diluted with unlabeled methylamine to the appropriate specific activities. Scintillation reagent chemicals were obtained from Amersham Corp. and were of scintillation grade. Sodium azide, carbonyl cyanide-m-chlorophenylhydrazone, and amino acids were obtained from Sigma Chemical Co. Amines and amides were obtained from Aldrich Chemical Co. Other common chemicals were obtained from commercial suppliers and were of reagent grade. MTHYLAMIN TRANSPORT IN PSUDOMONAS 787 RSULTS Temperature dependence. Suspensions of fresh cells in growth medium less methylamine were incubated with shaking in a water bath regulated at the required temperature until the cell suspension reached that temperature (usually within 15 min). At that time, the assay was started by the rapid addition of [1"C]methylamine solution that had also been equilibrated at the required temperature. The assay was then continued in the manner previously described. Methylamine transport was temperature dependent, with the optimum temperature occurring between 3 and 35C. Optimum ph. Assays were conducted with cells suspended in medium adjusted to the desired ph. The assays were started by the addition of ["4C]methylamine solution that had also been adjusted to the same ph. These experiments revealed that the system showed a ph optimum of 7.2 and that the rates dropped off sharply on either side of this value. Kinetic constants for methylamine transport. Uptake kinetics for methylamine varied slightly from culture to culture even though the cells were grown and harvested under identical conditions. However, Michaelis-Menten kinetics were followed, with the Km value for methylamine uptake in the range of 1 to 3 mm and the Vn. between 9 and 12 nrmol/min per mg (dry weight) of cells (Fig. 1). Uptake was also studied with methylamine concentrations in the micromolar range. These methylamine solutions had specific radioactivities in the 17-dpm/,umol range to detect entry into cells at low concentrations. Some uptake was observed under these conditions; however, the uptake rates were very low, and no evidence of a high-affinity transport system could be obtained. ffect of other carbon sources and ammonia. Pseudomonas sp. MA grows well on other carbon sources, such as glucose or succinate, when ammonium chloride is used as a nitrogen source. It is known that such compounds exert a catabolite repression effect on the enzymes of methylamine metabolism in this organism (2). Therefore, it was of interest to determine whether they also affected the transport of methylamine into methylamine-grown cells of the organism. Table 1 shows that no large inhibitory or stimulatory effect was observed when glucose or succinate was present in the assay medium. Only a weak inhibition was observed with ammonium chloride; succinate, together with ammonium chloride, likewise showed very little inhibition. This finding was in direct contrast to all previous work on methylamine transport, in which ammonia was a potent inhibitor of uptake. Methanol, which is not a growth substrate for the organism but is a Cl compound, also had no effect on methylamine uptake. C4 >1 [IMTHYLAMIN] 1MM- FIG. 1. Kinetics of uptake of methylamine and inhibition by ethylamine. Assay conditions were as described in the text. Symbols:, uptake of methylamine; A, uptake of methylamine in the presence of 5 mm ethylamine. TABL 1. ffects of alternative carbon and nitrogen growth sources on methylamine uptake Compound tested (5 Relative uptake mm) of methylamine Control... (1) NH4Cl... 9 Succinate Succinate + NH4Cl Glucose Methanol... 1 a Cells were grown on methylamine, and the substances to be tested were included in the transport assay medium at the indicated concentration. [14C]- methylamine was present at 1 mm, and standard assay conditions were used.

788 BLLION, ALI KHAN, AND ROMANO ffects of amines, amino acids, and amides. Table 2 shows the effects of these nitrogenous compounds on methylamine uptake.

3 788 BLLION, ALI KHAN, AND ROMANO ffects of amines, amino acids, and amides. Table 2 shows the effects of these nitrogenous compounds on methylamine uptake. The greatest inhibition was observed with alkylamines in the inverse order of chain length. The inhibition of ethylamine was studied further and shown to be competitive, with a Ki of 2.5 mm (Fig. 1). None of the amino acids and amides tested showed any large degree of inhibition. This was in contrast to previous studies on methylamine uptake, in which several of the amino acids were strong inhibitors (11). ffects of respiratory chain inhibitors and uncouplers. Azide, cyanide, and carbonyl cyanide-m-chlorophenylhydrazone completely abolished the uptake of methylamine when they were introduced into the transport assays (Fig. 2). This was the case whether the inhibitors were present at the beginning of the assay or whether they were introduced after transport had commenced. These results suggest that the transport of methylamine is coupled to energy production within the cell membrane. Although these inhibitors caused immediate cessation of methylamine transport, they did not cause the efflux of methylamine from the cells. ffects of ionic strength. The effects of ionic strength on methylamine uptake were studied by using increasing concentrations of potassium chloride in the standard transport assay. The transport rate was slightly decreased (15%) when the KCI concentration was increased to 1 mm, but a further fivefold increase in the KCI concentration produced only a further 3% decline in the transport rate. Thus, methylamine TABL 2. ffects of amino acids, amines, and amides on methylamine transportr Relative uptake of methylamine at following Compound added concn: 1 mm 5 mm None (control) (1) (1) L-Alanine 8 65 L-Arginine 7 5 L-Asparagine 8 68 L-Glutamine Glycine L-Histidine thylamine n-propylamine n-butylamine Benzylamine 67 4 Dimethylamine Trimethylamine Formamide Acetamide 'xperimental conditions were as indicated in Table 1, footnote a. wo YC _. = 2 U et w.-. i c n J. BACTRIOL TIM (min) FIG. 2. ffects of respiratory chain inhibitors on methylamine uptake. Transport was started with the addition of radioactive methylamine; after 1 min, 5- ml samples were removed and placed in flasks containing the inhibitors, and the incubation was continued. Symbols:, control; O, 1-3 M cyanide; A, 1-3 M azide; V, 1-' M carbonyl cyanide-m-chlorophenylhydrazone. transport is essentially unaffected by total ionic strength. Osmotic shock treatment. When cells were treated with the osmotic shock procedure of Neu and Heppel (9), transport of methylamine was reduced 5%. The same treatment, however, did not reduce the rate of transport of glycine in this organism (Fig. 3). Inducibility of the transport system. Cells of Pseudomonas sp. MA showed no transport of methylamine after growth on succinate plus ammonium chloride. However, cells grown on succinate but with methylamine as the nitrogen source were able to transport methylamine, albeit at a reduced rate (Table 3). When cells grown on succinate plus ammonium chloride were incubated in methylamine medium, transport of methylamine began after a period of 2 h (Fig. 4). DISCUSSION Previous studies of methylamine transport in microorganisms have actually been studies of ammonium transport systems, where methylamine was used as an analog of ammonia because of its availability labeled with "4C. Thus, in such previous work, methylamine was being transported as a nitrogen source, using the ammonium transport system of the organism concerned. Hence, it was not unreasonable to find that such transport was inhibited by ammonia and amino acids and that, in some organisms, methylamine uptake was dependent on the nature of the prior nitrogeneous growth source. In contrast to these previous findings, we found that ammonia was not an effective inhibitor of methylamine transport in Pseudomonas sp. MA. This finding indicates that methylamine was not being transported via an ammonium.7

4 VOL. 142, 198 (14 x 5 ~~~~~~~ LU 4c ~~~~~~ ~~~~TM (mn FIG. 3. ffect of osmotic shock on methylamine uptake. A culture ofcells was divided into two halves. One half was subjected to the osmotic shock procedure. Uptake assays were then performed on both shocked and unshocked portions. Symbols: O, methylamine uptake in shocked cells; *, methylamine uptake in unshocked cells;,1 glycine uptake in shocked cells;,g glycine uptake in unshocked cells. TABL 3. ffect ofgrowth conditions on methylamine uptakea Relative up- Growth condition take of methylamine Methylamine (1 mm) 1 Succinate (25 mm) + NH4Cl (1 mm) Succinate (25 mm) + 52 methylamine (1 mm) a Cells of Pseudomonas sp. MA were grown on the compounds listed, and the cells were collected and washed. Standard transport assays were then conducted on these cells with methylamine at 1 mm. Rates are expressed as a percentage of the rate observed with methylamine-grown cells. transport system. Also, the transport system was found only in cells grown on methylamine, and no methylamine transport was detectable in cells grown on succinate and ammonia. The transport system was induced by methylamine in cells previously grown on succinate and ammonia in about 2 h. This induction time corresponded well with the induction time of 22 to 24 h for the carbon-assimilatory enzymes under similar conditions (2). Although succinate repressed the transport system, neither it nor glucose inhibited methylamine transport. This means that induced cells will take up methylamine even in the presence MTHYLAMIN TRANSPORT IN PSUDOMONAS 789 of a preferred carbon source. The lack of inhibition by methanol is interesting in that it reveals that the transport system is not merely a methyl group uptake mechanism. The specific methylamine transport system of Pseudomonas sp. MA showed very little inhibition by amino acids or amides. However, it was competitively inhibited by short-chain alkylamines, indicating that perhaps the system is a short-chain alkylamine transport system. The transport is apparently driven by an energized membrane, as evidenced by the drastic inhibition observed by phosphorylation uncouplers, and the transport proteins involved were disruptible by osmotic shock, indicating their presence in the periplasn. Previous pulse-labeling studies have demonstrated that methylamine is fixed into various N-methylated amino acid derivatives very rapidly when incubated with methylamine-grown cells of Pseudomonas sp. MA (7, 8, 12). Accordingly, no efflux of methylamine could be ob- charged with ["4C]methyla- served when cells mine were exposed to alkylamines and amino acids. The Km of 1 to 3 mm observed for methylamine uptake was much higher than that reported in previous studies, in which values ranged from 1' M for P. chrysogenum to 2 x 1-' M for. coli and S. cerevisiae. Since the cells were grown in 1 mm methylamine medium, perhaps there was not the need for a highaffinity transport system. In contrast to this, however, the V,, of 9 to 12 nmol/min per mg (dry weight) of cells was 5- to 1-fold higher than that in previous studies. The organism can grow with methylamine as a nitrogen source when succinate is provided as a preferred carbon source. The cells transport 3 Co 2 1 v L--A-A--ȯ TIM((hours) FIG. 4. Induction of methylamine transport. Cells grown on succinate-nh4ci were washed and suspended in methylami.e medium containing [14C]- methylamine at 32C. Samples were removed, filtered, and counted as described in the text.

79 BLLION, ALI KHAN, AND ROMANO methylamine at a much lower rate under these conditions, and it is not yet certain whether the cells utilize the same transport system for the transport of methylamine

5 79 BLLION, ALI KHAN, AND ROMANO methylamine at a much lower rate under these conditions, and it is not yet certain whether the cells utilize the same transport system for the transport of methylamine under these conditions as they do when transporting methylamine as a carbon source. ACKNOWLDGMNTS This work was supported by grant Y-488 from the Robert A. Welch Foundation, Houston, Tex., and by The University of Texas at Arlington Organized Research Fund. M.J.R. was supported by URPP grant SPI from the National Science Foundation. We thank Robert Willcott for assistance with some of the experiments. LITRATUR CIMD 1. Bellion,., and L. B. Hersh Methylamine metabolism in a Pseudomonas species. Arch. Biochem. Biophys. 162: Bellion,., and Y. S. Kim Catabolite repression of isocitrate lyase in methylamine-grown Pseudomonas MA. ffect of carbon and nitrogen sources. Biochim. Biophys. Acta 541: Bellion,., and J. Woodson Two distinct isocitrate lyases from a Pseudomonas species. J. Bacteriol. 122: Colby, J., H. Dalton, and R. Whittenbury Biological and biochemical aspects of microbial growth on C, compounds. Annu. Rev. Microbiol. 33: J. BACTRIOL. 5. Hackette, S. L., G.. Syke, C. Burton, and I. H. Segel Characterization of an ammonium transport system in filamentous fungi with methylammonium-'4c as the substrate. J. Biol. Chem. 245: Hersh, L. B., J. A. Peterson, and A. A. Thompson An N-methylglutamate dehydrogenase from Pseudomonas MA. Arch. Biochem. Biophys. 145: Hersh, L. B., L. Tsai, and. R. Stadtman The enzymatic synthesis of 5-hydroxy-N-methylpyroglutamic acid. J. Biol. Chem. 244: Kung, H. F., and C. Wagner y-glutamylmethylamide. A new intermediate in the metabolism of methylamine. J. Biol. Chem. 244: Neu, H. C., and L. A. Heppel The release of enzymes from scherichia coli by osmotic shock and during the formation of spheroplasts. J. Biol. Chem. 24: Pateman, J. A.,. Dunn, J. R. Kinghorn, and. C. Forbes The transport of ammonium and methylammonium in wild type and mutant cells ofaspergillus nidulans. Mol. Gen. Genet. 133: Roon, R. J., H. L. van, P. Dunlop, and F. L. Larimore Methylamine and ammonia transport in Saccharomyces cerevisiae. J. Bacteriol. 122: Shaw, W. V., L. Tsai, and. R. Stadtman The enzymatic synthesis of N-methylglutamic acid. J. Biol. Chem. 241: Stevenson, R., and S. Silver Methylammonium uptake by scherichia coli: evidence for a bacterial NH4' transport system. Biochem. Biophys. Res. Commun. 75: Downloaded from on January 19, 219 by guest

Saccharomyces cerevisiae

JOURNAL OF BACTRIOLOGY, Oct. 1975, p. 325-331 Copyright 0 1975 American Society for Microbiology Vol. 124, Ng. 1 Printed in U.S.A. Inhibition of Amino Acid Transport by Ammonium Ion in Saccharomyces cerevisiae