WMINIMUMIHIHIWHIHIMWIWWI (ID—800 Date m mw1m“mumluuuwmgggxw 3 1293 100 ,_.-l A LIBRARY Michigan State University This is to certify that the thesis entitled TRANSPORT OF 2-METHYLALANINE IN AZOTOBACTER VINELANDII presented by Allan E. Kaufman has been accepted towards fulfillment of the requirements for M . S . degree in microbiology Wye W Major professor fly Novenber 8 , 1979 ovmumz FINES ARE 25¢ PER DAY _ PER ITEM Return to book drop to remove this checkout from your record. TRANSPORT OF 2-METHYLALANINE IN AZOTOBACTER VINELANDII BY Allan E. Kaufman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1979 ABSTRACT TRANSPORT OF 2-METHYLALANINE IN AZOTOBACTER VINELANDII. by Allan E. Kaufman The growth of Azotobacter vinelandii is impeded by 2-methyla1anine (MA) when cultures are growing on glucose, but not when they are growing on fructose. Cells growing on fructose and cells resistant to MA take up less 14 C-labeled MA than do cells growing on glucose. The wide range of structurally dissimilar compounds that can counteract the effect of MA suggests that MA enters cells by a nonspecific transport mechanism that somehow depends upon glucose. ACKNOWLEDGEMENTS I wish to thank Dr. Harold Sadoff whose patience, guidance and general good will enabled me to complete this work. Thanks are also due to Dr. William Colonna and Janet R. Nelsen who both contributed intellectual and emotional sustenance when they were needed. ii TABLE OF CONTENTS Page LIST OF FIGURES. . . . . . . . . . . . . . . . . .iv LIST OF TABLES . . . . . . . . . . . . . . . . . . V TRANSPORT IN AZOTOBACTER VINELANDII: A REVIEW Introduction . Transport Mechanisms . Diffusion Facilitated Diffusion. Active Transport . Group Translocation. Methods of Investigating Transport Processes Whole Cells. Cytoplasmic Membrane Vesicles. Membrane Binding Proteins. \DmO‘UIU'IUOUJNNNH Transport of Carboxylic Acids. '_I N Transport of Ions. |'—‘ U1 Transport of Glucose . H Ch References TRANSPORT OF 2-METHYLALANINE IN AZOTOBACTER VINELANDII: A PAPER. Introduction . . . . . . . . . . . . . . . . .21 Materials and Methods. . . . . . . . . . . . .22 Results . . . . . . . . . . . . . . . . . . .25 Discussion . . . . . . . . . . . . . . . . . .28 References . . . . . . . . . . . . . . . . . .40 iii LIST OF FIGURES Figure Page 1. Effect of MA depending on the age of the culture. . . . . 31 2. Growth of cells (A) and uptake of 14C labeled MA(B) in wild type cells growing on glucose, glucose + MA, fructose or fructose + MA and in mar-25 cells growing on glucose or on glucose + MA . . . . . . . . . . . . . . . . 33 3. Growth of wild type cells on media containing glucose, glucose + MA, glucose + L-serine, glucose + L-serine + MA, glucose + isocitrate, glucose + isocitrate + MA, isocitrate and L- serine. . . . . . . . . . . . . . . . . . . . 35 4. Growth of wild type cells growing on media con— taining: glucose, glucose + isobutyric acid, fructose, fructose + isobutyric acid, glucose + a-hydroxyisobutyric acid, and fructose + a-hydroxyisobutyric acid.. . . . . . . . . . 37 iv Table LIST OF TABLES Iniiial rate of uptake of 2-deoxy-D- - 4C] - glucose by washed suspensions o A. vinelandii in the presence of 2- methylalanine. Ability of compounds to counteract the effect of 2-methylalanine on A, vinelandii growing on glucose . Page . 38 . 39 I. TRANSPORT IN AZOTOBACTER VINELANDII‘ A Review INTRODUCTION Azobacter vinelandii is a large, gramrnegative rod shaped bacterium that is highly aerobic and found living freely in soil and water (11). It has been the object of scientific interest for several reasons, in- cluding: a. its ability to convert atmospheric nitrogen to ammonia (40); b. its unusual respiratory physiology (several uncommon respiratory proteins and electron carriers are present in significant amounts (40)); c. its unusual genetic properties and a recently discovered system for genetic exchange (27). Relatively litten attention has been focused on how A. vinelandii transports substances from its environment across the cellular envelope and into the cytoplasm. The purpose of this review is to summarize what is known about transport processes in A. vinelandii and, where appro- priate, supplement this information with studies which have been done with other organisms. TRANSPORT MECHANISMS Diffusion Transport by diffusion is the result of random molecular motion and occurs without the intervention of specific membrane components (19). A passive pro- cess, diffusion is the result of concentration differ- ences or gradients and generally is subject to such effects as solvent drag, membrane charge and hydropho- bicity of the membrane (20). The membrane of A. vine- landii, however is thought to be generally impermeable to solutes (35) and as such diffusion is not a major mechanism by which substrates gain entry into the cell. Facilitated Diffusion A special case of diffusion, facilitated diffusion occurs when specific membrane components, or "carriers", are involved in transporting substances down concentration gradients (19). As in passive diffusion, the transported substance can not move against concentration gradients. No metabolic energy is expended during transport by this process. Active Transport By expending metabolic energy, a cell can accomplish the work of concentrating within thevolume enclosed by its membrane substances in low abundance outside the cell. Active transport is the general term applied to a process that is initiated by the formation of a complex between a membrane or cell surface componenti and an external sub- strate; the process is completed when the substrate has been translocated into the cell, the "carrier" regenerated at the cell surface, and energy utilized in the process (16). A. vinelandii appears to depend on such processes to move most substances into or out of the cell (35). As is the case in many other bacterial species (19,20), the energy component of active transport resides in the proton gradient established across the cell membrane by respira- tion. "Symport" and ”antiport" mechanisms couple the entry of protons to the entry of external substrates or exodus of substances from inside the cell,respectively. Group Translocation Although this mechanism for transport appears to be absent from A. vinelandii (34), its prevalence in so many other organisms warrants brief mention. In this process a covalent change is exerted upon the transported molecule such that the reaction itself is simultaneous with its entry into the cell (19,20). The best understood example of this mechanism is the hexose:phosphenol- pyruvate (PEP) phosphotransferase system (13). Here PEP is used to phosphorylate glucose while also trans- porting it into the cell. Interestingly, both membrane- bound (intrinsic) as well as soluble (extrinsic) proteins participate in the reaction (13). METHODS OF INVESTIGATING TRANSPORT PROCESSES Whole Cells Intact cells are at first glance the choice system for study because the convenience of working with them is combined with the advantage of being assured that the transport system(s) under investigation has not been altered or damaged by fractionation procedures. However the redundancy and complexity of individual trans- port systems as well as the rapidity with which substrates are assimilated or transformed by cells,contribute to the difficulties of investigators using such systems (37). Some techniques used to counter such problems have been: the use of starved cells, inhibitors and non-metabolizable substrates; the use of mutants blocked in transport; the use of rapid spectrophotometric assays or assays using radioactive materials; and the use of kinetic techniques such as stopped flow dialysis (15,35). Early studies of transport in A. vinelandii relied on indirect indications of transport in whole cells; uptake of oxygen due to respiration of substrates entering the cell (31); changes in packed cell volume when starved cells were exposed to various permeable or impermeable substances (21) and changes in light scattering when spheroplasts were placed in solutions containing various electrolytes (38). Cytoplasmic Membrane Vesicles The development of the technique for forming small vesicles composed of cytoplasmic membrane and essentially devoid of cytoplasmic constituents revolutionized the study of transport phenomena (l6). Vesicles with "right side out" orientation are produced when osmotically sensitized cells are lysed with EDTA, DNAase, and RNAase, then sub- jected to extensive homogenization, washing and differential centrifugation (19). Such vesicles made from E. 99;; membranes, when provided with an energy source such as lactate, will take up a wide variety of substrates in- cluding galactose, B-galactosides, arabinose, peptides, nucleosides and dicarboxylic acids (20). Vesicles with reversed or "inside-out" topology have been obtained by the use of various non-penetrating buffers and alternative techniques for breaking cells, especially the French pressure cell (15). Inverted vesicles allow the study ii uptake of processes usually occurring as secretion or extrusion in whole cells. This is especially useful in the study of various membrane bound ATPases (5). In addition to confirming the "basic assumption of the inherent impermeability of lipid bilayers to hydro- philic molecules" (39), experiments with vesicles have contributed greatly to an understanding of transport at a fundamental molecular level. The basic mode of action for many active transport systems is now thought to be "symport", the transport of a substrate linked to the movement of protons or sodium ions down a concentration gradient. Although very few intact transport systems have been isolated and characterized £2 21359 (16), it now seems unlikely that "carriers" shuttle across membranes. Rather transport proteins somehow span the lipid bilayer and function by some sort of conformational change or, possibly, by the association of oligomers which form channels or pores in the bilayer (39). There are, however, questions raised by the work with membrane vesicles. For example, there seems to be no correlation between the oxidation of various energy substrates and their effectiveness in transport processes (20). In vesicles made from A. vinelandii membranes, lactate, malate and NADH are all oxidized at about the same rate. However, lactate serves as a poor energy source for supporting glucose uptake and as a good source for Ca++ uptake. Malate yields the opposite results. NADH, which is theoretically the best electron source, will not drive either process (1,8). Clearly there exist interactions between transport proteins, membrane dehydrogenases and electron transport proteins that are still poorly understood (l6). Membrane Binding Proteins Oxender has shown that when gram-negative bacteria are subjected to mild proteolytic treatment or osmotic shock in the presence of EDTA, a group of low molecular weight proteins are removed from the periplasmic region of the cell envelope (26). Since cells so treated no longer trans- port various substrates across the cell membrane, these proteins are implicated as being involved in transport processes. Known as binding proteins, a number of these proteins have affinities for substrates not transported by membrane vesicle systems. A few substrates, especially amino acids, do interact with both types Of transport sys- tems. Such redundancy may be required to allow substrates to pass across the inner and outer membranes of the cell. Binding proteins themselves are not thought to cross the cell membrane. Rather they may serve to enhance the uptake of substances having extremely low external concentrations. Generally, the Km for binding protein systems is much lower than the Km for membrane vesicle systems (39). Another major difference in the two kinds of transport is that vesicles depend on proton gradients to "drive" transport whereas binding protein systems appear to use the hydrolysis of ATP (16). To date no studies involving binding proteins in A. vinelandii have been published. TRANSPORT OF CARBOXYLIC ACIDS The earliest work concerning transport in A. vinelandii arose from attempts to measure the level of Krebs-cycle enzymes in cells growing on various media (31). It was observed that merely exposing a culture to a particular substance ("adaptation") did not insure that an appropriate "permease" system.would be synthesized and thereby allow the substance to enter the cell. By the judicious use of inhibitors of DNA, RNA and protein synthesis, it was shown that a culture fully "adapted" to malate or succinate had synthesized a specific "per- mease". In addition it was demonstrated that the pre- sence of glucose in low concentrations andllo_t_ serving as a carbon of energy source decreased the time required for the adaptation to occur. Subsequent investigations of transport of di-and tricarboxylic acids in A. vinelandii were carried out by P.W. Postma and his associates at the University of Amster- dam (29,32,33). By measuring the levels of specific enzymes,the presence of four separate translocation systems was inferred having specificities for'the following anions: 10 I. succinate, fumarate, malate, oxalacetate II. a-ketoglutarate III. citrate, isocitrate IV. malonate There was no cross specificity between groups although any of the group I acids would fully induce the trans- locator effective for the entire group (29). Pyruvate, lactate and acetate however, appeared to be transported regardless of which transport system was present and consequently it remains unclear by what means these com- pounds traverse the cell membrane. Although no specific inhibitors of transport were used, it was believed that all transport processes observed were by energy dependent mechanisms since anoxia led to their immediate cessation (29). The role of glucose in accelerating transport of carboxylic acids was further investigated (32). At high concentrations of glucose (50-lOOmM) catabolite repression of the malate translocator was observed. However the enhancement of malate transport by low concentrations of glucose was not fully explained. This "glucose effect" could be accomplished, though with lesser efficiency, by replacing glucose with glutamate, casamino acids or 14c-1abe1ed a-ketoglutarate (33). The disappearance of glucose was the same regardless of whether or not induc- tion of the translocators for malate or succinate occurred. 11 It was concluded that glucose was not serving as an energy source in stimulating carboxylic acid transport (33). Induction of the malonate transport system simul- taneously induces the appearance of enzymes concerned with its metabolism; Malonyl-CoA synthetase and malonyl-CoA decarboxylase (33). Cyclic AMP (3'5'cAMP) can derepress the synthesis of carboxylic acid translocators (32). Hence the regulation of these systems appears similar to the classic operon model in E. 991E (24,28). Greater understanding of the molecular basis of transport exists in E. ggl;_than is the case in A. gigg- landii at present."Porin", an outer membrane protein of 36,500 daltons molecular weight, appears to be required for the energy dependent transport of some carboxylic acids in E. 39;; (6). Transport function is restored when this protein is added to cells blocked in transport by treatment with proteolytic enzymes or to transport mutants (7). It appears that multimeric aggregates of the protein form channels that allow carboxylic acid molecules to pass into the periplasmic space (24) . However the energy dependence as well as the manner in which specificity is invoked are not yet‘understood. TRANSPORT OF INORGANIC IONS Studies of how inorganic ions enter cells have not, until recently, been in the forefront of membrane research. However, there has been a gradual confirmation of an underlying belief that separate, specific mechanisms exist for every inorganic cation or anion required for growth (36). Early studies of A. vinelandii using starved, washed cells in various salt solutions suggested that the cell membrane is generally impermeable to ions (22, 23). As the external concentration of LiCl, NaCl or KCl increased, the packed cell volume decreased as water was drawn out of the cell, presumably due to increased osmotic pressure. It was concluded that ions could not pass the membrane. Slow entry, however, was observed when NH4C1, NaCl and CaCl2 were tested. When pulsed with an energy substrate, the cells swelled and consumed oxygen. Since these events could be prevented by addition of various inhibitors and especially uncouplers of oxi- dative phosphorylation, it was concluded that ion trans- port was an active, energy requiring, process (23). Further studies using A. vinelandii protoplasts , + demonstrated that the membrane 18 impermeable to K , Na+, NH: , 01', P04: and $04- in the absence of energy sources (38). However, in contrast to findings in whole 12 13 cells (29), protoplast membranes were permeable to potassium acetate but not ammonium acetate. Although a metabolic energy requirement was demonstrated, it was not clear whether transport was the result of a specific translocator or due to some kind of ion exchange process (30,38). Another study of NH: transport was done by Kleiner (21) who measured NHZ- from cells grown with various amounts of ammonia as well as various counter ions. He concluded that several ammonium.ion translocators exist because an adaptive process was required prior to ammonium entry when the anion in the medium was changed. Again, studies with inhibitors indicated that energy was required to maintain a one hundred-fold difference be- tween external and internal ammonium ion concentrations. Several studies of ion transport in membrane vesicles from A. vinelandii have been carried out by E.M. Barnes and his associates (4,5,8,10). Previously, a membrane- associated, trypsin-activated ATPase had been described in A. vinelandii (l8). Barnes' treatment of membrane vesicles with trypsin followed by the addition of lactate led to the specific, ATP dependent uptake of Ca++ (4,8). Since the vesicles were prepared with inverted orientation (5) it was concluded that A. vinelandii is similar to many H other bacteria in actively extruding Ca from the cytosol despite its requirement for Ca++ as a growth factor. 14 Sodium ions also appear to be expelled from the cell in a energy dependent process (10). However, in con- ++, the Na+ exodus is independent of ATP trast to Ca hydrolysis and requires a proton gradient. Kinetic studies of Na+ uptake by everted vesicles,as well as mea- surement of the appearance of Na+ in the medium surrounding normally oriented vesicles,suggest a classic antiport mechanism is at work exchanging one Na+ for each proton entering the cell (10). 3 Transport of Fe+ in A. vinelandii has been inferred due to its production of a green, fluorescent pigment in response to iron deficiency (12,14). Although little is known about iron transport in A. vinelandii.the mechanism of iron transport is better understood from work in other species, especially from studies of mutants blocked in various aspeCts of the process (25,36). E. 99;; cells produce enterochelin, an iron chelator, in response to low iron stress. Iron-chelator complexes are then actively transported into the cell where hydrolytic enzymes release the iron and degrade the carrier molecule. Although receptors for the iron-chelator complex have been iden- tified on the cell surface, the exact mode of transport through the membrane is unknown. TRANSPORT OF GLUCOSE Most of the information available about glucose uptake in A. vinelandii comes from experiments with membrane vesicles (l,2,3,9). Vesicles with normal orien- tation were shown to take up 14C-labeled glucose when provided with malate as an energy source but not with lactate. It appears that glucose uptake in A. vinelandii is coupled to a flavin-linked malate dehydrogenase in the membrane (1). Difference spectra of various membrane preparations indicated that a second site for glucose uptake exists proximal to ubiquinone in the respiratory chain (3). Both sites function by active processes, i.e. as glucose-proton symports (9). The glucose transport system in A. vinelandii appears to be inducible since vesicles from acetate grown cells take up less than 1% of the amount taken up by glucose grown cells (2). Although little is known about how glucose is transported through the membrane, a very high specificity for glucose can be demonstrated by the use of analogs. Hopefully,this might lead to ultimate isolation of at least the receptor component of this transport system (42). 15 REFERENCES Barnes, E.M., 1972. Respiration-coupled glucose transport in membrane vesicles from Azotobacter vinelandii. Arch. Biochem. Biophys. I52z795—799. Barnes, E.M., 1973. Multiple Sites for coupling of glucose transport to the respiratory chain of membrane vessicles from Azotobacter vinelandii. J. Biol. Chem. 248:8120-8124. Barnes, E.M., 1974. Glucose transport in membrane vesicles from Azotobacter vinelandii. Arch. Biochem. Biophys. 163:416-422. Barnes, E.M., 1974. Respiration-coupled Ca++ transport by membrane vesicles from.Azoto- bacter vinelandii. Fed. Proc. Amer. Soc. Exp . Biol. __33 : 1:21:56 . Barnes, E.M. and Bhallacharyya, P., 1977. Isolation of membrane vesicles with inverted topology by osmetic lysis of Azotobacter vinelandii spheroplasts. J. Supramolec. Struct. Ez333-344. Bauoil, P. and Nikaide, H., 1977. Pleiotropic transport mutants of Escherchia coli lack Porin, a major outer membrane protein. MEIec. Gen. Genet. 158:23-33. Bewlick, M.A. and Lo, T.C.Y., 1979. Dicarboxylic acid transport in EsCheriChia coli K12 Can. J. Biochem.§l:653466l. Bhattacharyya,P. and Barnes, E.M., 1976. ATP-depen- dent calcium transport in isolated membrane vesicles from Azotobacter vinelandii. J. Biol. Chem. EEE:5614-5619. Bhattacharyya, P., Shapiro, S. and Barnes, E.M., 1977. Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrane vesicles. J. Bacteriol. I29z756-762. l6 10. ll. 12. 13. 14. 15. 16. 17. l8. 19. 20. 17 Bhattacharyya, P. and Barnes, E.M., 1978. Proton- coupled sodium uptake by membrane vesicles from Azotobacter vinelandii. J. Biol. Chem. 253: Buchanan, R. and Gibbons, N. (eds.), 1974. Bergey's Manual of Determinative Bacteriology. Eighth Edition, p. 255. Williams and Wilkins Co., Baltimore, Md. Corbin, J.L. and Bulen, W.A., 1969. The isolation of 2,3-Dihydroxybenzoic acid and 2-N, 6-N-Di-(2,3-di- hydroxybenzoyl)-L-1ysine formed by iron-deficient Azotobacter vinelandii. Biochem. Ez757-762. Cordaro, C., 1976. Genetics of the Bacterial phosphoenol-pyruvate: glucose phosphotransferase system. Ann. Rev. Genetics 19:341-359. Fukasawa, K., Goto,M., Sasaki, K., Hirata, Y., and Sato, S., 1972. Structure of the yellow-green fluorescent peptide produced by iron-deficient Azotobacter Vinelandii Strain O. Tetrahed. EE: 5369-5365 Futai, Masamitsu, 1978. Experimental systems for the study of active transport in bacteria. '_I_1}_Rosen, B. ‘(ed.) Bacterial Transport, pp. 7-42. Marcel Dekker, Inc., New York. Harold, F., 1977. Membranes and energy translocation in bacteria. Current Topics in Bioenergetics 6:84-149. Hirata, H., Sone, N., Yoshida, M., and Kagawa, Y. 1977. Isolation of the alanine carrier from membranes of a thermophilic bacterium.and its reconstitution into vesicles capable of transport. J. Supamolec. Stuct. Ez77-84. Jurtshuk, P. and McEntire, J., 1975. Characterization studies on the membrane-bound adenosine triphos- phatase (ATPase) of AzotobaCter vinelandii. Can. J. Microbiol. 2I7I807:I8I4 Kaback, H.R., 1972. Transport across isolated bacterial ctyoplasmic membranes. Bioch. Bioph. Act. 265: 367-416. Kaback, H.R., 1974. Transport studies in bacterial membrane vesicles. Science 186:882-892. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 18 Kleiner, D., 1975. Ammonium.uptake by nitrogen fixing bacteria. Arch. Microbiol. 104:163-169. Knowles, C.J., and Smith, L., 1971. Effect of osmotic pressure of the medium on the volume of intact cells of Azotobacter vinelandii. Biochim. Biophys. Acta. _2_3_4_:144-152. Knowles, C.J., and Smith, L., 1971. The relationship between substrate-induced respiration and swelling in Azotobacter vinelandii. Biochim. Biophys. Acta. EEA:153-161. Lo, T.C.Y., 1979. The molecular mechanisms of sub- strate transport in gram-negative bacteria. Can. J. Biochem. 21:289-301. Meyer, J., and Hornsperger, J., 1978. Role of pyoverdinepf, the iron-binding fluorescent pigment of Pseudomonas fluorescens. J. Gen. Microbiol. 107: 329-33I. ' Oxender, D.L. and Quay, S., 1976. Isolation and characterization of membrane binding proteins. Methods in Membrane Biology 9:183-242. Page, W., and Sadoff, H.L., 1976. Physiological factors affecting transformation of AzOtobacter vinelandii. J. Bacteriol. 125:1080-1087. Pastan, I., and Adhya, S., 1976. Cyclic adenosine 5' monophosphate in Escherichia coli. Bact. Rev. 59:527-551. Postma, P.W., and Van Dam, K., 1971. The inducible transport of diland tri carboxylic acid anions across the membrane of AzotobaCter vinelandii. Biochim. Biophys. Acta. Z§2:515-527. Postma, P., Visser, A., and Van Dam, K., 1973. The movement of cations connected with metabolism in Azotobacter vinelandii. Biochim. Biophys. Acta. '298z34l-353. Repaske, R., Shroat, J., and Allman, D., 1960. Permeability of Azotobacter to succinate and malate. J. Bacteriol. 12:394-405. Reuser, A.J., and Postma, P.W., 1972. The induction of a C -dicarboxylic acid anion translocator in Azotobacter vinelandii. FEBS. Lett. El:145-l48. 33. 34. 35. 36. 37. 38. 39. 40. 19 Reuser, A.J., and Postma, P.W., 1977. The induction of translocators for di and tricarboxylic anions in Azotobacter vinelandii. Eur. J. Biochem..EE: 584-592. Romano, A., Eberhard, S.J., Dingle, S.L., and McDowell, T.D., 1970. Distribution of the phosphoenolpyru- vate: glucose phosphotransferase system in bacteria. J. Bacteriol._EQA:808-813. Silhary, T., Ferenci, T. and Boos, W., 1978. Sugar transport systems in Escherichia coli. In Rosen, B.(ed.) Bacterial Transport, pp. I27-I69. MarceI—Dekker, Inc., New York. Silver, 8., 1978. Transport of cations and anions, IE Rosen, B. (ed.) Bacterial Trasnport, pp. 221-324. Marcel Dekker, Inc., New York. Simoni, R.D., 1972. Macromolecular characterization of bacterial trasnport systems. In Fox, C. and Keith, A. (eds.), Membrane MolecuIEr Biology, pp. 289-332. Sinauer Assoc., Inc., Stamford, Conn. Visser, A., and Postma, P., 1973. Permeability of Azotobacter vinelandii to cations and anions. Bio- chim; *Biophys. Acta. 298:333-340. Wilson, D.B., 1978. Cellular transport mechanisms. Annu. Rev. Biochem. 31:9339965. Yates, M., and Jones, C.W. 1974. Respiration and nitro en fixation in Azotobacter. Advances in ‘Micro iol Physiology.ll:97-135. TRANSPORT OF 2-METHYLALANINE IN AZOTOBACTER VINELANDII by A. E. Kaufman and H. L. Sadoff Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 A paper to be submitted to the Journal of Bacteriology. INTRODUCTION When the non-metabolizable analog of alanine, 2-methylalanine (also known as a-amino isobutyric acid), is added to a culture of Azotobacter vinelandii, inhibition of growth is observed if glucose or maltose is the sole growth substrate in the medium (13). Providing a source of fixed nitrogen partially prevents 2-methylalanine (MA) induced inhibition, although MA and ammonia are thought to exert dissimilar effects upon the regulatory mechanism for nitrogen fixation (4). Inhibition of growth does not occur when MA is added to a culture growing on alternate substrates such as acetate, galactose, fructose or sucrose (13). The experiments described herein were undertaken to explain why the effects of 2-methyla1anine upon A. vinelandii are limited to cells growing on glucose. 21 MATERIALS AND METHODS Bacterial Strains and Culture Conditions A. vinelandii (ATCC 12837) was used throughout this study. A 2-methylalanine resistant strain (mar-25) was derived from the parental strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, according to the method of Sorger (16). Cell stocks were maintained on modified Burk nitrogen free medium (9) containing 0.8 mM MgSOa, 0.58 mM CaSOA, 18.0 uM FeSOA, 1.0 uM NaMoOa, 5.0 mM potassium phosphate buffer (pH7 .4) and 1‘7o glucose . Growth Studies Exponential phase cells were washed twice in glucose-free medium and suspended in 50 ml of medium containing an appropriate carbon source at a concentration of 50 mM. Cultures were incubated on a water shaker bath (New Brunswick Scientific, New Brunswick, N.J.) at 30°C, and monitored with Klett-Summerson Photo Colorimeter (Klett Mfg., New York, N.Y.) equipped with a #54 filter. When the cell density reached 50 Klett units, MA and/or other compounds were added to a final concentration of 22 23 20 mM (pH 7.0-7.4). One Klett unit represents a viable cell count of 2.2 x 106 cells/ml as determined by plating on Burk Nitrogen free medium. Chemicals Isobutyric acid, 2-methylalanine and a-hydro- xyisobutyric acid were obtained from Sigma Chem. Co. (St. Louis, Mo.). Radiolabeled 2-methy1alanine [§.- (3-14C) - amino isobutyric acid:]was obtained from New England Nuclear Co. (Boston, MA); 2-deoxy-D- E1-14C] glucose was obtained from Amersham Corp. (Arlington Heights, 111.). All other materials were obtained from commercial sources and were of analytic or reagent quality. 14 Uptake of C MA Radioactive MA (0.1 uC/umole(was added to 10 ml cultures (final concentration: SuCi/ml) growing on glucose or fructose upon reaching a turbidity of 50 Klett Units. Samples (0.25 ml.) were removed in duplicate and filtered on a multisample vacuum manifold (Millipore Co., Bedford, MA) through glass microfibre filters (Whatman Ltd., Mailstrom, England). The filters were immediately rinsed with 5.0 m1 of iced, glucose-free medium, dried for 2-4 hours at 80°C and placed in scintillation fluid. Radioactivity was measured with a Packard 3000 Tricarb liquid scintillation 24 spectrometer. Protein was determined by the method of Lowry et a1. (8) with bovine serum albumin used as a standard. 25 RESULTS Inhibition of Growth The effect of MA Upon growing cells was most pro- nounced when it was introduced early in the exponential phase of growth (Fig. 1). Inhibition was marked when MA was added to cultures having a turbidity of 50 Klett units. The response of older cultures was not as great or un- detectable. In addition, the effect of MA appeared to be reversible. Exponential phase cultures (turbidity of 50 Klett units) were exposed to MA for 1,2,4 or 6 hours. When washed twice and suspended in MA-free medium at their original turbidities, these cultures grew, without noticeable lag, to final turbidities equivalent to control cultures unexposed to MA (data not shown). In order to ascertain whether MA interferes with the transport of glucose into cells, suspensions of glucose- grown cells were exposed to 2-deoxy-D-(l-14 C) glucose in the presence of MA (Table l). The initial rate of uptake increased with exposure to greater amounts of MA. The data were in a range similar to those of Barnes (2) who measured the uptake of glucose and glucose analogs by mem- brane vesicles prepared form A. vinelandii, strain 0. 26 Uptake of radiolabeled MA. Inhibition of growth was observed when MA was added to cultures of wild type cells growing on glucose (Fig. 2A); however similar effects were absent in cultures of mar-25 or wild type cells growing on fructose. After 24 hours the inhibited (glucose + MA) cultures contained three times the levels of 14 C-MA observed in the growing cultures (Fig. 2B). In addition the amount of radioactivity associated with the non-growing cells increased nearly eight-fold over the course of the experiment; the increase in radioactivity in the other cultures was only about two- fold. Using a t-test (14) the regression slopes of the uptake of radioactivity were compared. A nonsignificant difference (p<.001) was found between them. Therefore it is reasonable to assume that the uptake of MA in mar-25 and the wild type cells growing on fructose were equal. Prevention of MA effects Structurally similar amino acids, such as alanine or serine have been shown to compete with MA for transport into cells in several bacterial species (3,5,14,17). Conse- quently, these as well as other compounds were tested for ability to block the inhibitory effect of MA on growth. Serine was capable of counteracting MA while isocitrate 27 was not (Fig. 3). Neither serine nor isocitrate is capable of supporting growth of A. vinelandii independently. Additional substances that were tested are listed in Table 2. Effects of Isobutyric Acid and a-Hydroxyisobutyric Acid In order to determine whether the inhibitory proper- ties of MA might be attributed to any particular chemical group on the molecule, isobutyric acid and a-hydroxyisobu- tyric acid were added to cultures growing on glucose or fructose (Fig. 4). Isobutyric acid did appear to tran- siently affect the growth of only the culture growing on glucose. In contrast a-hydroxyisobutyric acid immediately stopped the growth of both cultures. Microsc0pic examina- tion of the culture revealed non-motile, refractile cells in large aggregates. When the cells were washed and plated on fresh medium not containing a-hydroxyisobutyric acid no growth was observed. DISCUSSION The cellular envelope of A. vinelandii has been shown to be ’ selectively impermeable . Ions such as Ca4+, K+, Cl' and NH4' (6,7,18) as well as carboxylic acids (10) enter the cell only by means of highly specific, energy dependent transport systems. In the case of glucose, an inducible, malate dehydrogenase-linked, carrier-mediated system appears to be responsible for moving glucose across the cell membrane (6,16). To date no group translocation (phosphotransferase) system has been shown to operate in A. vinelandii (17). The observation that the inhibitory action of MA is limited to early exponential phase cells metabolizing glucose suggested that glucose transport might be the site of MA interference with growth. However, our finding that MA enhanced Z-deoxyglucose uptake is not consistent with such an hypothesis. The significant increase in the amount of MA entering cells exposed to glucose is reminiscent of previous findings regarding carboxylic acid translocation systems in A. vinelandii (10,11). It was found that the presence of glucose facilitated the entry of succinate and malate prior to induction of the specific transport systems. 28 29 Our experiments also seem to indicate that glucose in- creases the permeability of A. vinelandii, although it is not clear whether MA is co-transported with glucose or is interacting withaapolyfunctional transport component at the cell membrane. Support for the latter notion comes fromtfluawide range of compounds that counteract the effect of MA. This is in contrast to the behavior of transport systems specific for alanine and serine, which are unaffected by molecules of disshmilar structure (5,15,17). The action of isobutyric acid upon A. vinelandii is similar to that of MA in that both affect only glucose growing cells. However, the brevity of its effect on growth as well as the failure of MA resistant cells to resist isobutyric acid (data not shown) suggests that isobutyric acid is exerting a different effect than MA. Similarly, a-hydroxyisobutyric acid appears to be acting in a different manner than either MA or isobutyric acid in immediately killing both fructose and glucose grown cells. Since the cells tend to agglutinate as if their surface had become "sticky" the toxic effect of a-hydroxyiso- butyrate may be due to some chemical action upon the cell surface rather than the result of entry into the cell. 30 Figure 1. Effect of MA depending on the age of the culture. Arrows and associated symbols indicate turbidity at the time of addition: A - 50 Klett units; 0- 100 Klett units; -- 150 Klett units; .- 200 Klett units; C]- control (no MA added). 31 r6 mi 0 A _ o A won a o I. I I II 1- 1 u 0 mm mm “w “w ”:2: :3: (hours) TIME 32 Figure 2. Growth of cells (A) and uptake of 14C-labled MA(B) in wild type cells growing on glucose (A), glucose + MA (‘), fructose (O), or fructose + MA (.) and in mar-25 cells growing on glucose (D) or on glucose + MA(I'). 33 4ooqt 200-- 0 5 100 -- 9:2: :3: 5 2 n _ 2 3 24 -- 16-. 5532.. :3 22.; .9: \ES 52:: <2 maz 34 Figure 3. Growth of wild type cells on media containing: glucose (A) , glucose + MA (A), glucose + L-serine (D) , glucose + L-serine + MA (I), glucose + isocitrate (O) , glucose + isocitrate + MA (.), isocitrate (O) and L-serine (.). KLETT UNITS 35 8 160- + 80- 40 T :Eo-ooo—o—o—e 4| 43- '" - : : 41 + 0 8 16 24 TIME (hours) 36 Figure 4. Growth of wild type cells on media containing: glucose (1:), glucose + isobutyric acid (‘), fructose (D) , fructose + isobutyric acid (I) , glucose + a-hydroxyisobutyric acid (.), and fructose + a-hydroxyisobutyric acid ((3). KlETT UNITS 37 3204 160 .. . . a» .. -. 5T 80 6“”.7‘ 40 TIME (hours) 38 Table 1 Initial rate of uptake of 2-deoxy-D-[1-14Q]glucose by washed suspensions of A. vinelandii in the presence of 2-methy1alanine.a 2-deoxyglucose: (mM) 2-methy1a1anine: (mm 4 8 l6 0 .021. 005 .021. 009 .0945. 002 5 -b .071. 001 151.001 10 - .11: 040 .1745. 006 20 - .101. 013 .271. 242 810 ml of washed cells in glucose-free Burk medium were incubated for 5 minutes in a shaker bath at 30°C with indicated amounts of MA and pulsed with indicated amounts of 2-deoxyg1ucose (0.15uCi/umole). values represent initial uptake rates at each concentration (3 replicates) and have units of m moles/min./mg whole cell protein. bnot determined 39 Table 2 Ability of compounds to counteract the effect of 2-methyl- alanine on A, vinelandii growing on glucose. Compound ability to serve as ability to prevent sole growth substrate MA inhibition acetate + ammonium chloride - alanine - arginine aspartate benzoate butyrate citrate gluconate isocitrate a-ketoglutarate lactate lysine malate oxalacetate proline pyruvate serine succinate .+.+..++++.. + u ++++++++II+++I++HE+ + , a: = partial prevention 10. 40 REFERENCES Barnes, E.M., 1973. Multiple sites for coupling of glucose transport to the respiratory chain of membrane vesicles from Azotobacter vinelandii J. Biol. Chem. EAEz8120-8124. Barnes, E.M., 1974. Glucose transport in membrane vesicles from Azotobacter vinelandii. Arch. Bio- chem. Biophys. I63z4I6-422. Edmondson, J.W., Lumeng, L., and Li, T., 1979. Comparative Studies of alanine andcx-aminoiso- butyric acid uptake by freshly isolated rat liver cells. J. Biol. Chem. 254:1653-1658. Gordon, J. and Brill, W.J., 1972. Mutants that produce nitrogenase in the presence of ammonia. Proc. Nat1.Acad. Sci.U.S.A. 92:3501-3503. 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