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The development of membrane associated functions in Micrococcaceae Groot Obbink, Derk Jan 1973

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THE DEVELOPMENT OF MEMBRANE ASSOCIATED FUNCTIONS IN MICROCOCCACEAE •by DERK JAN GROOT OBBINK B.Sc. (Botany) University of Melbourne, 1966 M.Sc. (Bacteriology and Immunology) University of Western Ontario, 1969. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1973 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f MICROBIOLOGY The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 26 July, 1973 i ABSTRACT Members of the Micrococcaceae were shown to have broad differences in the base composition of their DNA and to exhibit a strong correlation between aerobiosis and a high %GC. An analysis of the genome sizes of the DNA revealed a four-fold difference in the range. In general the organisms with a large genome had a high %GC. " P_. aerogenes, which had the smallest genome, was an anaerobe and had a genome comparable in size to that reported for mycoplasma. Hybridization studies between some organisms with small genomes showed that they were a genetically heter-ogeneous group. An examination of the amino acid requirements of the organisms showed that aerobic organisms with large genomes and high %GC were nutritionally independent while anaerobic organisms with small genomes and a low %GC had broad nutritional requirements. P_. aerogenes would grow only in complex medium. In an effort to observe any developmental changes, amino acid transport, as one type of membrane associated function, was studied in a series of micrococci selected for their differences in genetic complexity. With the exception of P_. aerogenes, which would grow only in complex medium, and with which no proline transport could be demonstrated, the transport of proline was a well developed process. It was shown to be energy dependent, specific for proline, to display saturation kine-tics and to be derepressed in organisms auxotrophic for, and starved for i i this amino acid. Transport for isoleucine and methionine were also shown to be well developed in M. varians, M.sp.250 and SL aureus and it was concluded that the development of active transport was an early function in bacteria which may have given them a selective advantage. A study of membrane bound oxidative and phosphorylative enzymes showed that there was a strong correlation between the number of mem-brane bound oxidative and phosphorylative enzymes and aerobiosis. Treatment of the membranes with a dilute buffer containing EDTA, which affected the hydrophilic interactions in the membrane, had variable effects on the solubilization and activity of membrane bound enzymes. Triton-X which affected hydrophobic interactions had less variable results. The effect on the level of activity and the pattern of solubilization of a particular enzyme was similar in the different membrane systems studied. Therefore, hydrophobic interactions may be stable characteristics that are conserved. A study of the protein structure of the membrane revealed an increase in number of protein subunits and a general increase in the molecular weight of the subunits with increasing genetic complexity. Although increasing genome size seemed to confer nutritional independence and increased number and size of membrane subunits, functional aspects of the membrane were well developed in organisms with a small genome size. TABLE OF CONTENTS Page INTRODUCTION AND LITERATURE REVIEW 1 MATERIALS AND METHODS 19 I. Organisms and Media 19 II. DNA 19 1. Isolation of DNA 19 2. Measurement of %GC 20 (a) Tm method 20 (b) Buoyant density 21 3. Measurement of genome size by the Cot. method 21 4. Cot. hybridization studies 22 III. Nutrition 22 1. Amino acid requirements 22 IV. Transport of Amino Acids 23 1. Medium for transport experiments 23 2. Uptake of labelled amino acids 24 3. Low temperature experiments 24 4. Energy dependence 24 5. Specificity 25 6. Induction or derepression of proline transport 25 7. Analysis of free amino acids in cells 25 V. Membrane Bound Enzymes 26 iv Table of Contents (continued) Page 1. Growth of cultures 26 2. Washing buffers 27 3. Preparation of membranes 27 4. Sucrose density gradients 28 5. Chemical assays 28 (a) Protein 28 (b) Phospholipid... 28 6. Enzyme assays 28 (a) NADH dehydrogenase 28 (b) NADH oxidase 29 (c) d- and 1-lactate dehydrogenase 29 (d) Mai ate dehydrogenase 30 (e) ATPase 30 (f) Succinate dehydrogenase 30 (g) Adenosine deaminase 31 (h) Fumarase 31 VI. SDS Gel Electrophoresis 31 1. Running buffer 31 2. Preparation of gels 32 (a) Stock solutions 32 3. Preparation of samples 32 V Table of Contents (continued) Page VII. Chemicals 33 RESULTS 34 I. Properties of DNA 34 1. Base ratios 34 2. Genome size 37 3. Cot. hybridization 39 II. Nutrition 41 III. Amino Acid Transport 43 1. Transport of proline 44 (a) Uptake of proline 44 (bj Effect of metabolic inhibitors 46 (c) Effect of temperature 46 (d) Kinetic analysis 48 (e) Specificity 48 (f) Induction or derepression of proline transport.. 48 2. Transport of other amino acids 49 IV. Properties of Membrane Bound Enzymes 55 1. Isolation of membrane preparations 58 2. Sucrose density gradients 60 3. Relative amount of lipid in membranes 62 4. Enzyme content in membranes 62 5. Wash treatments 64 vi Table of Contents (continued) Page (a) Effect of concentration of detergent 66 (b) Effect of removal of divalent cations 66 6. Sequential washings 69 (a) Sucrose density gradients 72 (b) Solubilization of protein 74 (c) Solubilization of phospholipid 78 (d) Solubilization of enzymes 78 (aa) NADH dehydrogenase 78 (bb) Succinate dehydrogenase 81 (cc) d- and 1-Lactate dehydrogenase 86 (dd) Malate dehydrogenase 89 (ee) ATPase 89 (ff) Adenosine deaminase 95 V. Membrane Subunit Structure 96 GENERAL DISCUSSION 100 LITERATURE CITED 110 LIST OF FIGURES Page Figure 1. Thermal denaturation profile of DNA M. varians, M. freudenreichii and S_. aureus 36 Figure 2. Time course of proline uptake and incorporation into protein by micrococci.. 45 Figure 3. Lineweaver-Burk kinetics of proline uptake by micrococci 50 Figure 4. Effect of proline starvation on the rate of up-14 take of C-proline 51 Figure 5. Lineweaver-Burk kinetics of methionine uptake by M.sp.250 56 Figure 6. Sucrose density profiles of membrane fractions of M. varians and P_. aerogenes 61 Figure 7. Flow chart for sequential washing procedure 71 Figure 8. Solubilization of protein from M. varians membr-anes by washing procedures 76 Figure 9. SDS gel electrophoresis of protein solubilized by DTE and Triton-X from anaerobically grown S_. aureus 77 Figure 10. Solubilization of phospholipid from M. lysodeikti-cus membranes by washing procedures 80 v i i i List of Figures (continued) Page Figure 11. Solubilization of NADH dehydrogenase from aerobically grown S_. aureus by washing procedures. 84 Figure 12. Solubilization of succinate dehydrogenase from M. varians membranes by washing procedures 87 Figure 13. Solubilization of 1-lactate dehydrogenase from membranes of aerobically grown ,S. aureus and M. varians by washing procedures 88 Figure 14. Solubilization of malate dehydrogenase from mem-branes of M. varians and M. lysodeikticus by washing procedures 92 Figure 15. Solubilization of ATPase from membranes of M. varians by washing procedure 94 Figure 16. SDS gel electrophoresis of SDS solubilized mem-branes of anaerobical ly grown S_. aureus and M. varians 98 ix LIST OF TABLES Page Table I. Comparison of %GC, genome size and relationship to in Micrococcaceae 35 Table II. Comparison between the buoyant density and the melting temperature of DNA for obtaining %GC 38 Table III. DNA homologies of selected organisms by Cot method 40 Table IV. Amino acid requirements of the Micrococcaceae Table V. Kinetic analysis of proline transport 47 Table VI. Effect of chloramphenicol on the derepression of proline transport 52 Table VII. Effect of metabolic inhibitors on transport of amino acids 53 Table VIII. Kinetic analysis of methionine transport Table IX. Kinetic analysis of isoleucine transport 57 Table X. Conditions for producing membrane preparations 59 Table XI. Relative amount of lipid in membranes of Microco-ccocaceae 63 Table XII. Enzyme complement of membranes of some Micrococcaceae 65 X List of Tables (continued) Page Table XIII. Effect of concentration of Triton-X on NADH dehydrogenase distribution 67 Table XIV. Effect of concentration of Triton-X on ATPase distribution 68 Table XV. Dissociation and reassociation of NADH dehydro-genase by dialysis against EDTA and magnesium 70 Table XVI. Effect of washings on membrane density 73 Table XVII. Protein solubilized by the washing procedure 75 Table XVIII. Relative amount of lipid in washings of membrane of M. varians 79 Table XIX. Specific activities of membrane bound enzymes in the presence of TKMG, DTE and triton-X 82 Table XX. NADH dehydrogenase solubilized by the washing procedure 83 Table XXI. Succinate dehydrogenase solubilized by the wash-ing procedure 85 Table XXII. d- and 1-Lactate dehydrogenase solubilized by the washing procedure 90 Table XXIII. Malate dehydrogenase solubilized by the washing procedure 91 Table XXIV. ATPase solubilized by the washing procedure 93 Table XXV. Subunits of micrococcal membranes as determined by SDS gel electrophoresis 99 ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. J.J.R. Campbell for his guidance and interest during the course of the work and for his perseverance during the editing of the thesis. I would also like to extend my thanks to Dr. A.F. Gronlund and Dr. G. Weeks for many worth-while discussions. I am especially grateful to my wife, Diane, for her interest, patience and good humour during the course of this research. Finally, I would like to express my appreciation to Mrs. Jeanette Davidson for typing the final manuscript. 1 INTRODUCTION AND LITERATURE REVIEW The expressions "unity in biochemistry" and "comparative biochemistry" were introduced in 1931 by Kluyver to emphasize the similarity of metabolic reactions in plants, animals and bacteria. As a bacteriolo-gist, Kluyver wished to draw attention to the contribution that the study of bacterial metabolism would play in the understanding of problems in plant and animal biochemistry. However, although the concept of comparative biochemistry was not abandoned by subsequent generations of bacteriologists, the emphasis in future work was towards the demonstra-tions of differences in the metabolic patterns of microorganisms and the use of these differences as bases for differentiation and classification. As our knowledge of metabolic pathways became essentially completed and concurrently our understanding of control mechanisms and bacterial genetics evolved, along with techniques such as those for sequencing proteins, the comparative aspects of the biochemistry and molecular biology of organisms became new and rewarding areas of study. The most revealing of the techniques for the determination of genetic relatedness have been DNA:DNA or DNA:RNA hybridization and the determination of the amino acid sequence in a specific protein such as cytochrome c. The determination of the amino acid sequence of the cytochrome c of a number of animals has been used to assemble a time scale for the evolution of species. The concept is based on the calculation that there was one stable mutational change in the DNA coding for cytochrome c 2 every 26 million years (Margoliash, Fitch and Dickerson, 1968; Nolan and Margoliash, 1968). The limited work carried out on the cytochrome c of bacteria indicates that there have been as many or more, mutations in the DNA affecting the amino acid sequence of cytochrome c of bacteria as there has been in the whole of the animal kingdom. This suggests the possibility of profound evolutionary differences in bacteria while still remaininq, in the main, as undifferentiated single cells. Bacteria were probably very early cellular forms and this fact, combined with their rapid rate of multiplication, has given them an unusual opportunity to develop certain characteristics to a very high level. On the other hand, because of the myriad of microenvironments available to bacteria, it is possible that some species have not been subjected to selective forces and have remained as relatively primitive forms, at least as far as certain characteristics are concerned. A com-parative study of a particular phenomenon, structure or enzyme system in a series of selected bacteria could, therefore, yield information about its evolutionary development and could provide useful information on the factors which are essential to its function. Lipmann (1971) supported this concept in his search for primitive mechanisms of peptide synthesis. The discovery that the peptide antibiotics were synthesized by bacteria without the involvement of ribosomes or the other usual components of the protein synthesizing apparatus confirmed Lipmann's proposal that "metabolic fossils" were present in some bacteria. The electron transport protein ferredoxin appeared to be another example of a living fossil, for the type of molecule present in higher plants was the same as in the 3 primitive blue green algae although more complicated than in the anaerobic Clostridia or in photosynthetic bacteria (Hall, et_ aJL 1971). The task of establishing a satisfactory basis for phylogenetically relating bacteria has achieved only partial success. The problem has been complicated because during the process of their development many transitional forms become extinct. There was, in addition, only a meagre fossil record. Hall (1971) has most recently attempted to phylogenetically relate the procaryotes on the basis of their ability to generate energy. In his scheme the most simple organisms were anaerobes which derived their energy from the transformations of intermediatary metabolism and it was the result of a series of mutations which led to the development of the obligate aerobes capable of oxidative phosphory-lation. This approach seemed a good one, since it embraced all procaryotes. Stanier and van Niel had presented a case as early as 1941 for a phylo-genetically based taxonomy. Klein and Cronquist (1967) and Aaronson and Hutner (1966) have used the approach of comparing metabolic traits of existing organisms in the hope of being able to establish features which might be considered primitive and then to use these to order different groups. This approach is complex however when a large number of diverse characteristics are considered. Studies on members of the Micrococcaceae based on physical and bio-chemical properties have been made by Silvestri and Hill (1965), Baird-Parker (1965) and Rosypal et al_. (1966). These authors have grouped representative Gram positive catalase producing cocci into two broad 4 groups; the Micrococci and the Staphylococci, based on their ability to ferment glucose. The groups have been further classified on physio-logical and biochemical criteria. These studies do not include the strictly anaerobic species, the Peptococci, described by Foubert and Douglas (1948), which are also Gram positive and catalase producing and included in the Micrococcaceae. The use of DNA base ratios, %GC, as determined from Tm by Marmur and Doty (1962) has become widely used in bacterial taxonomy and has been compiled for a wide range of organisms by Hill (1966). Rosypal et al. (1966), stated that a study of the phylogenetic relationship among bact-erial species made on the values of their base compositions, led to con-clusive results only when it concerned species of the same morphology, ie. usually a species of the same family. It is important then to use base ratios only in conjunction with other criteria. Base composition homologies have been extended to many groups of organisms. The lacto-bacilli have been shown to fall into three groups by Gasser and Mandel (1969) whereas the acetic acid bacteria fall into one large group with a %GC difference of less than 10% (DeLey and Schell, 1963). The base comp-psition of the Micrococcaceae has been reviewed by many authors; (Silvestri and Hill, 1965; Rosypal et al_. 1966; Bohacek, et_ a_l_. 1967; Garrity et al. 1969; Bohacek, et al_. 1970). These authors have shown that a wide range of %GC from 30% to 75% existed within this family. Those organisms classified as Staphylococci on the basis of their ability to ferment glucose showed a %GC range from 5 30-39%, but the aerobic Micrococci showed a wide range of %GC from as low as 49.8 to 75%. As a result of this wide range, Silvestri and Hill (1966) have suggested that the Micrococcaceae are heterogeneous and that the common characteristics of Gram reaction, catalase production and coccus morphology were the result of converging evolution. In the paper by Hill (1966) it can be seen from the organisms tested that there was a strong correlation between high %GC and aerobiosis. However, Singer and Ames (1970) have proposed that bacteria exposed to sunlight evolve a high %GC to avoid thymine specific damage by ultra-violet light. They have shown that a mutant of the yeast Saccharomyces  cerevisiae lacking an excision repair mechanism was far more sensitive to being killed by ultraviolet light. There are however, a number of excep-tions to this hypothesis as reported by Leth Bak ejt al_. (1972). A method for examining phylogenetic relatedness in more general terms involves measuring the genome size of an organism. Current methods for determining the molecular weight of bacterial genome DNA can be done chemically, by electron microscopy or by autoradiography of circular chromosomes (DeLey; 1971). However, the first technique is imprecise and the special techniques of the second and the slowness of the third probably prevented their wide application. Britten and Kohne (1968) described an optical method for determining genome size which is accurate and convenient to use. The procedure involved following the rate of reannealing of sheared denatured DNA under the appropriate conditions of temperature and concentrations of DNA and cations so that the reannealing 6 rate obeyed second order kinetics. The kinetics were thoroughly re-viewed by Wetmur and Davidson (1968). The product of the concentration of DNA and the time taken for the DNA to half reaneal (Cot. 0.5) was directly related to the genome size. Seidler and Mandel (1971) used Escherichia coli as a standard and derived an equation which could be used to calculate any unknown genome size and also made a correction for the effect of %GC on the rate of reannealing. The genome sizes of bacteria can differ by 7-fold. Gill is et al. (1971) have examined forty strains of bacteria and found a genome size 9 range between 1.4 and 3.6 x 10 daltons. They found organisms with limited enzymatic activity had smaller genomes. Escherichia coli and Neisseria were found to have larger genomes than the fastidious Chlamydia and Rickettsia, (Kingsbury, 1969),and Leth Bak et al_. (1970) reported a g genome size of 7 x 10 daltons for the biochemically sophisticated aerobe Pseudomonas aeruginosa. The information on genome sizes may create a basis for the phylo-genetic differentiation of microorganisms at a broad taxonomic level while DNA base ratios and DNA homology studies allow differentiation at a lower level. Homology studies have been performed by different techniques which involve the use of solid supports such as filters or agar gel and labelled DNA (Bolton, 1962; Gillespie and Spiegelman, 1965). However, the techniques of Seidler and Mandel (1971) which involved reannealing indi-vidual genome types and mixtures of genome equivalents, is now a preferred method for studying genetic relatedness. A knowledge of the relative level of sophistication of bacteria should be valuable since such informa-7 tion would permit an investigator to select, for experimental studies, bacteria that represent various stages in the development of particular structure, pathway or control mechanism. The cytoplasmic membrane, is of course, present in all bacteria and the fact that its function is intimately related to its structure makes it an interesting subject for the study of developmental changes in bacteria. One would expect to find certain essential characteristics that are common to all such membranes, but additional characteristics should be incorporated into the structure of most sophisticated species. One important and well developed function of the membrane is transport. Since all bacteria have the same naturally occurring amino acids in their proteins and since almost all bacteria incorporate amino acids from a growth medium, it was thought that a study of the relative sophistication of amino acid transport mechanisms would yield information which could be useful in assessing the relative development of members of the Micrococcaceae. A large numberof more or less specific transport systems for the incorporation of solutes into bacteria have been described. In recent years progress in the field has been rapid and well discussed in reviews (Holden, 1962; Kepes and Cohen, 1962; Britten and McClure, 1962; Kaback, 1970 and Oxender, 1972). The selective transport and accumulation of amino acids by bacteria were first shown by Gale (1947) working with staphylococci. He found that the processes were unidirectional and in some cases required a metabolizable energy source. The enzymatic nature 8 of metabolite incorporation into bacterial cells was first demonstrated for organic acids by Barrett and Kallio (1953) and was later extended to amino acids by Britten and McLure (1962). Cohen and Monod (1957) set forth the basic principles of nutrient transport and it has become evident to many investigators that the plasma membrane was largely impermeable to the majority of polar molecules and that an enzymatic process was required for entry into the cell. The kinetic and genetic analysis of many microbial transport systems gave rise to the belief that the substrate combines reversibly with a carrier and the resulting complex moves across the osmotic barrier. This has been well reviewed by Cohen and Monod (1957), Britten and McLure (1962), Kaback (1970), Kennedy (1970), Kepes (1971), Lin (1971) and Oxen-der (1972). Amino acid transport systems in bacteria, unlike sugar trans-port systems, are constitutive unless the amino acid serves as both a carbon and nitrogen source. Proline transport which can be induced in P_. aeruginosa (Kay and Grorilund, 1969), has been shown to be energy de-pendent in a number of organisms such as Escherichia coli (Britten and McLure, 1962). P_. aeruginosa (Kay and Gronlund, 1969) and S_. aureus (Groot Obbink and Campbell, 1973). On the other hand, anaerobes like Clostridium perfringens (Finch, 1969) exhibited no evidence of active transport for any substrate tested. So there is at least an indication that anaerobes may have poorly developed systems for amino acid trans-port while aerobes have highly developed systems. Kaback (1972) has divided the categories of solute transfer across the osmotic barrier into four categories. 9 (a) Passive diffusion. A substance crosses the membrane as a result of a random molecular movement. The transported solute is thought not to interact with any molecular species in the membrane. (b) Facilitated diffusion. The transported solute is presumed to combine with a specific carrier in the membrane and both the carrier and the carrier-solute complex oscillate between the inner and the outer surfaces of the membrane releasing and binding molecules on either side. Neither passive nor facilitated diffusion requires metabolic energy and neither process results in a concentration gradient. (c) Active transport. The solute is concentrated against a concen-tration gradient and the process requires metabolic energy and a specific membrane carrier molecule. The affinity of the carrier is modified on one side of the membrane so that the solute is released. (d) Group translocation. A covalent change takes place in the solute so that the reaction itself results in the passage of the molecule through the diffusion barrier. An example of this is the bacterial phos-phorylation of sugars. Although there have been numerous reports on the mechanism where-by energy is coupled to transport in bacterial membranes, the significant contributions have been made in recent years. One such advance has been the use of membrane vesicles to study transport processes in vitro which was introduced by Kaback and Stadtman (1966). They were able to show that vesicle preparations from £. coli were capable of actively trans-porting proline. These studies have been extended to S^. aureus (Short 10 e_t al_. (1972), B_. subtil is (Konings e_t al_. 1971) and Mycobacterium phi ei (Hirata et a_l_. 1971). A recent review by Kaback (1972) provides a de-tailed coverage of progress in the field. Transport of some sugars and amino acids is driven in these vesicles by respiration coupled reactions. In JL. coli the coupling substrate required for transport was d-lactate and its oxidation by a membrane bound d-lactate dehydrogenase was coupled to the transport of a variety of compounds. Other substrates such as succinate or NADH could replace d-lactate but were far less effective in driving transport. The nature of the most effective dehydrogenases varied from one bacterial system to another. a-Glycerophosphate dehydrogenase was most effective in S_. aureus vesicles (Short et al_. 1972) while NADH dehydrogenase was most effective in B_. subtil is (Konings et al_. 1971). The last workers also demonstrated that an artificial electron donor system of phenazine methosulfate-ascorbate could be coupled effectively to amino acid and sugar transport. By the use of electron donors, the site of energy coupling between transport and the respiratory chain in JE. col i was found to be between d-lactate dehydrogenase and cytochrome b-j. These authors showed that sulfhydryl inhibitors blocked transport and that this effect could be reversed by dithiothreitol, thereby implicating sulfhydryl proteins as carriers. Phosphorylative activity did not seem to be required for transport in E_. col i vesicles because added ATP or PEP did not stimulate transport nor did arsenate inhibit. DCCDI an inhibitor of ATPase, did not inhibit proline transport in M. phlei (Hirata et a_]_. 1971). On the other hand, Harold (1972) had found that Streptococcus 1 DCCD - N,N^-dicyclohexylcarbodiimide 11 faecal is, an organism that lacked both internal energy reserves and oxidative phosphorylation and was dependent on substrate level phos-phorylation, had its ability to transport amino acids inhibited by un-couples of oxidative phosphorylation. Other evidence pointed to ATPase as the energy donor for the transport of amino acids, K+ and phosphate by this organism. The ATPase system had been found to be a multienzyme system involving at least three components - the ATPase itself, a protein believed to link the enzyme to the membrane and an unidentified component which conferred DCCD sensitivity upon the enzyme and the inhibition of transport. It was suggested that an energized state of the membrane was necessary for transport. These differences were resolved by Klein and Boyer (1972), who showed that whole cells of JE. coli, which transported amino acids equally well aerobically as well as anaerobically, became arsenate sensitive under anaerobic conditions and DCCD, which inhibits ATPase prevented proline transport under these conditions. On the other hand, aerobically grown cells were unaffected. Compounds which uncoupled oxidative phosphory-lation inhibited transport under both conditions, thus suggesting that a similar or identical energy conserving state is formed from either energy source, oxidative or phosphorylative. Kaback (1972) had shown that transport activity in vesicles was inhibited by anoxia and Klein and Boyer (1972) showed that added ATP did not stimulate the anaerobic vesicles. It was therefore concluded that some coupling factors may be lost in the preparation of the vesicles. 12 Attempts have been made to dissociate transport systems. Dissociable water soluble binding proteins which can be released by osmotic shock (Neu and Heppel, 1965) or by Polymyxin B (Cerny and Teuber, 1971) are almost entirely the domain of Gram negative organisms and are associated with high affinity transport systems. Extensive reviews have been writ-ten about the proteins by Pardee (1968), Oxender (1972) and Lin (1971). They are not however, integral components of the membrane. Kaback (1972) has shown that the galactose binding protein from FE. coli showed no immuno-logical cross reactivity with purified membrane preparations and was without effect on galactose transport by vesicle preparations and thus was not considered an integral membrane component. An examination of the integral components of the bacterial cyto-plasmic membrane has been a much more difficult task. Only a limited number of studies on bacterial membranes have been undertaken and an accurate assessment of the number of different proteins in a cytoplasmic membrane has not been made. Part of the difficulty has been caused by the lack of techniques for studying proteins which are insoluble in an aqueous environment (Reave!ey and Burge, 1972). Although enzymes involved in such important cellular functions as oxidative phosphorylation, active trans-port and cell wall biosynthesis are membrane associated, remarkably little is known about how these enzymes fit into the structure of the membrane and how their function relates to their membrane association (Reaveley and Burge, 1972). Even in a single membrane system, there is a complexity and a variety of functions (Nachbar and Salton, 1970). A number of recent 13 reviews on bacterial membranes have been written (Salton, 1971; Reaveley and Burge, 1972). Razin (1972) has provided a comprehensive review on the reconstitution of biological membrane systems. In general, the problem of membrane isolation and purification has been a much simpler task for Gram positive organisms that for Gram nega-tive ones and cells of Gram positive organisms susceptible to peptidoglycan degrading enzymes yield protoplasts free of cell walls in a hypertonic medium (Weibull, 1968). The isolation of cytoplasmic membranes devoid of cell walls from Gram negative bacteria is extremely difficult. When Gram negative bacteria are broken by a French pressure cell, they yield two fractions on a sucrose density gradient; one which is cell wall en-riched and one which is cytoplasmic membrane enriched (Schnaitman, 1971). Osborn ejt al_. (1972) have described an isopycnic sucrose density centrifu-gation method which yields relatively pure cytoplasmic membranes, but these were still contaminated with outer wall material. The mechanical breakage of Gram positive cells such as S_. aureus also released cytoplasmic membranes (Mitchell and Moyle, 1957; Lengsfeld ejt al_. 1973), but fragment-ation of the membrane occurred by this method. Intact cytoplasmic mem-branes could only be produced when the cell wall is removed enzymatically (Reaveley and Burge, 1972). Recently, Braun and Bosch (1972) have shown and characterized a structural protein from E. coli which covalently binds to peptidoglycan. This protein is responsible for the intimate association between cytoplasmic membranes and cell walls of Gram negative organisms and thus for the inability to obtain pure membranes. Salton (1971) points 14 out some of the problems that arise during the characterization and iso-lation of bacterial membranes. The first was a general one of defining the conditions and environment for membrane isolation and separation from the cell sap and cytoplasmic proteins. The second problem was one of distinguishing between cytoplasmic membranes and mesosomes and the third problem was in defining structure in relation to residual or contaminating cell wall components. The cell walls of Gram positive bacteria are de-void of protein or 1ipopolysaccharide in contrast to those of Gram negative bacteria. The presence of divalent cations such as magnesium is of great im-portance during the isolation of intact cytoplasmic membranes. Although this requirement is a function of the particular organism being studied, its importance has been demonstrated many times (Rogers et al_. 1967; Ghosh and Carroll, 1968; Fitz-James, 1968). Some studies have been made on the protein and peptide compositions of cytoplasmic membranes by SDS gel electrophoresis. Reaveley and Burge (1972) found that membranes of Bacillus 1icheniformis contained about 36 major protein bands whose molecular weights ranged from 20,000 to 160,000 with the principal components having molecular weights of approximately 50,000. Patterson and Lennarz (1970) found that 90% of the membrane protein of a strain derived from B_. megaterium consisted of a single protein band while Schnaitman (1970) obtained from 20 to 30 well resolved bands of protein from E.. coli. He also found one major protein which had a molecular weight of 44,000 and comprised about 40% of the total. Changes 15 from aerobiosis to anaerobiosis and from fermentable to non-fermentable carbon sources produced relatively small differences in major proteins resolved on gels as compared to the large differences in membrane bound enzyme activities. Changes in the composition of membrane systems can occur with changes in growth conditions. Streptococcus faecal is membranes contain around 28% lipid in log phase cells but this amount can go as high as 40% in stationary phase (Shockman et al_. 1963). The phospholipid content of different membrane systems varied considerably and these have been ex-tensively reviewed by Op den Kamp et_ aj_. (1969). The resolution and isolation of membrane proteins and enzymes have been made difficult by the tendency of these proteins and associated lipids to coalesce and form aggregates. A review of the properties of these proteins have been made by Singer (1971) and Guidotti (1972). A variety of methods have been used in attempts to isolate and "solubilize" membrane proteins. These include the use of organic solvents, extremes of pH, detergents and chaotropic agents (Nachbar et al_. 1972). However, many of these treatments also inactivate enzymes thus limiting their usefulness (Rothfield and Romeo, 1971; Razin, 1972). Non-ionic detergents have been effective in solubilizing membrane bound enzymes in an active form. Using Triton-X-100, Fox and Kennedy (1965) solubilized the M protein of B-galactoside transport from cell envelope fragments derived from E. coli. Schnaitman (1971) showed that Triton-X selectively solubilized proteins from the cytoplasmic membrane of E. coli. A variety of other enzymes including NADH dehydrogenase and d-lactate 16 dehydrogenase have been solubilized from membranes by this detergent or by other mild detergents (Gordon ejt aj_. 1972; Eisenberg ejt al_. 1970). These latter workers have shown that low concentrations of deoxycholate increase the specific activity of NADH oxidase and succinic dehydrogenase. However NADH oxidase was unstable, requiring the addition of magnesium ions. Bishop ejt al_. (1967) showed that the process of solublizing comp-onents of the cytoplasmic membrane is not random, and these investigators showed that a low concentration of sodium dodecyl sulfate (0.05 - 0.1%) which was insufficient to completely solubilize B_. subtil is membrane could solubilize NADH dehydrogenase while succinic dehydrogenase remained mem-brane bound. The detergent differentially solubilized protein leaving a lipid rich residue. This was in contrast to the action of deoxycholate on M. lysodeikticus membranes which left a lipid depleted residue (95 -97% protein) (Reaveley and Burgess, 1972). Hendler and Burgess (1972) have attempted to solubilize active components from E. coli membranes under controlled conditions. They found that succinate dehydrogenase remained coupled to cytochrome c-| and the preparation retained succinate oxidase activity after treatment with deoxycholate. However NADH de-hydrogenase and d-lactate dehydrogenase were solubilized and their oxidase activities were lost. Gordon et al_. (1972) have solubilized components of the proline transport system from jE. coli with the non-ionic detergent Brij-36T and separated these components by gel filtration. The high molecular weight components showed proline specificity and d-lactate dehydrogenase activity and were inhibited by electron transport inhibitors. 17 The low molecular weight component was non-specific and inhibited by sulfhydryl inhibitors. This inhibition could be reversed by dithio-threitol. The differential affinity of Triton-X and deoxycholate for membrane and hydrophilic proteins was shown by Helenius and Simons (1972). They showed that radioactive deoxycholate and Triton-X would bind up to 70% of the weight of lipophilic proteins derived from erythrocyte membranes, but had little tendency to bind hydrophilic proteins. The magnesium requirement of cytoplasmic membranes has been established and the washing of these structures with low ionic strength buffers lacking magnesium resulted in the release from M. lysodeikticus membranes of NADH dehydrogenase which had lipid associated with the enzyme. Removal of this lipid by butanol extraction resulted in diminished activity which was recovered by the readdition of lipid. Membrane bound ATPase, which has probably been studied most extensively in a number of systems has been released from the membrane by a variety of methods (Razin, 1972). A larger percentage of the enzyme could be re-moved from the membrane of B_. megaterium by adjusting the pH to 8.5 and dialyzing against water. The solubilized ATPase differed from the membrane bound form with respect to calcium activation and cold inactivation. The re-addition of divalent cations resulted in the reconstitution of the enzyme with the membrane. Salton and Schor (1972) showed that calcium activated ATPase from M. lysodeikticus membranes was released in two forms: (i) a shock wash form that was trypsin stimulated, capable of rebinding 18 to ATPase depleted membranes in the presence of magnesium and possessing some additional minor protein components, (ii) a form extracted by butanol which was not trypsin stimulated nor could it be reconstituted to membranes by magnesium and did not possess any additional protein components but possessed the same major subunits as the shock wash form. Hanson and Kennedy (1973) purified ATPase from membranes of E_. coli to near homogeneity and reported that although ATPase has been solubilized into different forms with different methods of extraction, genetic and immunological studies revealed that it was the same protein in all cases. Current theories on membrane structure have been reviewed by Finean (1972) and Singer (1972). The favored model is the fluid mosaic model of Singer and Nicol son (1972). This model proposes a lipid bilayer which has a heterogeneous set of globular proteins embedded in i t . The proteins have hydrophobic and hydrophilic regions which are oriented into and out of the bilayer. Hydrophilic regions of the membrane are stabilized by divalent cations. We have chosen to study the Family Micrococcaceae with a view to examining some enzyme components of the cytoplasmic membrane of represent-ative species and correlating these with %GC, base ratio and nutritional dependency. 19 MATERIALS AND METHODS I. Organisms and Media. The following organisms were used in this study. Micrococcus  varians ATCC 19100, M. lysodeikticus (from R.A.J. Warren, University of British Columbia), M. salivarius ATCC 14344, M. roseus ATCC 418, Staphylococcus lactis, ATCC 15306, M. freudenreichii, ATCC 407, M.sp.250 Czechoslovak Collection of Microorganisms (CCM), M.sp.901, CCM, S_. aureus ATCC 6538P, S. aureus ATCC 12600, S_. aureus V8, M.sp. "Neufeld" (from N. Neufeld, Dept. of Fisheries, Vancouver), M. candidus ATCC 1482, Peptococcus saccharolyticus ATCC 14953, P_. aerogenes ATCC 14963, Veillonella alcalescens ATCC 17745. With the exception of P_. aerogenes and V_. alcalescens, these organ-isms were maintained on agar slants containing 0.5% NaCl, 0.2% cerelose, 0.3% beef extract, 0.5% pepticase and transferred once a month. The Micrococcaceae which excluded y_. alcalescens were checked for purity by their pigmentation colony morphology, Gram stain and their ability to produce catalase. II. DNA. 1. Isolation of DNA. With two exceptions, cultures were grown to stationary phase in a medium at pH 7.2 that contained 0.5% glucose, 0.5% pepticase and 0.2% yeast extract. P_. aerogenes was grown anaerobically at pH 7.2 in 20 0.5% glutamate, 2% pepticase, 1% yeast extract and 0.1% thioglycollate. Veillonella alcalescens was grown in a similar medium but with 1.5% sodium lactate being substituted for glutamate. The cells were washed with 0.15 M NaCl + 0.1 M EDTA pH 8.0, then frozen as a pellet overnight at -70 C. These cells were thawed, treated with 2 - 5 mg lysozyme (Nutritional Biochemicals) for 3 hours at 37 C. S_. aureus strains were treated with 5 units/ml lysostaphin (Schwartz/Mann) instead of lysozyme. In cases where lysozyme did not cause cell lysis, the cells were passed through a French pressure cell at 20,000 psi. Sodium dodecyl sulfate, which had been recrystallized, was added to a final concentration of 2%. After 2 hours at 37 C, pronase was added at a final concentration of 10 mg/ml for an additional 17 hours. DNA was then isolated by the phenol extraction method of Marmur and Doty (1962). DNA was checked for purity by assaying for RNA by the orcinol method (Ashwell, 1957) and for protein 230 280 (Lowry et al_. 1951). The OD^y, OD^ gQ, 0D30Q and hyperchromicity was measured according to the criteria of Marmur and Doty (1962). Purified DNA preparations were kept frozen at -70 C in 0.1 x SSC (0.15 M NaCl + 0.0015 M Na citrate, pH 7.8) until used. 2. Measurement of %GC (a) Tm method Purified DNA was melted in a thermostatically controlled automatic recording Gilford Spectrophotometer (Model 2400). The tempera-ture was raised at the rate of 0.2 C/min and was continued until there was no further increase in optical density. A final 0D9(-n was taken and 21 the ratio of the 0^260 at temperature T over the C^gQ at 25 C was plotted as a function of T. The mid-point of the increase is called the Tm and this is directly related to %GC as described by Mandel and Marmur (1968). (b) Buoyant density. The DNA from E_. coli B (Worthington) at a density of 1.710 3 g/cc was used as a standard marker. The sample was prepared according to the method of Mandel et al_. (1968) and contained 0.84 ml CsCl (13 g dissolved in 7 ml of H20), 0.18 ml H^, 0.01 ml E_. coli B DNA and 0.04 ml of test DNA (1 OD^ gg/ml for both DNA's approximately). A cell was set up as described in the Beckman manual and then centrifuged for 22 hours at 44,000 rpm in a Beckman Model E ultracentrifuge. Tracings of the devel-oped negatives were made with a Joyce-Loebel double beam recording densito-meter and the %GC was calculated by the equations of Mandel et al_. (1968). 3. Measurement of genome size by the Cot method. Purified DNA at a concentration of 200 to 300 ug/ml in SSC was sheared by two passages through a French pressure cell at 15,000 psi. This produced fragments of moderately uniform size in the molecular weight 5 range of from 3 to 4 x 10' daltons. The 0D260 of sheared DNA was recorded and the sample was then heat denatured in an oil bath at 10 C above the Tm for 10 minutes. It was then transferred to a thermostatically control-led automatic recording spectrophotometer maintained at 25 C below the Tm of the sample. The optical density of the sample after the temperature 22 drop was taken to represent 0% renaturation and the time was recorded. The 0D2gg was monitored until 50% renaturation occurred and this time was used to calculate the Cot. 0.5 and the genome size according to the method of Seidler and Mandel (1972). 4. Cot. hybridization studies. Cot. hybridization studies of organisms with closely related %GC were performed by determining the individual Cot. 0.5 values and genome size as above and then adding together equivalent weights of sheared DNA which corresponded to the respective genome sizes. The mixed sample was heat denatured in parallel with the individual samples which acted as internal controls. The degree of homology of the DNA was inversely proportional to the apparent additivity of the mixed genome. III. Nutrition. 1. Amino acid requirements. The amino acid requirements for members of the Micrococcaceae were investigated by a modification of the method of Zubrzycki et al. (1969). Aerobic cells were grown in 0.5% pepticase, 0.2% yeast extract and 0.5% glucose in a shaking water bath to mid-Togarithmic phase. Anae-robic bacteria were grown in de-aerated flasks supplemented with 0.1% sodium thioglycollate. Cells were washed twice with warm saline, resus-pended in saline and then inoculated in 7 duplicate molten agar tubes at pH 7.2, maintained at 45 C. The plating agar contained, per litre, 5 g glucose, 0.25 g MgS04-7H20, 1 g (NH4)2S04, 7.4 g K2HP04, 1 mg adenine, 1 mg uracil, 10 ml of Eagles Vitamin mixture and 2.0 g Difco-Agar. 23 Each tube contained combinations of L-amino acids, each at a con-centration of 30 mg/litre. The following combinations of amino acids were omitted from the medium when they were under investigation: 1. Glu, Tyr, Try; 2. Leu, Arg, Asp; 3. Ala, Glu, His; 4. Lys, Cys, Cyn; 5. Gly, lieu, Met; 6. Pro, Ser, Thr; 7. Val, Phe. After the pour plates had set, the missing amino acids were spotted on the plates with an inoc-ulating loop in the form of a triangle. The plates were read after 24 -48 hours incubation at 30 C. IV. Transport of Amino Acids. 1. Medium for transport experiments. Cells for transport experiments were grown in a liquid medium containing 0.5% pepticase, 0.2% yeast extract and 0.5% glucose. Incuba-tion was in a shaker water bath at 30 C or 37 C for 8 hours at which time the cells were harvested, washed with warm saline and used to inoculate a defined medium of pH 7.2 containing per litre: 5 g glucose, 0.25 g MgS04.7H20, 1 g (NH4)2S04, 7.4 g K2HP04, 3 g KH2P04, 1 mg adenine, 1 mg uracil, 1 ml Eagles Vitamin mixture (Microbiological Associates). Mineral supplement was added in the form of the nitric acid soluble fraction of the ash of 50 mg of yeast extract. When the organism required or was stimulated by amino acids, 50 mg of amino acid was added to the medium. The cultures were grown at 30 C or 37 C for 6-8 hours at which time they were in mid log phase. If the organism was auxotrophic for the amino acid under study, the cells were washed once with warm saline and then resuspended in a defined medium which lacked the amino acid. 24 2. Uptake of labelled amino acids. 14 The incorporation of C-amino acids into whole cells, protein and pools was determined by the Millipore filtration procedure of Britten and McLure (1962). Cells at the rate of 4 x 10 cells/ml were filtered onto a Tracer Lab E8B precipitation apparatus (TracerLab, Waltham, Mass.) and immediately washed with 2 ml of minimal medium. This procedure did not remove pool amino acids. Dried filters were placed in vials con-taining 5 ml of scintillation fluid (Liquifluor, New England Nuclear Corporation) and the vials were assayed for radioactivity in a Nuclear Chicago liquid scintillation spectrometer model 725. The incorporation of label into protein was followed by extracting the cells with cold 5% trichloroacetic acid filtering them as above and immediately washing them with 2 ml of cold 5% TCA. The dried filters were counted as before. 3. Low temperature experiments. Low temperature experiments were conducted in water jacketed reaction vessels maintained at the desired temperature with a Lauda K-2/R temperature controlled circulating pump (Brinkman Instruments Inc., Westbury, N.Y.). Cell suspensions were allowed to equilibrate at the desired temperature for 15 minutes prior to the initiation of the trans-it port experiment by the addition of C-amino acid. The reaction mixtures were continuously agitated with a magnetic stirrer. 4. Energy dependence. In experiments designed to determine whether or not energy gener-ation was essential to amino acid transport, sodium azide and iodoaceta-made at final concentrations of 30 mM and 1 mM were precincubated with 25 cells for 30 minutes prior to the start of the experiment. 5. Specificity. The specificity of proline transport was established by deter-mining in separate experiments, the effects on L-proline transport of 19 _5 L-ammo acids each at a concentration of 5 x 10 M. 6. Induction or derepression of proline transport. Cells that were not auxotrophic for proline were grown in the presence of 0.5% glucose supplemented with 0.1% proline or with 0.5% proline as the sole carbon and nitrogen source to mid logarithmic phase. These cells were centrifuged, washed with warm saline and then resuspended in glucose medium without proline at the rate of 4 x 10^  cells per ml. Cells that were auxotrophic for proline were grown to mid logarith-mic phase, washed once with warm saline and then resuspended in a medium minus proline. The cell suspension was distributed in 10 ml quantities into 50 ml Erlenmeyer flasks. When constant cell conditions were required in consecutive experiments, the cells were kept on ice. When cell sus-pensions were to be starved for proline, they were incubated at 37 C without shaking for periods of up to six hours. 7. Analysis of free amino acids in cells 14 A sample of C-labeled amino acid was added to 10 ml of resus-pended cells in a centrifuge tube at 37 C. After 2 minutes, the cells were centrifuged and washed with warm saline and then resuspended in 10 ml of cold.5% TCA. This suspension was left overnight, in the cold, centrifuged and the supernatant collected. The solution was extracted 26 with ethyl ether five times to remove the TCA and then concentrated under vacuum. The concentrate was applied to a Dowex 50 (H+ form) column. The column was eluted with water to remove inorganic salts and then with 200 ml of 4 M NH^ OH to remove amino acids. The amino acids were separated by two dimensional cellulose chromatography (Jones and Heathcote, 1966). Two plates were run in parallel one being used for autoradiography and the other for the detection of amino acids, by nin-hydrin spray. Autoradiography was carried out by exposing medical X-ray film to the plate for one week. Cold pool analyses were done by extracting non-labelled cells with TCA, carrying out the same purification steps as above and then applying aliquots in the range of .025 - 25 ymoles to a Beckman 120 C Amino Acid analyser. V. Membrane Bound Enzymes. 1. Growth of cultures. Membranes were prepared from cells grown in rich medium. M. varians, M. lysodei kticus and aerobic S^. aureus were grown in 250 ml volumes at pH 7.2 in 2 litre Erlenmeyer flasks at an agitation rate of 250 rpm in a medium that contained per litre: 20 g pepticase, 10 g yeast extract and 5 g glucose. Glucose was omitted from S_. aureus medium for the synthesis of glucose repressed enzymes. Anaerobic S_. aureus and P_. aerogenes were grown without shaking in 4 litres of deaerated medium at pH 7.2 in 4 litre flasks. The medium for S_. aureus contained per litre: 20 g pepticase, 10 g yeast extract, 5 g glucose, 1 g sodium thioglycol-27 late. Glutamate, at the level of 0.5% was substituted for glucose in the medium for P_. aerogenes. Cells were harvested and washed twice with saline. 2. Washing buffers. The following buffers were used in membrane experiments: (a) TKMG - 0.03 M Tris Hcl pH 7.5, 25 mM MgCl2, 10 mM KC1, 20% glycerol. (b) DTE - 0.003 M Tris HCl pH 7.5, 10 mM EDTA (c) Triton-X 100 - Detergent dissolved in TKMG at a concentration of 0.2% or 1.0%. 3. Preparations of membranes. Washed cells at a final concentration of 20 - 30 ODggg per ml were suspended in an incubation mixture that contained 0.03 M Tris HCl pH 7.5, 25 mM MgCl2 and a peptidoglycan degrading enzyme. Lysozyme at a concentration of 200 ug/ml was added to suspensions of M. varians and M. lysodeikticus and incubated at 30 C. Lysostaphin was added at a concentration of from 1 - 5 units/ml to S_. aureus and incubated at 37 C. Lysozyme at a concentration of 1 mg/ml was added to P_. aerogenes, incubated at 37 C and after approximately one hour the cells were passed twice through a French pressure cell at 20,000 psi. DNase was added at a concentration of 10 ug/ml. The lysate was centri-fuged at 35,000 x a_ for 30 minutes and some supernatant was kept for enzyme and protein assays. The pellet was washed twice with TKMG. Mem-branes were immediately used or stored at -70 C in TKMG. The washing procedures are described under Results. 28 4. Sucrose density gradients. Sucrose stock solutions 30% w/w and 70% w/w were made by dis-solving sucrose in buffer comprising 0.03 M Tris HCl pH 7.5, 25 mM MgClg and 10 mM KC1. Linear 4.5 ml gradients were constructed by a Buchler polystaltic pump in 5 ml cellulose nitrate tubes overlayed with 0.5 ml of membrane material (approximately 3-5 mg). Gradients were centri-fuged at 35,000 rpm for 3 hours in a Beckman SW 29.1 rotor. Gradients were unloaded by bottom puncture, collected in approximately 36 fractions of 10 drops each and analysed for refractive index with a Bausch and Lomb refractrometer. 0D£gQ readings of the fractions were taken after they were diluted with 0.5 ml of H^ O. 5. Chemical assays. (a) Protein. Protein was estimated by the method of Lowry et al. (1951). (b) Phospholipid. Phospholipid was extracted by the method of Bligh and Dyer (1959). The lipid was ashed by heating a small aliquot in the presence of 100 yl of 10% Mg(N03)2 1 n e t n a n o^ anc* Phosphate was assayed by the method of Ames (1966). An approximation of 25.5 was used to convert weight of phosphate to weight of lipid. 6. Enzyme assays. All spectrophotometry assays were carried out in a Beckman Model DBG-T double beam spectrophotometer, (a) NADH dehydrogenase. This enzyme was assayed using 2,6 dichlorophenolindophenol 29 (DCIP). The assay mixture contained in a volume of 2.9 ml; 300 umoles Tris HCl buffer, pH 7.5 6 umoles KCN 0.15 umoles DCIP Membrane or wash sample The reaction mixture was incubated at 30 C for 5 minutes and the reaction was started by the addition of 3 umoles NADH (0.1 ml). The decrease in absorbance at 600 nm was followed. (b) NADH oxidase. This enzyme was assayed by measuring the decrease in absor-bance at 340 nm. The assay mixture contained in a volume of 2.9 ml; 300 umoles Tris HCl buffer, pH 7.5 Membrane sample The reaction mixture was incubated at 30 C for 5 minutes and the reaction was started by the addition of 3 umoles of NADH (0.1 ml). (c) d- and 1-Lactate dehydrogenases. These enzymes were assayed using DCIP. The assay mixture contained in a volume of 2.9 ml; 300 umoles Tris HCl buffer, pH 7.5 6 umoles KCN 0.15 umoles DCIP Membrane or wash sample. The reaction mixture was incubated at 30 C for 5 minutes and the reaction was started by the addition of 4 umoles of d- or 1-lactate (0.1 ml). The decrease in absorbance at 600 nm was followed. 30 (d) Malate dehydrogenase. This enzyme was estimated in the same manner as lactate dehydrogenase but 4 ymoles of sodium malate were used as substrate. (e) ATPase. This enzyme was assayed by measuring the release of inorganic phosphate from ATP. The assay mixture at 0 C contained in 1 ml; 100 ymoles KC1 25 ymoles MgC^ 50 ymoles Tris acetate buffer, pH 6.0 Membrane or wash sample. The reaction was started by incubating a series of samples at 30 C for 0, 5, 10 and 15 minutes and the reaction was terminated by the addi-tion of 1.0 ml 5% sodium perchlorate. Membrane material was pelleted by centrifugation and inorganic phosphate in the supernatant was assayed by the method of Ames (1966). Activity was expressed as the change in absor-bance at 660 nm per 5 minutes. (f) Succinate dehydrogenase. This enzyme was assayed using DCIP. The assay mixture con-tained in 2.8 ml; 40 ymoles phosphate buffer, pH 7.6 60 ymoles KCN 300 ymoles EDTA 0.15 ymoles DCIP Membrane or wash sample. This reaction mixture was incubated at 30 C for 5 minutes. 60 ymoles 31 of sodium succinate (0.1 ml) was added to the reaction mixture and the reaction was started by the addition of 1 mg of phenazine methosulfate (0.1 ml). The decrease in absorbance at 600 nm was followed. (g) Adenosine deaminase. Adenosine deaminase was measured by the change in the ratio 250 of OD^-gQ- The assay mixture at 0 C contained in 1 ml; 25 umoles Tris acetate buffer, pH 6.0 Membrane or wash sample. Samples were incubated at 30 C for 0, 5 and 10 minutes. The reaction was terminated by placing the tubes in a boiling water bath for 5 min-utes. The membrane material was removed by centrifugation. Activity 250 was expressed as the change in ratio of OD^ gQ per five minutes. (h) Fumarase. This enzyme was measured using malate as substrate and measuring the increase in absorbance at 240 nm. The assay mixture con-tained in a volume of 2.0 ml; 40 umoles phosphate buffer, pH 7.6 Membrane or supernatant material. The reaction was started by the addition of 6 umoles malate and fol-lowing the increase in absorbance at 240 nm. VI. SDS Gel Electrophoresis. 1. Running buffer. Running buffer contained per litre; 28.8 g glycine, 6 g Tris and 1 g sodium dodecyl sulfate (SDS). 2. Preparation of gels, (a) Stock solutions. (aa) Acrylamide solution - contained per 100 ml; 22.5 g acrylamide and 0.6 g methylenebisacrylamide. Insoluble material was filtered through Whatman No. 1 filter paper. (bb) Running gel buffer - 3 M Tris HCl, pH 8.9 (cc) Stacking gel buffer - 0.6 M Tris HCl, pH 6.7 (dd) 1.5% NH4 persulfate - freshly made up each time (ee) N N N'N1 - tetramethylethylenediamine (TEMED) (ff) 10% SDS The glass tubes were 10 cm long, with an internal diameter of 6 mm and treated with "Photo Flo 200" solution. For a typical run of 12 gel 30 ml of running gel solution was made up by mixing 13.5 ml acrylamide solution, 4 ml of buffer, 0.3 ml SDS and 10.5 ml of H20. 0.1 ml NH4 persulfate and 25 ml TEMED were then added to the mixture and 2 ml was dispensed into each tube. A small volume of H20 was then layered over the gels which set in about 10 minutes. Stacking gel solution was prepared by mixing 1 ml acrylamide solu-tion, 1.25 ml stacking buffer, 0.1 ml SDS and 7.55 ml H20. The same volumes of NH^  persulfate and TEMED were added and 0.6 ml of gel was added to the top of the runs in gel and overlaid with H20 as before. 3. Preparation of samples. Samples containing approximately 2 mg/ml were dissolved in 1% SDS and then heated in a boiling water bath for 5 minutes. 50 yl Sampl were mixed with a drop of 70% w/w sucrose, 10 yl mercaptoethanol and a 33 drop of 0.05% bromphenol blue, then layered on the gels. Electrophoresis was performed at a constant current of 5 ma per gel and a run was com-pleted in 3 to 4 hours. The gels were fixed with 50% trichloroacetic acid. The gels were stained overnight in a solution that contained per litre; 2.5 g Coomassie brilliant blue, 450 ml methanol, 450 ml h^ O and 100 ml glacial acetic acid. They were destained in the same solvent without the dye. The gels were scanned at a wavelength of 600 nm in a linear transporter attached to a Gilford spectrophotometer. VII. Chemicals. Lysozyme was purchased from Nutritional Biochemical Corporation. Lysostaphin was purchased from Schwartz/Mann. Photo Flo 200 solution was purchased from Kodak. All other chemicals were reagent grade. Radiochemicals were purchased from Amersham/Searle Corporation. 34 RESULTS I. Properties of DNA. The DNA from members of the Micrococcaceae was isolated in order to determine the %GC and the molecular weights of the genomes. Also, some Cot. hybridization experiments were performed on the DNA of organisms which had similar %GC values. 1. Base Ratios. The range of %GC extended from 72% to 27% (Table I) and these values have been well documented by other authors (Auletta and Kennedy, 1966; Bohacek et aj_. 1970). M. freudenreichii and M.sp.250 had %GC values that were intermediate to the. clusters at the high and low extremes of the range. There was a positive correlation between high %GC and aero-biosis (Table I) and the range of %GC found for this family is close to the extreme values reported for all bacteria. Hill (1966) has reported a strain of M. luteus with a %GC of 75.5% while the lowest %GC was recorded for a Mycoplasma sp. (23.5%). The purity of the DNA was monitored by 230 2Rf) assaying for protein and RNA and measuring the OD^ gg and OD^ gg- . The values obtained were within the range specified by Marmur and Doty (1962) for pure preparations. The thermal denaturation profiles of S_. aureus  6538P, M. freudenreichii and M. varians are illustrated in Figure 1. The denaturation takes place within a temperature range of a few degrees and the absence of RNA is shown by the fact that the base line OD value does not increase while approaching the temperature of thermal denaturation. Table I. Comparison of %GC, genome size and relationship to 02 in Micrococcaceae. %G+C from Tm Genome size Dal tons x 10"9 Relation to 02 * M. lysodeikticus 72 2.82 Aerobe M. salivarius 71.8 3.25 Aerobe M. roseus 70.1 3.31 Aerobe M. varians 69.6 3.4 Aerobe S. lactis 69.0 N.T. Aerobe M. freudenreichii 58.9 2.58 Aerobe M.sp. 250 53 1.42 Fac. M.sp. 901 35.1 N.T. Fac. S. aureus 6538P 30.5 1.42 Fac. S. aureus 12600 30.5 1.18 Fac. S. aureus V8 30.5 1.05 Fac. Micrococcus sp. "Neufeld" 30.5 N.T. Fac. M. candidus 29.1 N.T. Fac. P. saccharolyticus 28.5 1.26 Fac. P. aerogenes 27 0.82 Anaerobe * Obtained from literature N.T. - not tested. Fac. - Facultative. 3£f Figure 1. Thermal denaturation profile of DNA of (a) S_. aureus 6538P, (b) M. freudenreichii and (c) M. varians. DNA was dissolved in 0.1 x SSC. 37 A comparison was made between the results obtained for %GC by the thermal denaturation (T ) method of Marmur and Doty (1962) and the dens-ity centrifugation method of Schilkraut et al_. (1962) (Table II). The values for %GC obtained from density centrifugation when E_. coli DNA was used as standard were lower in the high %GC region and higher in the low %GC region, than those obtained by denaturation studies. This may be due to the conversion factor which was used for correcting 0.1 x SSC to SSC, before calculating the %GC or due to the uncertainty of the linear-ity of the relationship between Tm and %GC at both very low and very high %GC values (Gasser and Mandel, 1968). 2. Genome Size. The data of Table I shows that there was a close correlation between the %GC and the genome size of members within this family. The aerobic micrococci had large genomes which were more than four-fold larger than that of Peptococcus aerogenes. aerogenes had a genome size simi-lar to the very fastidious organism Haemophilus influenza (Leth Bak et al. 1970) and to the mycoplasma PPLD-39. The values obtained for the other members were intermediate between the extremes. The value obtained for S_. aureus 6538P corresponded closely to that obtained by Leth Bak et al. (1970) for S_. aureus MMCA. Thus within a single family such as this, there is a tremendous range of genetic information as determined by gen-ome size. The method for determining genome size used the genome JE. coli as a standard and incorporated a correction for the %GC of the DNA. It has been successfully applied to a range of DNA isolated from T4 phage 38 Table II. Comparison between the buoyant density and the melting temperature of DNA for obtaining %GC. Organism £ T 3 from Tm %GC From buoyant density M. varians 69.6 65.8 M. freudenreichii 58.9 57.3 S. aureus 6538P 30.5 35.1 P_. saccharolyticus 28.5 33.9 39 to higher bacteria and the results have been confirmed by other methods (Seidler and Mandel, 1971). It was necessary to obtain DNA from station-ary phase cultures of organisms grown in a rich medium to avoid the pos-sibility of isolating DNA with multiple replicating forks, as this would yield Cot values which were falsely high. The genome molecular weights assume that there was no redundancy in the DNA. No strong support for intra-genome homologies in bacteria have been reported although some evidence exists for it in _E. coli (Cutler and Evans, 1967). The degree of homology however was not clear since the duplexes were not well characterized. 3. Cot Hybridization. Some Cot hybridization studies were performed with DNA isolated from organisms with low %GC to see if there was genetic relatedness. A narrow range of DNA is chosen because a difference in %GC greater than 10% makes it unlikely that fragments of the two bacteria will have comp-lementary base sequences. There was almost total homology between the three staphylococci (Table III) and there was up to 50% homology between P_. saccharolyticus and ;S. aureus 6538. However, there was no genetic simi-larity between Veillonella alcalescens, an organism previously declared to be a micrococcus and S_. aureus 6538P, nor was there any similarity between P_. saccharolyticus and P_. aerogenes. This method had been shown by Seidler and Mandel (1971) to be a sensitive and accurate method for hybridization and could be applied to DNA from a wide range of sources. 40 Table III. DNA homologies of selected organisms by Cot. method. Organism Cot. 0.5 % Homology 1. S. aureus 6538P 5.47 S_. aureus V8 4.14 100% Mixture 4.30 S_. aureus 6538 6.1 S_. aureus 1260 5.37 86% Mixture 6.59 3. S.. aureus 6538P 5.78 V_. alcalescens 4.37 0% Mixture 9.92 4. S_. aureus 6538P 5.4 P_. saccharolyticus 6.89 Mixture 9.17 5. P_. saccharolyticus 4.74 P_. aerogenes 3.45 0% Mixture 8.60 41 II. Nutrition It is generally accepted that the earliest bacteria were anaero-bic and that they used as nutrients, amino acids, peptides and other organic compounds that had been formed abiotically in the primordial soup. They therefore had limited synthetic powers and broad nutritional requirements. These organisms would have acquired pathways which could produce activated phosphorylated compounds from abundant intermediates. As these intermediates became limiting, some organisms probably acquired mechanisms for producing them. However, energy metabolism from substrate level phosphorylation was completely dependent upon the possibility of channeling any given organic compounds into a relatively few metabolic sequences in which phosphate activation reactions occurred. Aerobiosis and the development of oxidative phosphorylation increased the efficiency of energy generation and the biosynthetic ability of the organisms and this was reflected in a larger genome size. There was also an increase in the %GC although there does not appear to be a good explanation for this correlation. An examination of the amino acid requirement of members of the Micrococcaceae indicates that this generalization is applicable to them (Table IV). In accordance with their genome size and %GC, there is a direct correlation with their amino acid requirements. The aerobic organ-isms with high %GC could synthesize all their amino acid requirements from glucose and inorganic ammonium salts. Facultative organisms with a low %GC and genome size both required and were stimulated by amino acids and the requirements of P_. aerogenes were so complex that it would not 42 Table IV. Amino acid requirements of the Micrococcaceae. Organism %GC Genome size x 10-9 daltons Absolute Requirement Stimulated by M. lysodeikticus 72 2, .82 None None M. salivarius 71.8 3 .25 None None M. roseus 70.1 3. .31 None None M. varians 69.6 3 .4 None None i - lacti s 69.0 N .T. None None M. freudenreichi i 58.9 2, .58 None Met M. sp.250 53 1 .04 None Met M. sp.901 35.1 N .T. Arg None I- aureus 6538P 30.5 1 .42 Arg, Pro, Val, Cys lieu None s. aureus 12600 30.5 1 .18 Glu, Pro, Arg, Val Cys, None S_. aureus V8 30.5 1 .05 Arg, Val Leu, Cys, Pro, Phe lieu , Gly, M. sp. "Neufeld" 30.5 N .T. Arg, Val His, Pro, Leu, Met M. candidus 29.1 N .T. Try, Cys, Gly, Leu, Pro, Met Glu, Val, None P_. saccharolyticus 28.5 1 .26 Arg, lieu Leu, , Pro, His Val Glu, Cys, Tyr P_. aerogenes 27 0.82 CAN GROW ONLY IN COMPLEX MEDIUM N.T. - not tested. 43 grow in Eagles medium supplemented with glutamic acid. Other organisms with a low %GC have also been shown to be amino acid requiring, for example Clostridium perfringens (Finch, 1969) and Streptococcus agalactiae (Willett and Morse, 1966). III. Amino Acid Transport. The presence of a large number of more or less specific transport systems in the cytoplasmic membranes of bacteria has been established and it is characteristic that the systems for the transport of sugars and amino acids exhibit a high degree of substrate specificity. It is generally believed that transport systems are responsible for the rela-tive constancy of the internal environment of the cell and since transport is one of the highly developed functions of a bacterial membrane system, it was thought worthwhile to study comparatively some properties of amino acid transport in the Micrococcaceae. We chose amino acid transport be-cause unlike the transport of sugars, these were constitutive in most microorganisms unless the amino acid served as both a carbon and a nitrogen source. Sugar transport systems on the other hand were inducible and slight changes in the growth conditions affected the nature of transport systems. The proline transport was studied more intensely because in organisms like E_. coli and P_. aeruginosa, it has been shown that this system is specific and energy dependent (Britten and McLure, 1962; Kay and Gronlund, 1969), whereas in a simple anaerobe like Clostridium per- fringens , it and other amino acids have been shown to pass into the cell by simple diffusion (Finch, 1969). 44 1. Transport of Proline, (a) Uptake of proline. The patterns of amino acid uptake by some micrococci are shown in Fig. 2. M. varians established a constant pool size after ten minutes and maintained this constancy until all the proline in the medium had been removed. The label in the pool then decreased as proline passed from the pool and was incorporated in protein. The uptake of proline by M.sp.250 occurred at a much faster rate and all the external proline was removed from the medium by one minute. The uptake of label into TCA precipitable material was significant even at 15 seconds. S^. aureus was auxotrophic for proline and this system became derepressed when cells were starved for this amino acid. In unstarved cells, there was a low rate of uptake and proline was incorporated into protein in a sigmoidal pattern indicating that it was diluted in a preformed pool. Starved cells on the other hand, showed a rapid rate of uptake and label was linearly incorporated into protein. This indicated pool depletion and this was confirmed by amino acid analysis of the pools of starved cells. P_. saccharalyticus was also auxotrophic for proline and the pattern of uptake of starved and unstarved cells was similar to _S. aureus. It was not possible to grow P^. aerogenes in a chemically defined medium so two approaches were tried in an effort to show uptake. The first approach was to grow cells in a rich medium supplemented with glutamate, then to harvest these cells and resuspend them in a phosphate buffer supplement-ed with glutamate and follow the uptake of radioactivity after the addi-4-5 f Figure 2. Patterns of uptake of proline by (a) M. varians, (b) M.sp.250, (c) P_. saccharolyticus, (d) S_. aureus 6538P. (0), total uptake; (t), protein. f_. saccharolyticus was starved 6 hrs for proline in the presence of 19 other amino acids. S_. aureus was starved in the presence of required and stimulatory amino acids; (A), total in unstarved cells; (A) protein. External concen-tration of proline was 5 x IO"'' M. 46 tion of labelled proline. These results were negative, It had been established that M. varians which had been grown in a rich medium would 14 establish a pool of C-proline if after harvesting the cells were sus-pended in cold phosphate buffer supplemented with labelled proline. This approach was tried with IP. aerogenes, but pool formation could not be shown. M. varians, M.sp.250, :S. aureus and P_. saccharolyticus all showed ability to transport proline against a concentration gradient. (b) Effect of metabolic inhibitors. Most active transport systems in bacteria are sensitive to such metabolic inhibitors as azide and iodoacetamide. The effect of these inhibitors on proline transport was tested by preincubating cells for 30 minutes in the presence of 30 mM azide and 1 mM iodoacetamide and then measuring proline uptake. Transport of proline was completely abol-ished in M. varians, M.sp.250, S_. aureus and IP. saccharolyticus but these inhibitors caused only a partial loss of viability. (c) Effect of temperature. The similarities between the transport mechanisms in dif-ferent bacteria was investigated by conducting transport experiments at two temperatures and comparing the Q values. Table I shows Q values 10 10 for proline transport and these were remarkably similar. It was thought that Q-JQ values may give an indication of whether the system was enzyma-tic but recent evidence of De Gier et al_. (1971) showed that the perme-ability coefficients of non electrolytes through artificial membranes was in the same range as those found for biomembranes. Table V. Kinetic analysis of proline transport Organism K m molar V max moles min'^ mg"^  M. varians 1.3 2.5 x 10"6 2.1 x 10"9 M. sp. 250 1.4 1.7 x 10"6, 7.4 x IO"9, 1.85 x 10"6 9.1 x 10"9 S. aureus 1.5 5 x 10"6 10 x 10"9 Tstarved) S. aureus 1.4 5 x 10"6 5 x 10"9 Tjion starved) P. saccharolyticus N.T.2 2.5 x 10"6 5 x 10"11 Tnon starved) 'Measured at 30 C and for S. aureus. 15 C for M. varians, M. sp. 250 at 37 C and 15 C Not tested. 48 (d) Kinetic analysis. The proline transport systems for four systems displayed saturation kinetics confirming their enzymatic nature. The K^ 's were in the range of 10"^  molar and M.sp.250 showed the highest affinity constant and the fastest V (Table I). Unstarved cells of P. saccharolyticus max — had the lowest V . Figure 3 shows three double reciprocal plots: max 3 . K M. varians shows a linear plot, but M.sp.250 showed split kinetics. The K^  values that were extrapolated for M.sp.250 were close together and it is possible these could have the same value, thus indicating the same transport system was involved. Both starved and non-starved Sj. aureus cells had the same K^  value and were therefore part of the same system. (e) Specificity. The presence of D-proline in the medium or of nineteen other amino acids at one hundred-fold higher concentration than L-proline was without effect on the rate of L-proline transport. The system was there-fore specific for L-proline in all organisms tested except P_. aerogenes where no uptake was shown. (f) Induction or derepression of proline transport. Cells of M. varians which were grown in a glucose, medium sup-plemented with 0.1% proline or in a medium where 0.5% proline was the sole source of carbon and nitrogen did not induce a faster rate of proline transport. On the contrary, proline uptake was repressed by these condi-tions. jS. aureus and P_. saccharolyticus both of which were auxotrophic for proline were derepressed when starved for this amino acid. The pattern of derepression for S_. aureus is shown in Fig. 4. In this case, 49 derepression was maximal after two hours. This derepression required protein synthesis since chloramphenicol prevented further derepression (Table VI). S_. aureus showed more biochemical sophistication than P_. saccharolyticus since S_. aureus cells were able to derepress the proline transport system when they were resuspended in minimal medium supplemented with glucose. In P_. saccharolyticus, however, there was no derepression of transport when cells were resuspended in minimal medium. However, the presence of both required and stimulatory amino acids in the medium resulted in derepression and this effect was maximal when nineteen other amino acids were present in the medium. A twenty-fold increase in rate could be observed under these conditions. A determination of proline in the pool by autoradiography showed that this compound was not chemically modified in S_. aureus or in P_. saccharolyticus. 2. Transport of other amino acids. The transport of some other amino acids was studied comparatively in M. varians, M.sp.250 and S_. aureus. f_. aerogenes was not studied be-cause of difficulties in growing the organism in defined medium. Table VII shows the effect of azide and iodoacetamide on the transport of some other amino acids. All systems studied displayed active transport except the transport of glutamate by M. varians and of histidine by S_. aureus. In these two cases, there was no measurable uptake of these amino acids. Table VIII.shows the kinetic data for methionine transport of M. varians, M.sp.250 and S_. aureus. The 1^  for these transport systems were again in the same range as for proline transport. The double re-50f Figure 3. Lineweaver-Burk kinetics of proline uptake by (A) M. varians, (B) M.sp.250 and (C) S_. aureus 6538P. Rates were calculated at 15 seconds in the presence of increasing concentrations of proline. M_. varians and M.sp.250 were incubated at 30 C. S_. aureus was incubated at 37 C. 50 Figure 4. Effect of proline starvation on the rate of uptake 14 -7 of C-proline. External concentration was 2.34 x 10 M. 1 0 1 1 1 ! L I I 1 2 3 4 STARVATION PERIOD HRS 52 Table VI. Effect of chloramphenicol on the derepression of proline transport. Starvation conditions L-proline uptake moles min'^ mg"^  dry wt cells 0 hours 2 hours 6 hours 6 hours (100 ug/ml CM added after 2 hours) 2.6 x 10"10 7.5 x 10"10 11.7 x 10"10 7.2 x 10"10 Table VII. Effect of metabolic inhibitors on transport of amino acids. Organism Pro lieu His Met Glut Arg M. varians + + + + - N.T. M.sp. 250 + + + + + + S. aureus + + - + + N.T. P. saccharolyticus + + N.T. N.T. N.T. N.T. P. aerogenes - N.T. N.T. N.T. N.T. N.T. + Active transport - No active transport N.T. - Not tested. Table VIII. Kinetic analysis of methionine transport. K V „ m max Orgam sm , ., Molar moles min" mg" M. varians  M.sp.250 S. aureus 2.9 x 10"6 5.5 x 10'6, 6.7 x 10"6 1.2 x 10"6 2.64 x 10 * 1.82 x 10"8, 1.25 x 10"8 6.4 x 10"9 55 ciprocal plots were linear for M. varians and S_. aureus but methionine transport in M.sp.250 showed split kinetics (Fig. 5) similar to proline transport by these organisms. The kinetic data for isoleucine transport are shown in Table IX. The values for M_. varians and _S. aureus were one order of magnitude higher than the K^ 's obtained for proline or methionine transport. Linear double reciprocal plots were obtained in each case this time. _S. aureus was also auxotrophic for this amino acid and when cells were starved for this compound a derepression of transport was observed. The amino acid transport systems of aerobic and facultative micrococci are in the main active transport systems. However, within this group there are signifi-cant differences in the patterns of uptake of amino acids, in the Vmax> and the K 's as well as in the control mechanisms. There was not direct m correlation between these properties and %GC and genome size. Proline transport was specific in each case, except for P_. aerogenes. The in-ability of S_. aureus to actively transport histidine was reflected in an almost negligible amount within the pools of these cells. P_. aerogenes may only transport peptides since it requires a rich growth medium which contains peptides and will not grow in a medium consisting of all known amino acids and vitamins. IV. Properties of Membrane Bound Enzymes. The energy producing systems that drive active transport involve oxidative and phosphorylative enzymes that are membrane bound. Under Figure 5. Lineweaver-Burk kinetics of methionine uptake of M.sp.250. Rates were calculated at 15 seconds in the presence of increasing concentrations of methionine. Incubated at 30 C. 57 Table IX. Kinetic analysis of isoleucine transport. K V m max Organism , , molar moles min" mg" M. varians 12.5 x 10"6 1.5 x 10"10 M.sp. 250 3.9 x 10"6 3.1 x 10"9 S.. aureus 13.4 x 10"6 6.7 x 10~9, 5.0 x 10"9 58 aerobic conditions many organisms including E. coli, B_. subtilis and S_. aureus utilize oxidative enzymes to drive the active transport of amino acids whereas in anaerobically grown E. coli and aerobically grown Streptococcus faecal is phosphorylative processes have been implicated. The Micrococcaceae include species that are aerobic, facultative and anaerobic and it was therefore of interest to study the association of some of these enzymes with the cytoplasmic membrane and to determine some conditions that affect their release. 1. Isolation of membrane preparations. Table X summarizes the treatments used to obtain membrane fractions from four different species. M. varians, M. lysodeikticus and S_. aureus were completely susceptible to peptidoglycan degrading enzymes. The extent of cell wall hydrolysis could be followed by phase-contrast microscopy and the Gram stain. Under phase contrast microscopy the cells lost their retractility and a Gram stain revealed Gram negative debris. DNase was added to reduce the viscosity of the preparation. S_. aureus and M. varians formed vesicles but M. lysodeikticus membranes became frag-mented. Cells of P_. aerogenes did not lyse when treated with lysozyme but they were, however, far more susceptible to lysis by passage through a French pressure cell after pre-treatment with this enzyme. Therefore, there was some evidence that structural components of the cell wall had been attacked. Very few whole cells remained after the pressure cell treatment and these could be removed by low speed centrifugation. The membrane containing pellets from these treatments were washed twice with Table X. Conditions for producing membrane preparations. Organism Treatment M. varians Lysozyme 200 yg/ml, up to 90 min at 30 C Centrifuge 2x at 35,000 x £ M. lysodeikticus Lysozyme 200 yg/ml, up to 30 min at 30 C Centrifuge 2x at 35,000 x q_ S.. aureus Lysostaphin 1 - 5 units/ml, up to 60 min at 37 C. Centrifuge 2x at 35,000 x q_ JP. aerogenes Lysozyme 1 mg/ml for 60 min at 37 C, French pressed 2x at 20,000 p.s.i. Low speed centrifugation then 2x at 35,000 x £ 60 TKMG buffer to remove cytoplasmic material and ribosomes. Analysis of the washings showed that additional washings with this buffer removed very little protein. When peptidoglycan degrading enzymes act on the cell walls of Gram positive cells, the wall is entirely degraded to low mole-cular weight soluble components and membrane preparations have negligible cell wall material associated with them (Weibull, 1968). An added advan-tage to working with Gram positive cells is that the cell walls are devoid of enzymes and so even if contamination occured, it is not likely to be of importance. There is likely to be cell wall associated with some of the membrane preparation of P_. aerogenes, since lysozyme did not completely degrade the wall. Many of the membrane bound enzymes of Gram negative organisms have been studied under conditions where cell wall material was still bound to the cytoplasmic membrane. 2. Sucrose Density Gradients. Equilibrium density centrifugation in sucrose of washed mem-brane fractions showed one major band for M_. varians, M_. lysodei kticus and for aerobically and anaerobically grown S_. aureus. Figure 6 shows the equilibrium density profile for the membrane preparation of M. varians after these membranes had been washed six times with TKMG. Only a single band was obtained and thus suggested that the material was homogeneous. IP. aerogenes on the other hand, banded as two major peaks. An SDS gel profile of material from both peaks revealed similar profiles and there-fore the peak which banded at 63.2% sucrose probably had cell wall mat-erial associated with i t . S_. aureus showed one major band at 59.60% sucrose and one minor band at a lighter density. This lighter band prob-Figure 6. Sucrose density profiles of membrane preparations of (A) M. varians and (B) P_. aerogenes. Sucrose gradients were from 30 - 70% w/w (see Materials and Methods). 62 ably corresponded to the mesosomal upper band described by Popkin et al. (1971) for S_. aureus 6538P. M. lysodei kticus showed one major band at 60% sucrose and a number of minor bands at lighter densities as was shown for M. lysodeikticus by Sal ton et al- (1968). 3. Relative Amount of Lipid in Membranes. Gram positive bacteria, unlike Gram negative, have all of their phospholipid associated with the cytoplasmic membrane. Table XI shows the relative amount of lipid in membranes. The lipid was determined as phospholipid, by estimating the inorganic phosphate and using an approxi-mate conversion factor of 25.5. M. lysodeikticus had the highest ratio of lipid to protein. This value was higher than the maximum of 30% ob-tained by Salton (1966) using a different strain of M. lysodei kticus. Patterson and Lennarz (1971) have reported that membranes from Gram posi-tive bacteria could have from 20% to 50% of the dry weight of their mem-branes consisting of phospholipids. Both anaerobic and aerobic staphylo-cocci have less than 20% phospholipid in their membrane preparation, however some of the lipid could be glucolipid and would not be determined by this procedure. Although there was a positive correlation between high %GC and phospholipid content, both the content of total lipid and the degree of extraction can vary with different membrane systems. 4. Enzyme Content in Membranes. Known membrane markers such as NADH dehydrogenase and ATPase were strongly associated with the membrane and negligible activity was found in the supernatants of the lysates. On the other hand, the soluble enzyme fumarase was present in the supernatants of the lysates of M. varians and M. lysodei kticus but was absent from the corresponding membrane prepara-Table XI. Relative amount of lipid in membranes of Micrococcaceae. Organism Ratio Lipid + protein^ M. varians 0.27 M. lysodeikticus 0.37 S_. aureus (aerobic) 0.17 S_. aureus (anaerobic) 0.19 P_. aerogenes 0.18 Lipid was extracted by the method of Bligh and Dyer (1958). Inorganic phosphate was measured by the ashing method t>f Ames (1966). Protein was determined by the method of Lowry et al_. (1951). Average of 3 determinations. 64 tions. The distribution of eight enzymes, some of which could be related to transport are shown for some of these membrane systems in Table XII. It is apparent there is a strong correlation between high %GC and aerobiosis and the number of functions present. P_. aerogenes in accordance with its low genome size and anaerobic state has only ATPase and NADH dehydrogenase as membrane bound enzymes from this group. Membrane bound enzymes in S_. aureus varied with the growth conditions. Under anaerobic conditions only ATPase and NADH dehydrogenase were present but under aerobic conditions, membrane bound oxidative enzymes were present. These cells were also subject to catabolite repression since succinate dehydrogenase and malate dehydrogenase were detected only during aerobic growth in the absence of glucose. 5. Wash Treatments The generally accepted model of membrane structure is the fluid mosaic model of Singer and Nicolson (1972). The model consists of a lipid bilayer which has proteins embedded in it at various depths. Those proteins at the surface are stabilized by hydrophilic interactions which in some cases,involve divalent cations while regions of proteins deeply embedded in the bilayer have been found to have regions of hydrophobicity. In addition, divalent cations stabilize the polar heads of phospholipids. Membranes were treated with low-ionic strength buffers containing EDTA to see if the removal of divalent cations would result in the release of proteins of lipoprotein complexes from the membrane structure. Non-ionic detergents such as Triton-X are known to break hydrophobic protein-protein and protein-lipid bonds. They bind to membrane proteins but not Table XII . Enzyme complement of membranes of some Micrococcaceae. n . ATPase NADH NADH Malate Succinate l-lactate d-lactate Adenosine u r g a n i s m Oxidase DeH DeH DeH DeH DeH deaminase M. varians M. lysodeikticus + + + + + trace 2 2 _S. aureus (aerobic) + + + + +a +^  S_. aureus (anaerobic) + + P_. aerogenes + - + -M^embrane material suspended in TKMG. 2 Grown in the absence of glucose. 66 cytoplasmic proteins and they change the phase characteristics of the medium. A number of detergents such as Lubrol-WX, deoxycholate and Brij 53 were tried, but in preliminary experiments, Triton-X-100 was found to be the detergent of choice in solubilizing NADH dehydrogenase activity. This detergent was then used in subsequent treatments. (a) Effect of concentration of detergent. The effect of concentration of detergent on the solubiliza-tion of NADH dehydrogenase and ATPase was examined. The total activity and specific activity of the solubilized enzyme is recorded in Table XIII. The membrane preparation was suspended at room temperature in TKMG, with and without the addition of detergent and then centrifuged at 108,000 x a_ for 30 minutes. The pellets were resuspended in an equal volume of TKMG. These results were expressed in arbitary units and the same amount of membrane material was used in each assay. 0.2% Triton-X was as effect-ive as 1% in solubilizing activity except for S_. aureus (anaerobe). In the case of P_. aerogenes and S_. aureus (aerobe), the high concentration inhibited the enzyme. Salton (1971) had reported that some enzymes were stimulated by particular concentrations of detergent and when this con-centration range was exceeded, inactivation occurred. The specific act-ivity of Triton-X washed membranes was increased in all cases. Triton-X had a marked stimulatory effect on the activity of ATPase and the 1.0% concentration was effective in releasing activity into the supernatant.(Table XIV). (b) Effect of removal of divalent cations. Divalent cations were removed from isolated membrane prepara-Table XIII. Effect of concentration of Triton X on NADH dehydrogenase distribution. Control 0.2% Triton X-100 1.0% Triton X-100 Organism Membrane Supernatant Membrane Supernatant Membrane Supernatant M. varians Total1 Activity .28 0 .13 .09 .13 .11 2 Specific Activity 2.04 0 1.15 1.03 1.1 1.1 S. aureus "(aerobic) Total Activity .47 .04 .16 .54 .16 .43 Specific Activity 3.2 0.7 1.07 7.5 1:1 5.2 S. aureus "(anaerobic) Total Activity .02 0 .02 .01 0 .02 Specific Activity .11 0 .12 .16 0 .23 P. aerogenes Total Activity .39 .01 .25 .07 .18 .07 Specific Activity 2.0 0.17 1.3 .7 .94 .7 1 Total activity as AODggg/min "Specific activity as AOD^ QQ/min/mg protein Membranes were suspended in TKMG, TKMG +0.2% Triton X, TKMG + 1% Triton X at room temperature for 15 minutes, then centrifuged at 4 C for 30 minutes at 108,000 x g_. Membranes were resuspended in an equal volume of TKMG. Equal aliquots of membrane and supernatant were assayed for activity. Table XIV . Effect of concentration of Triton-X on ATPase distribution. Organism M. varians Control 0.2% Triton-X 1.0% Triton-X Membrane Supernatant Membrane Supernatant Membrane Supernatant Total 1 Activity Specific'1 Activity 0.055 0.62 0 0 0.175 1.6 0 0 0.33 3.1 0 0 S. aureus (aerobic) Total Activity Specific Activity 0.18 0.87 0 0 0.47 2.8 0 0 0.12 0.94 0.24 1.2 S. aureus Tanaerobic) Total Activity Specific Activity 0.14 0.79 0 0 0.12 9.72 0 0 0 0 0.47 5.5 P_. aerogenes Total 0.17 Activity Specific Activity 0.87 0 0 0.3 1.56 .1 1.0 ^Total activity as A0DggQ/5 min. 2 Specific activity as A0DggQ/5 min/mg protein 3Method as for Table XIII. 0.42 2.2 0.19 1.9 69 tions in order to test for the dissociation and reassociation of NADH dehydrogenase from the membranes. Membrane preparations containing approx-imately 90 mg of membrane protein were dialyzed in the cold against two changes of dilute buffer containing EDTA (DTE) for 36 hours and then centrifuged at 108,000 x £ for thirty minutes. Total activity of the membranes and supernatant fluid was measured. The fractions were then recombined and dialyzed against 0.03 M Tris buffer, pH 7.5 containing 25 m MgCl2 for 12 hours and the activity of the pellet and the supernatant were again assayed (Table XV). There was no activity in the supernatants of TKMG washed cells. The removal of divalent cations resulted in the solubilization of a substantial amount of activity in M. lysodeikticus and S.. aureus (aerobically grown). There was very little NADH dehydrogenase in this preparation of S_. aureus membranes from anaerobically grown cells. No activity was lost from P_. aerogenes and there was little solubilization from M. varians. After twelve hours of dialysis against a 25 mM MgCl^  considerable activity for M. lysodei kticus became reassociated with the membrane, but reassociation was not complete. There was a small loss of total activity during the dialysis procedure. 6. Sequential washings. Once it was established that DTE and Triton-X were capable of solubilizing enzymes from membranes, it was of interest to examine the patterns of release, by sequential washings, of the membrane bound enzymes listed in Table XII. A flow diagram of the procedure used is shown in Figure 7. The membrane pellets were washed repeatedly until little additional protein was being removed. The washing procedures Table XV. Dissociation and reassociation of NADH dehydrogenase by dialysis against EDTA and magnesium. Dialysis against. Dialysis against Organism dilute Tris EDTA Tris + Mg2+ 3 % Membrane Supernatant Total Membrane Supernatant Total l_oss_ M. varians 82 9 91 73 6 79 13 M. lysodeikticus 242 445 687 360 260 620 10 S. aureus (aerobic) 34 142 176 32 68 150 15 S. aureus (anaerobic) 7 4 11 8 3 11 0 P. aerogenes 91 0 91 90 0 90 0 Activity expressed in arbitrary units as total AODggg/min. Preparation dialysed against 0.003 M Tris HCl pH 7.5 + 10 mM EDTA for 36 hrs in the cold. Preparation dialysed against 0.03 M Tris HCC pH 7.5 + 25 m'M MgCl. for 12 hours in the cold. 71 Peptidoglycan degrading enzymes French pressure cell (if necessary) DMase 10 yg/ml Centrifuge at 35,000 x q_ for 30 minutes Resuspend pellet in TKMG (same volume) Centrifuge at 35,000 x g for 30 minutes Resuspend pellet in TKMG (same volume) resuspended in TKMG (control) (2-8 mg/ml) Divide into 3 equal fractions Centrifuge at 108,000 x £ for 30 minutes (check supernatant fluid for protein) resuspend in DTE resuspend in 0.2% Triton X-100 in TKMG Continue washing until no more protein is solubilized. Keep samples of membrane and supernatants for assays. Figure 7. Flow sheet for sequential washings. 72 changed the appearance of the membranes. Neither TKMG nor DTE solubil-ized anypigment from M. varians membranes, however most of the pigment was removed by the first Triton-X wash and successive washes removed all the pigment. TKMG did not appear to change the physical appearance of M. lysodeikticus membranes but both DTE and Triton-X had a marked effect. DTE removed some of the pigment and the membrane became "sticky" on the first wash and a large amount of material was solubilized leaving behind small fragments. Triton-X almost completely solubilized the membrane. DTE and Triton-X again removed material from membranes of aerobically grown _S. aureus, but the membrane was not solubilized to the extent of M. lysodei kticus. Neither S_. aureus membranes (anaerobically grown) nor those of f_. aerogenes were pigmented but washing with Triton-X gave these membranes a lighter appearance, (a) Sucrose density gradients. The effect of washing the membranes with DTE or Triton-X was examined by sucrose density centrifugation. In all the aerobic merTH brane systems examined, the washings with DTE resulted in a less dense membrane indicating that the washings had removed relatively more protein than lipid (Table XVI). However, membranes of anaerobically grown S_. aureus and the lighter peak of P_. aerogenes became more dense indicating that relatively more lipid was lost than protein. Lukoyanova ejt al_. (1961) have reported destruction of M. lysodeikticus membranes by washing with EDTA and Munoz et al_. (1968) reported the loss of 85% of the original starting protein after treatment with EDTA and dilute Tris buffer. With Table XVI. Effect of washing on membrane density . TKMG3 DTE Triton-X 0 r g a m s m % Sucrose % Sucrose % Sucrose M. varians M. lysodeikticus .S. aureus (aerobic) S. aureus (anaerobic) 60 60 59.2 59.6 54.7 41.5 55.6 61.8 66.2, 63.8 2 30^  62.8 61.4 P_. aerogenes 63.2, 56 61.5, 60.4 62.7, 60.5 Density expressed as % sucrose w/w from the refractive index of the fractions. All the OD^ gQ absorbing material occurred at the top of the gradient. 'All systems were compared after an equal number of washings. 74 the exception of the more dense peak of P_. aerogenes and M. lysodei kticus, all the membranes became more dense after repeated washings with Triton-X. M. lysodeikticus membranes became very light after one wash treatment and C^gQ material was found at the top of the gradient. The density of the other membrane preparations indicated that relatively more lipid than protein was removed by Triton-X. This finding has been shown for other strains of M. lysodei kticus (Salton,ejt al_. 1968) and S^. aureus (Lengsfeld, et.^al 1973). (b) Solubilization of protein Triton-X was the most effective agent in solubilizing protein from five membrane preparations (Table XVII). The amount of protein sol-ubilized varied slightly in similar treatments of the same preparation, but the patterns of release were similar in each case. The five membrane systems showed heterogeneity with respect to the amount of protein re-leased. In these experiments, M. lysodeikticus membranes were most readily solubilized and even the amount of protein solubilized in the control wash was significant. However, the amount of protein released by DTE or Triton-X was significantly higher. After two washings with Triton-X, there was little further solubilization of protein. A profile for the release of protein from membranes of M. varians is shown in Fig. 8, as an example. The pattern obtained for the other membranes showed a difference only to the extent that DTE could solubilize protein. An analysis of the solubilized protein by SDS gel electrophoresis showed that there was similarity in the profiles. All the major protein bands that Table XVII. Protein solubilized by the washing procedure. Organism Wash Wash number Total recovered Total left on Treatment 1 2 3 4 5 6 in supernatant membrane Percent M. varians TKMG 2 1 1 0 0 - 4 92 DTE 2 2 2 1 1 - 8 92 Triton-X 16 11 11 6 0 - 44 61 M. lysodeikticus TKMG 20 8 2 - - 30 74 -DTE 33 9 1 - - 43 25 Triton-X 76 21 11 - - 108 18 S. aureus TKMG 4 2 2 2 1 11 78 (aerobic) DTE 9 3 1 1 0 - 14 70 Triton-X 22 5 2 1 1 - 31 50 S. aureus TKMG 2 0 1 1 1 2 7 63 (anaerobic) DTE 6 8 5 4 2 1 26 82 Triton-X 21 5 1 1 0 0 29 54 P. aerogenes TKMG 3 2 2 1 0 - 8 85 DTE 5 5 1 0 0 - 11 100 Triton-X 7 4 4 1 2 - 18 78 Starting material equals 100%. 1G ? Figure 8. Solubilization of protein from M. varians membranes by washing procedures. Concentration of protein approxi-mately 8 mg/ml. TKMG, • = not solubilized, • = solu-bilized; DTE, 0 =• not solubilized, I = solubilized; Triton-X, A = not solubilized; A = solubilized. 40 0 1 2 3 4 5 WASH NUMBER 7 7 f Figure 9. SDS gel electrophoresis of protein solubilized by (a) DTE and (b) Triton-X from anaerobically grown S_. aureus. Scan speed = 1 cm/min. Chart speed = 2 inches/min. Absorbance 60 Onm Absorbance 60 0nm were present in the DTE wash were present in the Triton-X wash but there were additional bands in the latter. The profile of S_. aureus and M. lysodeikticus were most nearly alike. Figure 9 shows the profiles of solubilized membrane protein from S_. aureus grown anaerobically. (c) Solubilization of phospholipid. The pattern of solubilization of phospholipid varied for the different membranes and the only correlation that could be made was that organisms with high %GC appear to have more phospholipid (Table XVIII). Triton-X was most effective in the extraction of lipids from the mem-branes. All the lipid was solubilized from M. lysodeikticus membranes after three washes with Triton-X (Fig. 10). DTE and TKMG were also ef-fective in solubilizing lipid from M. lysodei kticus but not to the extent of Triton-X. In contrast to the small amount of protein solubilized from M. varians, membranes by TKMG and DTE, substantial phospholipid was solubilized by these washes (Table XVIII), and this resulted in lipid enriched fractions. It should be noted that the ratio of protein to lipid in the first Triton-X wash was similar to the ratio that existed in the membrane. This enrichment was not as marked for M. lysodei kticus or for either aerobic and anaerobic S_. aureus. The extraction of phos-pholipid from P_. aerogenes membranes gave variable results between dif-ferent extractions. (d) Solubilization of enzymes, aa. NADH dehydrogenase. This enzyme had the highest specific activity of any of the membrane bound enzymes studied and was present in each membrane 79 Table XVIII. Relative amounts of l i p i d in washings of membranes of M. variansJ Wash Treatment Wash number 2 3 Percent TKMG 72 83 88 100 DTE 85 72 68 47 Triton-X 30 43 30 49 Results expressed as 1 ipid 1ipid + protein percent. 3 Of Figure 10. Solubilization of phospholipid from M. lysodeikticus by washing procedure. TKMG, • = not solubilized, • = solubilized; DTE, 0 = not solubilized, • = solubilized; Triton-X, A = not solubilized; A = solubilized. 60 1 2 W a s h N u m b e r 81 system. In the presence of Triton-X, its specific activity was in-creased by as much as 50% (Table XIX). DTE on the other hand, had a var-iable effect on activity. It caused a marked stimulation in the case of P_. aerogenes on the one hand, but inhibited the activity of anaerobic S_. aureus membranes on the other. The addition of magnesium did not reverse this inhibition. Triton-X was effective in solubilizing the enzyme in all cases and with the exception of P_. aerogenes solubilized the bulk of the enzyme in the first wash (Table XX). DTE was effective in solubilizing this activity in three membrane systems but was less effective than Triton-X. There was some loss of activity which was unaccounted for and part of this may be due to loss between the homogenization steps. It appears that the activity of solubilized enzyme is increased in the case of P_. aerogenes. The profiles of solubilization were similar and Figure 11 shows the profile obtained for S_. aureus (aerobe), bb. Succinate dehydrogenase This enzyme has been reported to be firmly bound to the membrane in a number of systems (Salton, 1971) and to be present usually in the membranes of aerobic organisms. Neither TKMG nor DTE solubilized the activity in any of three bacteria (Table XXI), but Triton-X was effective. The greatest amount of enzyme was solubilized after the second wash in two cases, and this was in contrast to NADH dehydrogenase which was solubilized to the greatest extent in the first wash procedure. Even with M. lysodeikticus where the bulk of the succinate dehydrogenase was solubilized in the first wash, a substantial amount of activity was still released in the second wash, again in contrast to NADH Table XIX. Specific activities of membrane bound enzymes in the presence of TKMG, DTE and Triton-X. Organism Treatment ATPase SDH NADH Deh d-LDH 1-LDH MDH Aden-D Percent varians TKMG 100 100 100 100 100 100 100 DTE 41 72 105 0 0 58 0 Triton-X 177 72 142 60 39 38 96 M. lysodeikticus TKMG 100 100 100 100 100 DTE 70 74 135 N.P. N.P. 68 0 Triton-X 130 104 125 49 72 S. aureus TKMG 100 100 100 100 (aerobic) DTE 107 95 139 N.P. 100 N.T. N.P Triton-X 230 68 n o 58 S. aureus TKMG 100 100 (anaerobic) DTE 59 N.P. 48 N.P. N.P. N.P. N.P Triton-X 240 107 P. aerogenes TKMG 100 100 DTE 200 N.P. 188 N.P. N.P. N.P. N.P Triton-X 260 150 N.P. - not present N.T. - not tested. Table XX. NADH dehydrogenase solubilized by the washing procedure. Or ism Wash Wash number J q U 1 r e c o v e r e d T o t a 1 l e f t o n ^ Treatment -, 0 . n c c in supernatant membrane Percent M. varians TKMG 0 0 0 0 0 - 0 20 DTE 7 0 - 0 0 0 - 7 98 Triton-X 84 11 7 5 0 107 2 M. lysodeikticus TKMG 7 7 0 - - - 14 75 DTE 22 11 6 - - - 39 23 Triton-X 100 6 0 106 3 S. aureus TKMG 0 0 0 0 0 - 0 85 (aerobic) DTE 15 15 7 8 5 - 50 27 Triton-X 72 9 6 6 8 106 3 S. aureus TKMG 4 3 0 0 0 0 7 88 (anaerobic) DTE 10 10 10 10 10 0 50 58 Triton-X 62 10 7 5 4 0 88 0 P. aerogenes TKMG 0 0 0< 0 0 - 0 89 DTE 0 0 0 0 0 - 0 115 Triton-X 19 15 13 13 13 - 73 60 Total activity on the starting material in each wash treatment was set at 100%. Equal aliquots were used for each preparation. &4f Figure 11. Solubilization of NADH dehydrogenase from membranes of aerobically grown S_. aureus. TKMG, • = not solubilized, • = solubilized: DTE, 0 = not solubilized, • = solu-bilized; Triton-X, A = not solubilized, • = solubilized. IP — A WASH NUMBER Table XXI. Succinate dehydrogenase solubilized by the washing procedure. Organism Wash Treatment Wash number Total recovered in supernatant Total left on membrane Percent M. varians TKMG 0 0 0 0 0 0 59 DTE 0 0 0 0 0 0 54 Triton-X 24 62 19 0 0 105 0 M. lysodeikticus TKMG 0 0 0 - - 0 95 DTE 0 0 0 - - 0 35 Triton-X 46 27 0 - - 73 7 S. aureus TKMG 0 0 0 0 0 0 65 (aerobic) DTE 0 0 0 0 0 0 85 Triton-X 14 26 6 6 4 56 5 86 dehydrogenase. Triton-X was inhibitory to the enzyme in two cases (Table XIX), but this was a variable effect and in some experiments, this inhibition was not evident. DTE had a marked inhibitory effect on M. varians and M. lysodeikticus which was not reversed by the addition of magnesium ions. The amount of activity recovered from the membranes of M. varians was quantitative but in most other cases, there was as great a loss of activity as 65% and it is possible that some enzyme is re-leased in an inactive form. The profile of release of the enzyme is shown for M. varians (Fig. 12). cc. d- and 1-Lactate dehydrogenase. These membrane bound, NAD independent enzymes have been associated with amino acid transport systems in such organisms as E_. coli and B_. subtil is (Kaback, 1972). The enzyme in M. varians showed a marked requirement for magnesium and resuspension of this membrane system in DTE resulted in a total loss of enzyme activity which could be completely recovered by the re-addition of a constant amount of magnesium (80 mM) to the reaction mixture after each successive wash. This procedure re-sulted in an increase in activity on the membrane that reached a maximum after two to three washings (Fig. 13). The enzymes of S_. aureus did not show a similar requirement for magnesium and after repeated DTE washings, there was some loss of activity which was not recoverable in the superna-tant. Common to these enzymes and in marked contrast to NADH or succinate dehydrogenase, was the fact that Triton-X almost totally inactivated the enzyme. This inhibitory effect could be partially overcome by the Figure 12. Solubilization of succinate dehydrogenase from membranes of M. varians by washing procedures. TKMG, • = not solubilized, • = solubilized; DTE, 0 = not solubilized, • = solubilized; Triton-X, A = not solubilized, A = solubilized. TOTAL ABSORBANCE C H A N G E / Ml N U TE (60 0 n m) iss <<n -co ,o O O O O o 88 F Figure 13. Solubilization of 1-lactate dehydrogenase from membranes of (a) aerobically grown S.. aureus and (b) M. varians by washing procedures. TKMG, • = not solubilized, • = solubilized; DTE, 0 = not solubilized, • = solubilized; Triton-X, A = not solubilized, A = solubilized. 80 mM MgCl5 was added to assay mixture of DTE washed cells. 88 89 addition of 20% glycerol to the buffer system. There was from 40% to 60% inhibition of activity when the membranes were suspended in 0.2% Triton in TKMG (Table XIX). However, the detergent readily solubilized the enzyme as is shown in Table XXII and in each case the activity of the solubilized enzyme was greater than it was in the presence of membranes dd. Malate dehydrogenase. This membrane bound enzyme showed similarities to d- and 1-lactate dehydrogenase. Triton-X markedly inhibited at very low concen-trations and this effect could be partially reversed by 20% glycerol. There was an inhibition of activity from 51% to 62% (Table XIX). Triton-X readily solubilized enzyme after the first wash and the activity of the solubilized form was similar to membrane bound lactate dehydrogenase in that it was enhanced when solubilized (Table XXIII). Magnesium did not appear to play a role in the activity of M. lysodeikticus since the loss of activity over three washes was not recovered by the addition of magnesium ions. DTE did however solubilize some activity from these membranes. M. varians appeared to have a critical requirement for magnesium ions which was much lower than ford- and 1-lactate dehydrogenase on the same mem-brane system. Repeated washings resulted in a very marked increase in the total activity on the membrane (Fig. 14). ee. ATPase. ATPase in common with NADH dehydrogenase is present in all of the membrane systems studied. Its role has been concerned with energy mechanisms in both aerobes and anaerobes and in the transport of ions and amino acids (Harold, 1972). Triton-X has a stimulatory effect Table XXII. d- and 1-Lactate dehydrogenase solubilized by the washing procedure. Organism Wash Treatment Wash number Total recovered in supernatant Total left on membrane 1 2 3 4 5 Percent M. varians TKMG 0 0 0 0 0 0 114 d-lactate DTE 0 0 0 0 0 0 176 Triton-X 130 0 0 0 0 130 0 M. varians TKMG 0 0 0 0 0 0 91 1 -lactate DTE 0 0 0 0 0 0 120 Triton-X 125 0 0 0 0 125 0 S. aureus TKMG 0 0 0 0 0 0 64 1-lactate DTE 0 0 0 0 0 0 67 (aerobic) Triton-X 135 0 0 0 0 135 0 U3 O Table XXIII. Malate dehydrogenase solubilized by the washing procedure. Wash number Organism Wash Total recovered Total left Treatment 1 2 3 4 5 in supernatant on membrane Percent M. varians TKMG 0 0 0 0 0 0 105 DTE 0 0 0 0 0 0 200 Triton-X 145 0 0 0 0 145 0 M. lysodeikticus TKMG 0 0 0 - - 0 83 DTE 17 6 3 - - 26 23 Triton-X 106 0 0 _ 106 0 3ZP Figure 14. Solubilization of malate dehydrogenase from membranes of (a) M. varians and (b) M. lysodeikticus by washing procedures. TKMG, • = not solubilized, •= solubilized; DTE, 0 = not solubilized, • = solubilized; Triton-X, A = not solubilized, • = solubilized. E .c. j 3 0 0 h O 03 U O .to -Q < O 2 0 0> 1 0 0 h —1$- 43 til 53 a 1 2 3 4 5 60 0 40 0 W ASH NUMBER WASH NUMBER Table.XXIV. ATPase solubilized by the washing procedure. Organism Wash Wash i lumber Total recovered Total left on Treatment 1 2 3 4 5 6 from supernatant membrane Percent M. varians TKMG 0 0 0 0 0 - 0 90 DTE 0 0 0 0 0 - 0 63 Triton-X 12 25 67 15 0 - 119 9 M. lysodeikticus TKMG 0 0 0 - - - 0 75 DTE 31 0 0 - - - 31 0 Triton-X 60 83 30 - - - 175 0 S. aureus TKMG 0 0 0 0 0 _ 0 75 (aerobic) DTE 38 0 0 0 0 - 38 0 Triton-X 12 6 12 24 12 - 66 17 S. aureus TKMG 0 0 0 0 0 0 0 100 (anaerobic) DTE 0 0 0 0 0 0 0 0 Triton-X 4 4 4 5 8 7 32 58 P. aerogenes TKMG 0 0 0 0 0 - 9 100 DTE 0 0 0 0 0 - 0 47 Triton-X 42 0 0 0 0 - 0 62 94f Figure 15. Solubilization of ATPase from membranes of M. varians by washing procedures. TKMG, • = not solubilized, • = solubilized; DTE, 0 = not solubilized, • = solu-bilized; Triton-X, A = not solubilized, A = solubilized. 94 95 on this'enzyme in all cases and this is in common with NADH dehydrogen-ase (Table XIX). The total amount of material solubilized by Triton-X over a series of washings varied (Table XXIV), and the least activity was solubilized from S-. aureus and P_. aerogenes. In each case except for P_. aerogenes, the enzyme was solubilized to the greatest extent after more than one wash. It had been shown in earlier experiments that ATPase was not always released after the first wash for P_. aerogenes. DTE was effective in solubilizing activity from M. lysodei kticus and S_. aureus. In all cases, successive washings with DTE resulted in a loss of acti-vity and divalent cations could not restore this loss. The pattern of solubilization of M. varians is shown in Figure 15. ff. Adenosine deaminase. This enzyme was found associated with membranes of M. varians and M. lysodei kticus, but was absent in other organisms.. The enzyme has been described as a cytoplasmic enzyme by Sal ton (1971). Triton-X did not stimulate but repressed the enzyme and solubilized it after one wash treatment. DTE totally inactivated the enzyme and control washes resulted in less than a 10% loss of activity (Table XIX). Of the complement of enzymes studied, organisms which had a high %GC had more enzymes present in their membranes. However, when grown aerobi-cally the facultative organism S_. aureus which has a low %GC was able to synthesize a number of membrane associated oxidative enzymes. There was a great deal of heterogeneity in terms of specific activities of particular enzymes and in terms of the relative activity to each other in a single mem-brane system. Nevertheless, some pattern does emerge with respect to the 96 washing of membranes with Triton-X. Firstly the solubilization of enzymes by this treatment was not random and in every membrane system, ATPase was firmly bound and although some activity was solubilized by the first Triton-X wash, most of the enzyme remained bound to the membrane. NADH dehydrogenase was relatively less tightly bound and the greatest amount of activity was released in the first wash. However, in general, more than one wash was required to solubilize most of the enzymes. In common with all the membrane enzymes, ATPase and NADH dehydrogenase were stimulated by Triton-X and the degree of stimulation was greatest for ATPase. Thus, in terms of these properties which may point to the location of this function in the membrane, there is a similarity between an anaerobe like P_. aerogenes and an obligate aerobe like M. varians. Succinate dehydrogenase of the various organisms also shows similarities in its reaction to Triton-X. Adenosine deaminase, lactate and malate dehydrogenase showed the same properties with respect to Triton-X in different membrane systems, namely they were quantitatively solubilized in the first wash and lactate and malate dehydrogenase were markedly inhibited by the detergent. DTE was heterogeneous in its pattern of solubilization and the effect on different enzymes. V. Membrane Subunit Structure. The membranes were dissolved in 1% sodium dodecyl sulfate and then run on 0.1% SDS gels to determine the total number of subunits and to estimate the approximate size range of the subunits. These values were 97 then correlated with genome size to establish whether increasing genome size conferred a possible increased role for proteins in the cytoplasmic membranes. The subunit profiles for M. varians and anaerobically grown S_. aureus are shown in Fig. 16. Schnaitman (1971) reported that E_. coli grown anaerobically and aerobically gave the same basic gel pattern. In the present study, it was found that the patterns obtained for anaero-bic S_. aureus was different from that obtained for aerobic aureus. P_. aerogenes had three subunit bands which accounted for the majority of its membrane protein. Table XXV lists the total subunits counted when approximately 100 yg of membrane protein was applied to each gel. The number of subunits observed in the gels of membranes from the obli-gate aerobes was about double the number observed for both aerobic and anaerobic S^. aureus. When catalase was used in a split gel as a 60,000 M.W. marker, most of the subunits in the aerobic membranes had a molecular weight greater than 60,000. In contrast, the protein subunits of the organisms with a small genome size were less than 60,000 in molecular weight. There was thus a positive correlation between increased genome complexity and the size and the number of protein subunits which comprise the cytoplasmic membranes. 93P Figure 16. SDS gel electrophoresis of membranes of (a) anaerobically grown S_. aureus and (b) M. varians. See Materials and Methods. Scan speed = 1 cm/min. Chart speed = 2 inches/ min. Arrow shows catalase marker. 00 Table XXV. Subunits of micrococcal membranes as determined by SDS gel electrophoresis. Source of membrane Subunits M.W. less than 60,000 M.W. greater than 60,000 Total M. varians 16 23 39 M. lysodeikticus 15 22 37 S. aureus (aerobic) 13 8 21 S. aureus (anaerobic) 14 9 23 P. aerogenes 10 9 19 In each instance, approximately 100 ug of membrane protein was dissolved in 1% SDS + 10 ul mercaptoethanol and applied to the column. 100 GENERAL DISCUSSION Data concerning the developmental relationships between species have been obtained by a variety of methods. These include a comparison of their modes of enzyme regulation (Canovas et aj_. 1967). Primary structures of proteins such as cytochrome c (Nolan and Margoliash, 1968), patterns of their biochemical pathways and the structure and chemistry of their cell walls. However, very little is known about the relationships of the cytoplasmic membranes of procaryotic cells and although there are conceptual features of the structure which are generally becoming accepted there is still controversy over details. A comparative approach to studying some properties of protein func-tions in bacteria along with developmental lines seemed timely and the family Micrococcaceae was chosen because within a single group of organ-isms there were members which were aerobic, facultative and anaerobic. In spite of these differences, certain characteristics such as morphology, Gram stain, mode of cell division and ability to produce catalase were common. Canovas et al_. (1967) have pointed out that a morphologically uniform group of bacteria such as Pseudomonas, although far more re-stricted than the Micrococcaceae, had close similarities in the control mechanisms of the 3-keto-adipate pathway even though there were signifi-cant differences in the %GC of their DNA. A structurally different organ-ism, Moraxella calcoacetica had an identical pathway but the control mechanisms were entirely different. They concluded that control mechanisms 101 might be relatively stable characters which tend to be conserved during the evolution of a biological group. The functions of a membrane system appear infinitely complex. Therefore it is possible that within a group of organisms some features may be conserved. In order to establish a basis for selecting representative organisms, DNA was isolated from a range of organisms and the %GC was checked where possible against the values reported by other authors (Bohacek et al. 1970). There was a strong correlation between high %GC and aerobiosis. The %GC of an organism is a valid criterion for grouping organisms only when it is used in conjunction with other properties. The genetic complexity of the bacteria was assessed by measuring their genome size. A number of methods such as autoradiography and chemi-cal determination have been reported in the literature (Gillis ejt al_. 1970) but the optical renaturation method of Seidler and Mandel (1972) was found to be the most satisfactory to perform and the method and results could be related directly to E. coli as standard. There was a large range of complexity within this family and the values that were obtained for S_. aureus strains were close to those obtained by Leth Bak (1970). The largest genomes belonged to organisms that had a high %GC and were aerobic. So far the genome size of bacterial DNA is known for very few organisms but the values for aerobic micrococci were similar to some other aerobes, yet smaller than the biochemically sophisticated obligate aerobe Pseudomonas  aeruginosa. On the other hand, Peptococcus aerogenes had a very small genome size which corresponded to the genome size of fastidious organisms like Haemophilus influenzae. Since the genome sizes of Staphylococcus and 102 Peptococcus were not greatly different, Cot hybridization studies were carried out according to the method of Seidler and Mandel (1971). As expected, the organisms classified as Staphylococcus showed genetic similarity. P_. saccharolyticus showed partial similarity to S_. aureus  6538P and was probably more similar to Staphylococcus than Peptococcus in other ways in that it was microaerophil ic, fermented glucose and was susceptible to lysostaphin. P_. aerogenes and V_. alcalcalescens (used as a control) showed no genetic similarity to any other organism tested. The amino acid requirements of these organisms were now examined to determine if these correlated with genome size and it was found there was a strong correlation. P_. aerogenes had very complex nutritional require-ments and could not be grown in a defined medium containing all known amino acids and vitamins. This confirmed the finding of Rogosa (1969). It was of interest that in closely related strains of S_. aureus there were variations in the amino acids required from one strain to another which would indicate the influence of different habitats at possibly a later stage in the development of these species so that auxotrophs could become esta-blished. The nutritional requirements of M. candidus and S_. lactis corresponded to their %GC in that with a low %GC they had broad nutrition-al requirements. These data provided a basis for selecting organisms for the study of some membrane related functions. Since transport was one of the highly developed functions of the membrane, it was thought that the same function studied over a range of genome size would reveal various degrees of devel-opment. However, this was not found. We chose to study the transport of 103 proline since it was highly specific in a number of organisms (Kay and Gronlund, 1969;.Britten and McLure, 1962) in contrast to some other amino acids that showed competition with each other in the transport process. In spite of profound differences in genome complexity and nutritional requirements, the transport system for proline in all cases except P_. aerogenes was found to be specific, energy dependent and enzymatic. There was no correlation between genome size and maximum velocities for these transport systems. However, the K^  values for the "permeases" and the Q-JQ values for the enzyme reactions were similar. Under the condi-tions of our experiments, we were unable to show any transport activity for P_. aerogenes and since the organism will grow only in complex medium containing peptides there is circumstantial evidence that it may require peptides in place of some amino acids. Both saccharolyticus and jS. aureus were auxotrophic for proline and the induction of synthesis of the transport enzyme was derepressed during starvation for this amino acid. Thus there is another indication for the close relationship between these two organisms. The transport systemsfor two other amino acids, isoleucine and methio-nine were well developed in three organisms and the passive transport of glutamate by M. varians and histidine by S_. aureus illustrate the hetero-geneity of the systems. One might expect that the development of a transport system would have been one of the earliest functions acquired by an organism since this would confer a definite selective advantage. 104 A study of some membrane bound oxidative and phosphorylative enzymes showed that there was a progressive development of function from anaero-biosis to aerobiosis in the Micrococcaceae. S_. aureus, in seeming con-tradiction to its small genome size, had a complement of enzymes similar to the aerobic micrococci when it was grown aerobically. It also showed regulatory properties in the synthesis of some enzymes when it was grown aerobically in the presence of glucose and this was not found in the obligate aerobes. Under anaerobic conditions, the membrane bound enzy--mes were similar to those found for P_. aerogenes. Frerman and White (1967) had shown that a shift from an anaerobic state to an aerobic state resulted in the formation of a membrane bound functional electron trans-port chain in S_. aureus which involved the synthesis of protoheme, cyto-chromes, cytochome oxidase, phospholipids, glucolipids and Vitamin K2 isoprenologues. Thus, in terms of protein in the membrane, one might expect to see large differences between an aerobe and a fermentative anaerobe. Schnaitman (1970) showed that in aerobically and anaerobically grown E_. coli, only a minor difference appeared on SDS acrylamide gels. The solubilization of membrane proteins has been achieved in the past few years by the use of detergents and by changing the ionic strength of the environment in the absence of divalent cations. A study of the re-lease of these proteins and their behaviour in different washing systems could reflect some of the properties of their integration into the mem-brane structure. We chose to use Triton-X 100 because this detergent has been widely used to solubilize membrane proteins without affecting 105 the activity of a number of enzymes. Although the precise way in which it acts on membranes is not yet known, it is known to break protein-pro-tein and protein-1ipid hydrophobic bonds and to selectively bind to membrane proteins (Helenius and Simons, 1972). In some cases, it has been shown to partially replace the lipid requirement of enzymes such as Enzyme II of the phosphotransferase system (Rothfield and Romeo, 1971) and to cause a stimulation of some enzymes such as succinate dehydrogen-ase (Pollock ejt aj_. 1971). Salton (1972) postulated that this stimulatory action may be due to exposing active sites or by binding hydrophobic in-hibitors. It could change the phase characteristics of the medium so that the more hydrophobic enzymes could be active in an aqueous environ-ment. Lengsfeld ejt a]_. (1973) found that Triton-X at a relatively high concentration caused marked swelling in jS. aureus vesicles and that the membranes became very asymmetric, eventually becoming lipid free. The use of different strength Tris buffers, lacking divalent cations, to release enzymes was preferred by Abrams and Baron (1968) because they felt this treatment was less likely to destroy native membrane struct-ures than were detergents. The solubilization of ATPase from membranes has generally been achieved with low ionic strength buffers. In the case of Streptococcus  faecal is an "all or none" release phenomenon occurred after six or seven washes (Abrams and Baron, 1968). ATPase was present in an active form in all the membranes studied and there was considerable heterogeneity with respect to the effect of the wash treatments. DTE generally inacti-106 vated the enzyme and caused the solubilization of an active enzyme in only two systems. There were differences between different strains of the same organism. Huang et al_. (1968) pointed out that magnesium was inhibitory to ATPase solubilized from M. lysodeikticus; however this was not found in our experiments. We were able to. solubilize ATPase from the strain of S_. aureus used in the present studies in contrast to the reported findings of Gross and Coles (1968). The effect of Triton-X was different from DTE. It markedly stimulated the enzyme in all membrane systems and the pattern of solubilization was similar in each case indi-cating that it was firmly bound to the membrane. Since ATPase was found in a wide variety of membrane systems and has been associated with energy generation in aerobes and anaerobes as well as with transport in anaerobic systems, it would appear that this function arose very early in the development of the bacterial cell and because of its vital role, it remained relatively unchanged in its relationship to the membrane. Cross reactivity studies with purified antiserum to M. lysodeikticus ATPase to other micrococci showed that antigenic relatedness existed in only a very limited range of bacteria.,. M. varians showed a low level of cross reactivity and thus at an antigen level, there were large differences between the ATPase of M. lysodei kticus and M. varians. NADH dehydrogenase was also present to a high level in all membrane systems studied and was stimulated in each case by Triton-X as well as showing a similar pattern of solubilization. This enzyme was involved in oxidative phosphorylation and amino acid transport (Kaback, 1972) but 107 its role in anaerobic organisms was less clear other than to possibly recycle NAD for substrate level phosphorylation. Washing with DTE again revealed heterogeneity and did not solubilize NADH dehydrogenase from all membranes. Nachbar and Salton (1970) showed that NADH dehydrogenase solubilized by Tris EDTA treatment from M. lysodeikticus membranes was lipid enriched. In general, succinic dehydrogenase had been reported to be firmly bound (Salton, 1972) and Kung and Henning (1972) were unable to solubil-ize this function from membranes of E_. coli with Brij 58. The enzyme had certain characteristics that were common to as wide a range of cells as yeast and mammalian tissue. This enzyme was solubilized from the membranes of some micrococci and the pattern of solubilization showed it was more firmly bound to the membrane than some other enzymes. The solubilization patterns of malate and lactate dehydrogenase were found to be different from the other enzymes studied in that both were readily solubilized and inhibited by Triton-X. The behaviour of these enzymes to DTE was again variable. Kung and Henning(1972), using mutants of E_. coli showed that d- and 1-lactate dehydrogenase and a-glycerophosphate dehydrogenase shared common binding sites on the membrane but that suc-cinate dehydrogenase had a different association with the membrane. An estimate of the total number of proteins present in a membrane system is a difficult task. SDS gel electrophoresis shows only the major subunits'. present and may mask many minor proteins. Only a few bacterial membrane systems have so far been examined. The cell envelope of E. coli had been resolved into 20 - 30 well defined bands (Schnaitman, 1970). An 108 examination of the Micrococcaceae showed that there were more subunits in organisms with a larger genome size and when split gels were run there were more subunits in the higher molecular weight range. Our study of the Micrococcaceae has shown that in the few character-istics investigated, a great deal of heterogeneity occurred within the group. There was a direct correlation between aerobiosis, genome size, %GC and nutritional requirements. However, some organisms with similar %GC and genome size showed no genetic relatedness and closely related organisms showed different nutritional requirements which suggested they had come from different habitats. A study of some properties of the cyto-plasmic membrane over a range of organisms showed that transport was well developed for a number of amino acids in all systems except one. These developed transport systems had similar properties but differed in a number of characteristics. Although P_. aerogenes did not appear to active-ly transport proline and had the smallest genome size, S_. aureus had only a slightly larger genome size and the properties of its transport system were as well developed as for the obligate aerobes which had larger genomes, emphasizing that the acquisition of an active transport system was an early event. In addition, aerobic Staphylococcus is highly devel-oped in its membrane bound functions and these are subject to catabolite repression. Under anaerobic conditions, it more closely resembles P_. aerogenes in the membrane enzymes studied. When the properties of some of these enzymes were studied by a wash treatment that removed cations, a similar enzyme in different membrane systems, showed different properties and no correlation to any other property could be shown. However, treat-109 ment of the membranes with Triton-X showed that similar enzymes in dif-ferent membrane systems responded in a similar way to washing with Triton-X. This leads us to speculate that the hydrophobic interactions of a particular enzyme may be conserved while undergoing changes in its hydro-philic interactions. An increase in the number and size of the. membrane proteins correlated with the increase in genome size and may indicate a development in the complexity of a particular membrane function. no LITERATURE CITED Aaronson, S. and S.H. Hutner. 1966. Biochemical markers and microbial phylogeny. Q. Rev. Biol. 41_: 13-36. Abrams, A. and C. Baron. 1968. Reversible attachment of ATPase to streptococcal membranes and the effect of magnesium ions. Biochemistry 7_:501-506. Ames, B.N. 1966. Assay of inorganic phosphate, total phosphate and phosphatase. J_n Methods in Enzymology, VIII. Edited by Colowick, S.P., N.O. Kaplan. Academic Press, New York, (115-118). Ashwell, G. 1957. Colorimetric analysis of sugars. J_n_ Methods in Enzymology, III. Edited by Colowick, S.P., N.O. Kaplan. Academic Press, New York, (87-90). Auletta, A.E. and E.R. Kennedy. 1966. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92: 28-34. Baird-Parker, A.C. 1965. 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