“0 ——-—v~—v ALTERATIONS IN THE MEMBRANE PROTEINS 0F FLA, CHE, MOT AND HAG MUTANTS 0F ESCHERICHIA COLI Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY CHWEN LIN LIAO 1976 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MTE DUE I MTE DUE DATE DUE , 429 2 2 19 8 1/98 mm.“ ABSTRACT ALTERATIONS IN THE MEMBRANE PROTEINS OF 1%. gig, N91 AND 1mg MUTAN‘I‘S OF W (Lop; By Chwen Lin Liao This research is an attempt to determine how a Specific cellular process such as motility can be controlled by membrane associated proteins and to use this system as a simple model to study how membrane associated proteins interact with the cellular environment to regulate cellular activities. Similar control at the membrane level is very important for eucarotic cell differentiation, intercellular communication and intracellular metabolism. The f;§_and m2; mutants were isolated from E. 22;; Aw #05. The cell envelOpes of E, cg;i_wild type. f;§, egg, m2; and hag mutants were isolated and separated into outer and cyt0plasmic membrane fractions. The proteins composition of these two fractions of mutants was compared to that of wild type cells by SDS electr0phoresis gels. The observed differences among wild type and mutants were noted. For {lg mutants, no Specific difference was found in outer membrane protein composition while the 81 K, 76 K, 70 K, 65 K and 38 K dalton proteins of cytoplasmic membrane fractions were found Chwen Lin Liao to be reduced in amount. Some of these proteins may be involved in the synthesis or assembly of flagella. For Egg mutants, the 50 K and 69 K dalton proteins of the outer membrane fractions were found to be reduced in amount and the 90 K, 8# K, 81 K, 76 K, 65 K and 51 K dalton proteins of the cytoplasmic membrane fractions were found to be reduced in amount. Some of these reduced proteins may be associated with the mechanism of chemotaxis. For m2: and hag mutants, the main outer membrane protein bands were found to be more intense and the 49 K and 38 K dalton proteins of the cytOpl- asmic membrane fractions were also more intense. The increase in the amount of these bands may reflect the need of these proteins to maintain structural stability or for the motility process. For the f;a_§_mutant, the protein pattern was quite different, the changes in the level of the different major proteins of the different major proteins of the outer membrane may be involved in maintaining and regulating the structural stability. ALTERATIONS IN THE MEMBRANE PROTEINS 0F m, mg. mg; AND EEC; MUTANTS 01" W 9.9M BY Chwen Lin Liao A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Department of BiOphysics 1976 To my parents ii ACKNOWLEDGEMENTS I wish to express my sincere thanks to my research adviser, Dr. Estelle McGroarty. for her patient guidance. advice and encouragement during the course of this research and the preparation of the thesis. Thanks are also extended to the thesis committee members, Dr. A. Haug. Dr. G. HOOper and Dr. G. Kemeny for their constructive criticism and advice. Finally, I like to express my thanks to my husband for his encouragement. typing and invaluable helping during the preparation of the thesis. This research was supported by the College of Oste- opathic Medicine, with the cOOperation of the Biophysics Department, Michigan State University. iii TABLE OF CONTENTS LIST OF TABI‘ES I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I LI ST OF FIGURES I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ISOIATI ON 0F NiiIJ‘I‘AN‘II‘S I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IntrOduCtion I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I cultures I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I media IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII methods I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I (A) Isolation of E. goli mutants ............... (B) Characterization of genetic phenotype of mutants I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I (C) Characterization of mutants by complementation teSt I I I I I I I I I I I I I I I I I I I I I I I Results I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I DiscuSSj-on I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Bibliography I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II. COMPARISON OF MEMBRANE PROTEINS AMONG ESQHERIQHI COLI WILD TYPE, ELA,MUTANTS, MOT MUTANTS AND Qflfi_ MUTANTS IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IntrOduc-tion IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Materials and NiethOdS IIIIIIIIIIIIIIIIIIIIIIIIIIII 1. organisms 0000000000000...cocoon-000000.00... 2. Growth conditions and media ................. 3. Preparation of cell envelOpe membrane fraCtj-ons IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 4. LipOpolysacchride content ................... SI PrOtein assay IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 6. Polyacrylamide gel electrOphOresis ......... 7. EleCtron microscopy coo00000000000000.0000... Results IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII EleCtron microscopy IIIIIIIIIIIIIIIIIIIIIIIIIIII Separation of outer and cytoplasmic membranes .. ElectrOphoresis of membrane fractions .......... iv Page vi vii FW*\JO\ kn -¢4?¢\JP‘ I‘ Hwa 15 15 3O 30 31 33 35 36 36 44 Page Comparison in outer membrane protein composi- tion between wild type and mutant strains ...... 52 Comparison in cytoplasmic membrane protein composition between wild type and mutant Strains IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 55 DiscuSSj-On I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 60 Appendix I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 67 Bibliography I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 73 LIST OF TABLES Table Page 1. Buoyant density of the outer and cytoplasmic membrane fractions from various organisms ........ N2 2. The result of protein and lipOpolysacchride assay of various organisms ....................... 43 3. Comparison of protein bands of outer membrane fractions of mutants to those Of wild type A!” 405 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 58 4. Comparison of protein bands of cytOplasmic membrane fractions of mutants to those of Wild type Aw 405 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 59 vi LIST OF FIGURES Figure Page 1. The result of complementation test. The number stands for 29 generated mutants named as CL 1. CL 2 and so on. w.t. is wild type AW #05. strong complementati on C::]””weak or no complementation. 0000000000000... 9 2. Electron micrographs of g. goli Aw #05 and its matants CL 3, CL 5 and CL 17 0.0.0.0.0000000.00000 12 3. Model of flagellum of E, 92;; (A) and its attachment to the cell envelope (B) ......................... 2R 4. Electron micrographs of g, ggli, gag mutant. fla E mUtant and EOt mutant IIIIIIIIIIIIIIIIIIIIIIIIIIII 37 5. Sucrose gradient centrifugation of total membrane fractions from E. 92;;_AW 405. Absorbance at 280 nm was measured across the gradient to locate the major bands. The activity of succinic dehydrogenase (SDH) marks the location of cytoplasmic membrane IIIIIIIIIIIIIIIIIIIIIIIIIIIII 39 6. SDS polyacrylamide slab gel electr0phoresis of the outer membrane proteins of (1) wild type AW 4053 (2) CL 3: (3) CL 5: (A) che__A_: (5) gh__e__B.: (6) fl_a _Ar (7) fla E; (8) mot; (9) hag mutants and (10) standard proteins Of bovine serum albumin. catalase, IgG-H, ovalbumin, lactate dehydrogenase and IgG-L. About 150 ug of protein was applied to each well IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ”6 7. SDS polyacrylamide gel electrOphoresis of the cytOplasmic membrane proteins of: (1) CL 3: (2) wild type AW 405: (3) CL 5: (N) A: (5) Q€£_§J (6) fl§_As (7) fl§_Er (8) male (9 has: (10 standard proteins of bovine serum albumin, catalase, IgG-H. ovalbumin and lactate dehydrogenase and IgG-L. About 150 ug of protein was applied to each well IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII “7 vii Figure 8. 9. SDS polyacrylamide tube gel electrophoresis of the outer membrane proteins. A: from left to right are: wild type AW 4053 CL 3: CL 5; CL 17: che A and standard proteins of bovine serum albumin, catalase, IgG-H. ovalbumin. lactate dehydrogenase and IgG-L. B: from left to right are: che B; fla A; fla g; @933 h§g_and also 6 standard proteins. About 500‘ug of protein was applied to the gel. .............................. SDS polyacrylamide tube gel electrophoresis Of the cytoplasmic membrane proteins. A: from left to right are: wild type AW 4053 CL 3; CL 5; CL 17; che A and standard proteins of bovine serum albumin, catalase, IgG-L. B: from left to ri ht are che B; fla A; fla E; mpg; hag and also standard proteins. About 500 pg of protein was applied to the gel. .................. 10 - 14. Scans of SDS polyacrylamide tube gel electrophoresis of membrane proteins shown in Figures 8 and 9. Sacns a to j are from outer membrane fractions; scans a' to j' are from cytOplasmic membrane fractions. The gels were scanned at 550 nm using a Gilford Spectrophotometer 240 IIIIIIIIIIIIIIIIIIIIIIIIIIII viii Page 48 50 67 I. ISOLATION OF MUTANTS Mutants of E, 9911 defective in motility or chemotaxis are of the following types: (1) Flagellin mutants - nag.gene mutants are those altered in the structural gene for flagellin. the protein component of the flagellum external to the bacterial body. Such mutation can lead to changes in the shape of the flagella as well as total loss of flagella. For example. curly mutants have an altered flagellin (1) which results in flagella with a wavelength about half that found normally. Such bacteria show only rotational movement. Altered flagellin can also lead to serological changes in the flagella (2). (ii) Paralyzed mutants ( mgt_mutants ) have flagella that look normal, but the bacteria are not motile; there appears to be only one m2: gene (3). (iii) Non-flagellated mutants ( £1§.mutants ) have no flagella and are therefore nonmotile. The genetic defect lies in one of 16 fla genes (35). (iv) Non-chemotactic mutants ( gag mutants ) are fully motile, but they fail to carry out chemotaxis toward any substance tried. Three genes are involved; ghg_A, ghe_£, and 2M (1"). 2 We attempted to isolate several of these types of mutants and study them as an approach to the understanding of the mechanism of motility. BacteriOphage x was discovered by Sertic and Boulgalsov (5) and was shown by them to attack only strains of Salmgggllg that are flagellated (6). Afterward. Meynell (7) showed that not only must flagella be present. but the flagella must be actively moving in order for infection to take place. Also, Schade and Adler (6) isolated a host-range mutant which attacks E. 2211. This phage x'contains deoxyr- ibonuoleic acid ( DNA ) which is double-stranded. The DNA has a molecular weight of 42 x 106 . The phage x particle weight is about 90 x 106. They also showed that the bacter- iophage x attaches to the filament of a bacterial flagellum by means of a tail fiber. but the ultimate receptor site for the phage is located at the base of the bacterial flagellum. Here. the phage injects its deoxyribonucleic acid into the bacterium. leaving the empty phage attached at the base. It is suggested that x phage slides along the filament of the flagellum to the base. owing to the movement of the flagellum. It is supposed that the rotating flagellum propels the phage‘x rapidly to the adsorption site at the base of the flagellum. Chi phage can thus be used to isolate mutants and these mutants then can be characterized by using complementation tests. In transduction. a bacteriOphage can carry small fragments of the donor chromosome to the recipient bacteria. In complete transduction, donor genes transferred by the phage replace their alleles on the recipient genome. Stocker et a1 (8) studying the transduction of flagellar characters by the Salmonella phage P22. observed not only swarms, but also trails: linear groups of microcolonies extending out from the site of inoculation. A trail marks the path of a bacterium made motile by abortive transduction. In such an abortive transduction. the wild-type motility gene introduced by the phage neither recombines with the recipient genome. nor multiplies. It is. however. dominant, and capable of determining a wild-type gene product. The bacterium containing the fragment is therefore motile and will swim away from the inoculum. but will leave a trail of colonies of its nonmotile progeny behind it. If the donor and recipient are defective in the same gene, a wild-type gene product can be made only after recom- bination. and trails can arise through intragenic complemen- tation. If they are in different genes, and the defective gene products do not interfere with the functioning of the wild-type products, then normal intergenic complementation will occur and trails should be seen. For studies of E, 2211, phage P1 can be used to carry out the abortive transduction complementation test. 931m (1) The strain used was E, ggii,K12 AN #05 which is thr', leu', his', vit B“. strr and Tér. (ii).X phage (iii) P1 phage All of these were obtained from Dr. John Armstrong. Media One percent ( w/V ) Difco tryptone broth was supplemen- ted with 0.2 fl ( W/V ) yeast extract and 1 fl ( W/V ) NaCI. Tryptone plates were prepared by the addition of 1 % ( w/V ) agar ( Difco ) to the tryptone broth. Semi-solid tryptone plate was prepared by the addition of 0.35 % agar ( Difco ) to the tryptone broth. L~broth was made by adding 10 g tryptone ( Difco ), 5 g yeast extract. 5 g NaCl. 10 ml sterile 10 % glucose and 10 ml sterile 0.25 M CaClz to H20 making a total volume of 1000 m1. Dilution buffer was made by adding 7 g.N32HP04, 3 g Kthoh, u g Naci. 0.2 g MgSOh.7H20 to 1000 ml H20. Minimal media was made by adding 100 ml minimal salts ( 60 g NaZHPOu. 30 g KH2P04, 5 g Naci, 10 g NHuCl in 1000 ml H20 ), 20 ml 20 % glucose. 10 ml 0.1 M MgSOu. 10 ml 0.01 M CaCIZ. 10 ml 1 % thr, 10 m1 1 % Ian, 10 m1 1 % his and 1 m1 100 mg/100 ml B ( thi ) to 82k ml H20. Msiheds (A) Isolation of E, £211 mutants Colonies of E, 9211 AN #05 grown on tryptone plates were picked and grow overnight in a 2 m1 tryptone broth. 5 One-tenth ml was mixed with 2 ml “5°C 0.35 % agar-tryptone broth, then overlayed on tryptone plates. A dr0p of chi- phage at 108/m1 was spotted on the plate. which was then incubated overnight. An inoculating 100p was scraped across the lysed area, and the bacteria were suspended in 2 ml tryptone broth. The suspension was streaked on a tryptone plate. and after incubation, isolated colonies were picked and characterized. (B) Characterization of genetic phenotype of mutants First of all. all mutants were grown in minimum media to test for the same genetic character as wild-type E, 9211 Aw #05, i.e.. they were all shown to be thr'. leu-, his-. vit B'. strr. Then, each mutants was grown on soft agar. An overnight culture of each mutant in 2 ml tryptone broth was epotted on a semi-solid tryptone plate and incubated for 16 to 18 hrs. at 37 °C to determine its motility. Finally. each mutant was examined using the electron microscOpe. Each mutant was grown overnight in 2 ml tryptone broth, and then spun at 5000 x g for 10 min. The pellet was resuspended in 1 ml of dilution buffer, and applied to collodian covered grids stained with PTA and examined using the electron microsc0pe to observe whether it has flagella or not. 6 (C) Characterization of mutants by complementation test P1 phage was used to carry out the transduction complementation test. It was grown on a liquid culture. T0p and bottom L-agar was prepared by adding 0.7 fl and 1.5 % agar respectively to L-broth. One tenth ml of P1 at 106/ml was preincubate with 0.1 ml of a fresh overnight culture of the bacterium in a small test tube for 15 min at 37°C. Then 2.5 ml of melted top agar was added ( which had been held at 45 to 50°C ) mixed quickly and poured uniformly over the bottom agar layer. The plates were incubated at 37°C for approximately 8 hours for maximum lysis. After overnight incubation the top layer was scraped off into a 50 ml centrifuge tube with the end of a sterile microscope slide. Five ml of L-broth was added and the agar-broth mixture was carefully homogenized by blowing it in and out of a 10 ml pipette. The mixture was centrifuged at 8000 rpm for 10 min in an IEC centrifuge. The supernatant was then carefully decanted into a sterile screw cap tube for storage. One or two drape of chloroform was added to prevent growth of contamination bacteria. The same procedure was used with suitable dilutions of the P1 stock to obtain isolated plaques to assay the titer of the P1 phage. The same procedure was also used to grow P1 on all mutants and to assay P1 on these different stocks. 7 The procedures described by Lennox ( 1955 ) was followed to carry out the transduction complementation test. A 0.5 ml sample of a freshly grown L-broth culture of the recipient strain, at approximately 2.5 x 108/ml ( 0.D. 0.1 ) was mixed with 0.02 ml of 0.25 M CaClZ at 37°C. About 0 x 109 infective units per ml of P1 grown on the appropriate donor were added. and the final volume was brought to 2 ml with L-broth. After 15 min incubation at 37°C to allow adsorption of the phage. the mixture was centrifuged at 5000 x g for 15 min. at room temperature. The bacteria were resuspended in 0.5 ml of 0.15 M NaCl, and 0.1 ml of the resuspended mixture was streaked across a semi-solid tryptone plate. The plates were incubated 2h to #8 hrs at 37°C and then examined for the presence of swarms or trails. The appearance of swarms indicated that recombination had occured between the two mutant alleles and therefore that the alleles were not identical. 3.29m; By suing x phage to isolate E, ggli_mutants. 30 mutants which were nonmotile and could grow in the supplemented minimum media were obtained. When checked with electron microscope only one of them was shown to have flagella, i.e.. to be a mg: mutant; all the others were nonflagellated. i.e.. :1; mutants. Also. the mg; mutant had less flagella than its parent - wild-type E, 9211 AW #05. 8 In genetic studies of motility in both galmgggllg species and in g, g2;1_the method chosen for complementation has been abortive transduction. In an abortive transduction the donor mutant gene is introduced into a nonmotile recipient by a phage. but fails to be incorporated or to multiply. If complementation occurs. the bacterium carrying the fragment will be motile. but as it swims out from the site of inoculation it leaves a trail of microcolonies containing its nonmotile descendants. If the donor is defective in the same gene as the recipient. trails usually do not form and complementation is said not to occur. Complementation tests on mutants were carried out using P1 grown on 29 nonflagellated donors and also on.E, 2211 Aw #05. A positive response always consisted of more than 50 trails and swarms. In a negative response no trails or less than 10 were observed. A slightly beaded edge to the side of inoculation was usually obtained. probably as a result of the formation of abortive transductants. The results are shown in Figure 1. The CL 5 is a mgt_mutant and the others are all 11; mutants. We can see that complement- ation was obtained between two mutants with a different phenotype. Thus these results agree with what has already been reported in that the flagellar genes ( {lg ) are distinct from the motility gene ( 223,). Some of the nonflagellated mutants complement with each other. i.e.. they belong to different complementation groups - roughly group I ( CL 1. CL 2, CL 3. CL 4. CL 6. CL 7. CL 8, Figure 1. The result of complementation test. The number stands for the 29 generated mutants. The mutants were named as CL 1. CL 2, and so on. w.T. represents wild-type Aw 405. strong complementation. E223" weak or no complementation. 10 H onsmam 0“03 HNMQ‘M‘OI‘mG NNNNNNNNN OI-INMQ‘Inkol‘mO‘O HI-II-lI-CI-IHI-ll-II-lt-IN Hva-mxohoom o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 00 O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 0 case 0 o o o o o o o o o o o o o o o o o o o o o o 0 o 0.0 Glunu. O O O o o o o o o o o o o o o o 000 00000000000 0 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o lblblblblblb 0 note 0 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o d o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 0.0 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 0 o o o o o o o o o o o o o o o o o o o o m o o o o o u o o o o o o o o o o .u.3 mm mm 5N mm mm vN MN NN HN ON mH mH NH mH mH vH MH NH HH OH m m h w m v m N H MOZOQ LNHIdIDHH 11 CL 9 ) and group II ( CL 11. CL 12. CL 13. CL 14. CL 15. CL 16. CL 17 ). The others - CL 10. CL 18 - 29. did not show strong complementation. so they are not easy to classify. This may be due to some of them being revertants or to the fact that two genes are mutated. I chose CL 3 and CL 17 to study two types of £1; mutants and CL 5 to study the mg; phenotype. These mutants were used to study their membrane protein components and compared to those of wild-type g, ggli,AW #05. D 880 For this research. I intended to isolate the membrane of two classes {lg_mutants generated in this study and the one mg; mutant. Presumably, there exists at least one 323 gene and 16 {lg_genes for E, 2211. The chance of obtaining a fig mutant is much higher than that of a 221 mutant. It is reasonable that only one mutant was obtained compared to the 29 1;; mutants isolated. It appeared that many of the mutants isolated at the same time belonged to the same complementation group. This may be due to the probability that different 11; genes have different mutation frequencies. One m2},mutant ( CL 5 ) and two groups of £1§_mutants ( CL 3. CL 17 ) as well as several revertants have been obtained. Figure 2 shows the electron micrographs of cells of wild-type g, ggl1,AW #05, CL 3. CL 5 and CL 17. 12 Figure 2. Electron micr0graphs of E. 2211 AM #05 ( A, X 40,000 )3 CL 3 ( B, X 16,000 )3 CL 5 ( C. X 25,000 ) and CL 17 ( D. X 25,000 ). C‘sLm 13 B IBLI OGRAPHY BIBLIOGRAPHY Iino, T.C. J. Gen. Microbiol. g2. 167-175 (1962). Joys. T.M. and B.A.D. Stacker J. Gen. Microbiol. ii. 121-138 (1966). Armstrong, J.B. and J. Adler Genetics, 56, 363-373 (1967). Armstrong, J.B. and J. Adler Genetics, 61. 61-66 (1969). Sertic, V. and N. Boulgakov Compt. Rend. Soc. Biol. 112. 1270-1272 (1935). Schade, 8.2. and J. Adler J. of Viral. 1. 591-598 (1967). Meynell. E.w. J. Gen. Microbiol. 25. 153-290 (1961). Stacker. B.A.D.. N.D. Zinder and J. Lederberg J. Gen. Microbial. 2, #10-033 (1953). 14 II. COMPARISON OF MEMBRANE PROTEINS AMONG E§§EEBIQ£IA.§QLI WILD TYPE. ELé.MUTANTS. m0: MUTANTS AND gun MUTANTS o t on The cell envelope of the Gram-negative bacterial cell is unique in several respects, and the structure which contributes most to this uniqueness is the lip0polysaccharide ( LPS ) - containing outer membrane of the cell wall; this layer delimits a zone outside the cytOplasmic membrane and controls the passage of molecules into and out of this ”periplasmic Space" (1). The function of the outer membrane is in turn, modified by other cell wall components which influence both its penetrability and its vulnerability to attack by external agents. The complex. multilayered cell wall is made up of a variety of structural macromolecules which can trap free molecules and ions so that each structu- ral "layer" defines a zone of particular'physicochemical attributes. The cell envelOpe of E, lei,is both chemically and morphologically complex (2). Electron micr0800py of thin- section preparations reveal the cell envelOpe as a multilay- ered structures, in which the mast peripheral component is the triple-layered, roughly 7.5 to 9.0 nm thick outer membrane 15 16 - the lip0polysaccharide or L membrane - made of lip0polys- accharide. protein and phospholipid. This outer membrane is separated from the cytOplasmic membrane by the thin ( 1.0 nm thick ) murein layer (3) - the intermediate peptid0glycan layer - made of peptid0glycan, digestible with lysozyme, and possibly some protein. The cytoplasmic membrane - an inner membrane - is made of lipid and protein. The outer membrane and the peptid0glycan layer are so closely associated that these two are often collectively referred to as the outer membrane or cell wall. At present, there is great interest in the cell envelOpe. especially the cytoplasmic membrane, which functions as the major permeability barrier in the cell and as the site of organization of many membrane-bound enzymes and multienzyme complexes. including the reapiratory electron tran3port system. The cell envelope of several different Gram-negative organisms have been isolated and separated into cytoplasmic and outer membrane fractions with relatively high purity ( h. 5. 6. 7. 8 ). The separation of the two membranes is based on isOpycnic sucrose fractionation of cell envelOpes obtained by lysis usually of lysozyme - ethylenediaminetetr- aacetic acid ( EDTA ) induced spheroplasts. The membrane containing fraction with the higher density is enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes and the similar morphology to intact cell walls. 17 The less-dense fraction is enriched in vesicles derived from the cytOplasmic membrane as indicated by the enrichment of cytochromes. the enzymes lactic and succinic dehydrogenase and nitrate reductase. and the similar morpholOgy to intact cytoplasmic membrane. Both fractions are rich in phospholi- pid (4). It is normally observed that the fragment of the cytOplasmic membrane are attached to the outer membrane fraction. DePamphilis and Adler (9) utilized extraction with Triton X-100 in the presence of Mg+2 to isolate the outer or L membrane from E, 2211 epheroplasts. Schnaitman (10) indicated that treatment of a partially purified preparations of cell wall of E. 2211_with Triton x-100 at 23°C resulted in a solubilization of 15 to 25 % of the protein. Examina- tion of the Triton-insoluble material by electron microsCOpy indicated that the characteristic morpholOgy of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100 treatment. Extraction with Triton x-100 thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. 9211. The cytOplasmic and outer membrane fractions differ markedly in over-all protein composition as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate as well as in Specific enzyme activities. Using electr0pho- resis on polyacrylamide gels. the proteins of the inner membrane of 2, £211 have been resolved into approximately 56 distinct bands. It has been estimated that there are on 18 the order of a hundred protein species bound to the inner membrane. including the many permease systems, the DNA replication enzymes and the electron transport complexes (11). On the other hand. there are only several different proteins in SDS polyacrylamide gel pattern of outer membrane of E. 9211 (4. 11). Schnaitman (4) has compared the protein composition of the two membranes by mixing the cytOplasmic membrane- enriched fraction from a 3H-labelled culture with the cell wall-enriched fraction from a 1l'ic-labelled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved. 27 bands were enriched in cytOplasmic membrane fraction. whereas 6 were enriched in cell wall-enriched fraction. One protein whose molecular weight is about 44,000 was reported as the major component of the envelOpe and was clearly localized in the cell wall. This protein accounts for 70 % of the total protein of the cell wall. Furthermore. Schnaitman (12) showed that it was possible to separate this 42,000 dalton major protein from.E. 9211 0111 into four distinct protein fractions by chromatography of the solubili- zed outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS. It is of interest to try to identify the specific function of membrane proteins visualized on polyacrylamide gels. The protein composition of the outer membrane is simplier than that of cytoplasmic membrane. Also, it is 19 easier to obtain purified outer membrane. For example, outer membranes can be purified from total envelOpe of lysozyme- EDTA induced SpherOplasts without centrifuging on sucrose grandients, by using Triton x-100 to solubilize cytOplasmic membranes from the preparations of total envelOpes (10). In addition, outer membranes can be isolated from lysozyme- EDTA induced spherOplasts of E, g211,K-12 by lowering the pH to 5.0 to aggregate this negatively charged membrane (13). It has been reported that the outer membrane of most Gram- negative bacteria contains a set of proteins which have an approximate molecular weight of 38.000 to 44.000 dalton. Schnaitman has shown that outer membrane of different species and strains, as well as a single species grown under differ- ent culture conditions can contain differences in the outer membrane protein profiles (12). Hans Wolf-Wat: et al (13). showed that a chain-forming antibiotic-supersensitive gay; strain had a slightly modified outer membrane protein pattern and a lower relative content of phosphatidylglycerol when compared to that oflgnyg: strain. Mutants of E, 9211 and fi, typhimuzigm have been isolated in.which the protein composition of the outer membrane is different from that of wild type. Wu (14) has isolated a mutant from E. ggli,K-12 which is more sensitive towards dyes and detergents. This mutants has a significant difference in outer membrane proteins when compared to wild type by SDS polyacrylamide gel electrophoresis as well as by 20 anion-exchange column chromatography of membrane proteins rendered soluble in 6 M urea (15). Also. a bacteriocin resistant, dye sensitive mutant of E, 9211 has been shown to have some reduced amounts of outer membrane protein (16). Mutants of §, typhimuzigm_which have defective lip0polysacc- harides ( LPS ) have been shown to have reduced amounts of outer membrane associated proteins by SDS slab gel electrop- horesis (17). These mutants are known to be sensitive to a variety of dyes and detergents such as crystal violet and dexycholate and to a variety of large polycyclic aromatic compounds. which suggests an increased permeability to these toxic compounds. The decrease in level of the major protein components of the outer membrane upon alteration of the LPS structure in deep rough mutant strains suggests a very close relationship between these components in the architecture of the outer membrane. The proteins might need the LPS structure in order to be anchored to the membrane. The outer membrane of a heptose-deficient lip0polysaccharide mutant of ‘ E. 921; GR 467 had a lower density than that of its parent strain, CR 34. It was found that major envelOpe proteins which were localized in the outer membrane were greatly diminished in this mutant GR 467. This mutant is also more permeable to dyes. detergents. This indicated the importance of LPS for the penetration of low molecular weight molecules through the outer layers of the cell envelope and the outer membrane protein may have an important role in these phenomena. 21 Because a large number of different cytOplasmic proteins are visualized on SDS gels. it is more difficult to determine the functions of specific cytOplasmic membrane proteins. Alterations in the cytOplasmic membrane proteins of various chlorate-resistant mutants of E, 2211 have been reported (18). Some proteins were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase. Spencer and Guest (11) reported that a succinate dehydrogen- ase mutants ( sdh ) of E, 221i was isolated, and the cytopl- asmic membrane proteins were compared with the wild type. t It was shown that one band was greatly reduced in the mutants. They (19) also isolated another mutant of E, 2211 lacking fumarate reductase and observed that inner membrane of this mutant was deficient in a major component of 75.000 daltons molecular weight and possibly a minor one of 87.000 daltons. The former band is more likely to correspond to fumarate reductase. Thus several researchers have used this kind of approach to determine the function of a specific membrane protein by isolating mutants lacking some specific enzymatic activity which is membrane associated and then comparing the membrane protein pattern with that of the wild type. Another approach is to study the membrane protein changes due to differences in cell growth conditions. A change of membrane protein synthesis at 41°C was recently observed in a temperature-sensitive DNA synthesis mutant of E. 2211 K-12 (20). This mutant grows normally below 30°C, 22 but at 41°C its ability to synthesize DNA stops immediately. Similar changes of membrane proteins have also been reported by Shapiro (21). These results suggest that the membrane proteins might be associated with DNA synthesis or cell division which normally depends on DNA synthesis (22. 23). Spencer and Guest (19) have compared the inner membrane protein between E. 2211 grown anaerobically and aerobically on SDS gels. They reported that the pattern of bands in the two types of preparation differed considerably and changes in approximately 20 components were observed. In particular. the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. McGroarty (24) has reported that few changes occur in the protein composition of both the cytoplasmic and outer membranes when cells of E. 2211_are switched from 37 to 43°C. She suggested that the changes in the two proteins of the cytoplasmic membrane may reflect the decreased need for one enzymatic function and the increased need for a second at elevated temperatures. Mizuno et al (56) reported that in sucrose-dependent spectinomycin-resistant ( sued-spar ) mutants of E, 2211 an alteration in the ribosomes caused the loss of one major protein in cytoplasmic membrane preparations. These researchers suggested a structural and functional interaction between ribosomes and cytOplasmic membrane. The structure of the bacterial flagella - the organelles of locomotion has been extensively described by high 23 resolution electron microscopy (1. 2. 3. 4). It consists of three morphologically distinct parts. the filament. the hook and the basal region ( Figure 3 ). The major portion of the organelle is the filament which has been studied most extensively and accounts for more than 97 f of the mass of intact flagella. The filament. external to the cell wall. consists of a helical microtube structure. It is composed of a monomeric protein-flagellin. which in E. 2211_has an apparent molecular weight of 52.000 daltons. The hook is a morpho10gically distinct unit. generally hook-shaped. attached to the proximal end of the filament and terminating at the cell wall. The hook structure differs from the filament in its antigeneticity (29. 30). solubility in acid and other reagents (31). and electrOphoretic mobility on polyarylamide gels (29). It is composed of a single protein subunit (32. 33). In E. 2211 this subunit has an apparent molecular weight of 42.000 daltons. It is also more stable to denatu- ration with acid. heat and urea. When treated with these denaturing agents under mild conditions. the filament will disintegrate but the hook usually is left intact (28. 31. 34). The basal components account for 1 % or less of the weight of the total structure. and they probably contain a large variety of protein subunits (35). According to DePamphilis and Adler (27. 34). the Basal region is composed of four distinct rings and a rod-like structure and is associated with or embedded in the cell envelope. The two outer rings are connected by the short rod to the two inner rings which 24 FIIAMENT ‘- L V<— P RING ROD -—> V“ S RING g”)? <— m RING A HOOK L RING LIPOPOLYSACCARIDE MEMBMNE CYTOPLASMIC MEMBRANE B Figure 3. Model of flagellum of 35202210012 9211 ( A ) and its attachment to the cell envelope ( B ). 25 are in close association with the cytoplasmic membrane ( Figure 3 ). It has been shown that flagella are bound to purified lip0polysaccharide membrane specifically at the basal body ring closest to the ( the L ring ) (27). The cytoplasmic membrane in preparations from osmotically lysed E. 2211 spherOplasts or E2211122_22231112_prot0plasts was specifically attached to flagella at the basal region ring farthest from the hook ( the M ring ). Also. lip0polysacch- aride containing membrane was found attached to the L ring of isolated intact flagella of E. 2211,(27). The flagellar organelle from Gram-negative organisms probably contain structural proteins other than flagellin and the hook protein. It is probable that the complex basal region consists of several different protein subunits. Furthermore. in genetic study. there are a number of genes which may be involved in controlling flagellar formation. In E. 2211. it is known that at least 20 genes are involved in the synthesis. assembly. regulation and function of bacterial flagella (36). In §21m222112. the genetic studies (37) also indicated that 10 genes are involved in flagellar formation. Probably some of these genes are only necessary for flagellar function and are not involved in controlling the synthesis of the basal region. For example. 222_A and 222_§ genes are involved in chemotaxis and mutation in these genes do not affect flagellar structure. 222 mutants have normal flagella and fully motile. but fail to carry out 26 chemotaxis toward any substance tried. 922_Q gene is identical with £12_A gene and mutation in this gene results in either a defect in chemotaxis or absence of flagella structures. This suggested that the f12_g gene controls the synthesis of a component which is necessary for the formation of the flagellar structure as well as for chemotaxis (33)- The 112_§ gene controls the length of the hook. and mutation in this gene results in the cells having long curly polyhooks (33). The £12_1 gene is involved in regulating the synthesis of the entire structure. and mutations close to {12_1 ( cfs mutations ) release flagellar synthesis from control by catabolite repression. The 22; gene controls the flagellar movement and mutations in this gene result in normal flagella that are paralyzed. i.e.. nonmotile. The 22g gene is the structural gene for the major protein subunit. flagellin. Mutations in 22g gene result in the synthesis of intact basal structure and hooks but no flagellin and therefore no flagellar filament (3). Thus. it was suggested (40) that there might be 15 genes involved in the synthesis of the basal components. In addition. it has been reported that isolated hook and basal rings are composed of at least six structural polypeptide components (35). The ring structures may be integral components of the two membranes of Gram- negative bacterial and may be identifiable in studies of the protein components of purified membranes. Therefore it is postulated that the flagellar basal 27 region might be an integral component of the membrane layers. The correlation between the flagellar synthesis and cell membrane synthesis has been reported by Ames et al (17). They found that a deep rough strain mutant of §. ty2212221nm which is defective in lip0polysaccharide synthesis not only has reduced amounts of membrane proteins. but also does not synthesize flagellin. In addition. McGroarty (40) reported that the regeneration of flagella from nonflagellated cells of ontggs 121g2312,needs not only RNA and protein synthesis. but also the presence of an intact cell envelope or concurr- ent synthesis of cell wall. She has shown that the regener- ation of flagella can be inhibited with penicillin and cyclaserine. antibiotics which inhibit synthesis of the murein layer. According to McGroarty. in nutrient medium containing enough NaCl. E. 2211 cells can be induced to produce 20 to 30 flagella per cell and 2, vulgaris can produce ten times more. When cells of E, 2211 and 2. 221ga2is are grown at elevated temperatures ( above 42°C ). flagella formation was repressed (40). A comparison of the membrane proteins of E, 2211 of cells grown at 37°C and 43°C. indicates that a group of proteins are missing or are reduced in amount in cells grown at 43°C when compared to cell grown at 37°C. Several of these temperature sensitive proteins which are repressed during growth at the high temperature correspond to minor protein components of intact flagella of E. 2211 A014. These results suggest that minor flagellar components can be identified on electr0phoresis polyacrylamide 28 gels of purified membranes of E, 2211. It is likely that the location of the 222 gene product is in or near the cytOplasmic membrane (42). Since it is reported that the energy for motility is derived from an intermediate in oxidative phosphorylation (42). The 22; gene product may couple the energy of electron transport to rotation of the basal region. Adler et al ( 43. 44 ) showed that chemotaxis is due to the binding of specific chemotaxis molecules to Specific receptors. These specific receptors then interact with a generalized chemotaxis system to cause movement toward an attractant. There are various specific receptors which lies close to the cytoplasmic membrane or in the periplasmic space. It seems that the energy for chemotaxis is derived from ATP. may be through an S-adenosylmethionine intermediate (42. 45). Larsen et al (42) have pr0pased the following model describing the source of energy for motility and chemotaxis: electron transport oxidazable substrates s>»H20 ) intermediate form E of oxidative ... nergy for phosphoryLation motility H i T Mg++. Ca . -ATPase anaerobically utiliz- ATP ... Energy for able substrates chemotaxis 29 The mechanism of chemotaxis in E. 2211 can be explained by temporal gradient of an active compound which affects the direction of rotation of bacterial flagella (46). Counterc- lockwise rotation of flagella produces smooth swimming and clockwise rotation caused cells to tumble resulting in a randomization of direction when the bacteria swims again (39. 41. 47. 48). Increasing the attractant concentration results in smooth swimming (48. 49). whereas decreasing the attractant concentration shifts it to tumbling (50). In contrast. the effect of increasing or decreasing a repellent concentration is apposite to that of an attractant (46. 51. 52). Thus. bacteria tend to move up a spatial gradient of attractant and move down that of a repellent. In the chemotaxis response chemoreceptors detect the change in the concentration of a chemotactically active compound and recognizes the molecule. Then the receptor in turn activates a common chemotaxic pathway through which recognition is translated into an affect on the flagellar rotation (43). In other words. the specific chemoreceptors receive the stimulus of active compounds which convey the stimulus to the generalized chemotaxis system products of 22222. 222_§ and 222_Q genes. which in turn affect the flagellar rotation. Due to the location of the receptors and flagellar effectors. it seems likely that the products of 222_2. 2n2_§. and 222_Q genes are located in the cytOplasmic membrane. Thus the isolated cell envelope should contain all the protein components necessary for motility. 30 Mat ’al and et a 3 1.03m The strain 2. 221.1 K-12 AW 405 is thr', leu'. his". vit B-. strr. and T6r. The strain.fi12 mutants ( CL 3. CL 17 ) and strain 221 mutant ( CL 5 ) were isolated from E. 2211 K-12 AN 405. The MS strains ( ELLA MS 371. {L5 MS 694. M MS 797 and hag MS 912 ) were obtained from M. Simon's Lab.. they are derivatives of MS 1350 which is his-. thy-. arg E-. gal U-. uvr C-. strepr. hag 207 and sup+. The 222 strains ( 222_A_ e14q1. and M e13p1 ) were obtained from J.S. Parkinson they are F-. sup+. B1-. his-. thr . leu . gal E . rec and strep . 2. o t o s 'a For membrane preparation. cells of g, 2211 were grown in nutrient broth containing 1 % ( W/W’) tryptone ( Difco ). 0.2 fl ( W/V ) yeast extract ( Difco ). and 1 % ( W/V ) NaCl. The cells were incubated at 37°C in shaking water baths until late in logarithmic growth phase ( 0.D. = 1.2 ). Further protein synthesis during cell isolation was inhibited by the addition of 20 ug/ml chlorampheniool ( final concent- ration )e 31 3. Eggpapgtiog of 2211 22221022 22222222 12a2t10n§ Outer and inner membrane were isolated essentially as described by Osborn et al (53). with slight modifications. One liter of cells grown in nutrient broth to °°D°560= 1.2. were harvested by centrifugation at 7.000 rpm in a sorvall RC2-BSS34 rotor for 25 min. The cells were resuspended in 10 ml of 0.02 M Tris ( hydroxymethyl aminoethane ) and 10 ml of 1.5 M sucrose kept at 4°C. The cells were lysed by adding 4 mg lysozyme. Then after 2 min. 40 ml of 1.5 mM ethlenedi- aminetetraacetic ( EDTA ) PH 7.5 was added over a 10 min period. and the mixture was incubated at 30°C for 20 min. Thelysate was sonicated at 40-50 w for 30 sec and 0.75 mg DNase was added along with 0.5 m1 of 0.2 M MgClZ. The cell lysate was incubated at 4°C for 30 min. After incubation. the unbroken cells were removed by centrifugation at 5.000 rpm for 10 min. The pellet was washed once with 25 ml of 10 mM Tris. 5 mM EDTA buffer pH 7.8. and the suspension was centrifuged using the same conditions. Both extracts were combined and centrifuged at 30.000 rpm in a Spino type 30 rotor for 90 min. The pellets were washed with 10 mM N-2 hydroxyethyl-piperazine-N'-2'-ethanesulfinic acid ( HEPES ) buffer. pH 7.4. The pellets thus obtained were suspended in 1.5 ml of 10mM HEPES. Suspensions thus prepared will be referred to as total membranes. To separate outer membranes and cytoplasmic membranes. total membrane suSpensions were layered onto a discontinuous 32 sucrose gradient. The gradient contained 4.8 ml of 2.02 M. 16.8 ml of 1.44 M and 12 ml of 0.77 M sucrose made in 10 mM HEPES buffer pH 7.4. The gradient were centrifuged at 25.000 rpm in a Spino SW 27 rotor for 16 to 18 hours. Fractions were collected by pumping from the bottom of the centrifuge tubes and pumping the contents into test tubes. The absorbsnce of each fraction.was read at 280 nm. Each fraction was also assayed for succinate dehydrogenase using the procedure described by Osborn et al (53). and the density of each fraction was determined by using an Erma Abbe's Refractometer. Peak fractions from themembrane bands were pooled and centrifuged at 30.000 rpm in Spino type 30 rotor for 90 min. The pellet of cytoplasmic membrane fraction was resuspended in 1.5 ml Of 0.0625 M Tris ( pH 6.0 ) for measurement of protein concentration and analysis of the membrane protein composition. Outer membrane fractions. which were contaminated by cytOplasmic membrane. were resuspended in 2 ml HEPES ( pH 7.4 ). and 0.2 ml of 20 % Triton x-100 was added and the sample was incubated at room temperature for 15 min to solubilize the cytoplasmic membrane. After that. theouter membranes were centrifuged at 30.000 rpm in Spinco type 30 rotor for 90 min. The pellet was resuspended in 1.5 ml of 0.0625 M Tris ( pH 6.8 ). and ready for analysis as described for cytOplasmic membrane. 33 4. L120201ysa2222122 gongggt Lip0polysaccharide content was estimated by determina- tion of ketodeoxy-octanoic acid ( KDO ) in isolated outer and cytOplasmic membranes. For KDO determinations. the pellet of membrane fractions was washed twice by centrifugation at 4°C for 90 min at 29.000 x g with distilled water in order to remove residual sucrose. The pellet was resuSpended in a small amount of distilled water. The Lowry assay (54) was used to estimate the protein concentration of each sample. A small volume of each sample ( total protein approximately 0.12 mg ) diluted to a total volume of 200 ulwith 0.25 N H2804 was boiled for 8 min. then cooled down to room temper- ature. One hundred ul of 4 % NaAsO3 in 0.5 N HCl and 1.6 ml of 0.6 % thiobarbituric acid was then added. This mixture was boiled for 10 min and then two ml of butanol ( 5 % in conc. HCl ) was added. and the sample was centrifuged. The absorption of the butanol phase was read at 552 nm and 508 nm. A standard curve of known KDO concentrations was run together with the samples to determine KDO content. A value of 0.45 umoles of KDO per mg of lipopolysaccharide was used to calculae lip0polysaccharide content (63). 50 a sa The method of Lowry (54) was employed with bovine serum albumin as standard to assay the protein concentrations of 34 outer and cytoplasmic membranes. We diluted the sample to 1/100 or 1/200 dilution. read the optical density at 560 nm and 620 nm. 6. 22lxa2rxlamids_sel_2lssir22hsresi§ The purified membrane fractions and a set of standard moleculs'weight proteins - Bovine serium albumin ( BSA ) ( 68.000 ). catalase ( 60.000 ). IgG-H ( 50.000 ). Ovalbumin ( 43.000 ). lactate dehydrogenase ( LDH ) ( 36.000 ) and IgG-L ( 25.000 ) were incubated at 100°C for two minutes in the presence of 2 fl ( W/V ) sodium dodecyl sulfate ( SDS ). 5 s ( w/v ) mercaptoethanol. 10 z ( w/v ) gyceroi. 0.001 % bromphenol blue and 0.0625 M Tris ( pH 6.8 ). Samples were applied to SDS polyacrylamide slab gels prepared according to the procedures described by Ames (55). The gels were cast in a model 220 Bio-Rad cell and contained 6 % acrylamide stacking gel and 12 % acrylamide running gel. The gels were electrOphoresed at a current of 30 mA. with circulating tap water for cooling. In addition. using the same gel system as with the slab gels. SDS tube gels were run. After electrop- horesis. the gels were stained with 0.2 % coomassie blue in 45 % ( V/V ) methanol. 10 % ( V/V ) glacial acetic acid and destained by diffusion in 45 fl ( V/V ) methanol. 10 % ( V/V ) glacial acetic acid. Tube gels were scanned at 550 nm using a Gilford spectrophotometer 240. 35 fiW Each organism was grown overnight in 2 ml tryptone broth. The cells were spun at 5.000 x g for 10 min. The pellet was resuspended in 1 ml of dilution buffer. and applied to carbon coated collodian covered grids and stained with 1 % phosphotungstic acid ( pH 7.0 ). The samples were examined by transmission electron microscopy. Also membrane fractions were applied to carbon coated collodian covered grids and stained with 1 % phosphotungstic acid ( pH 7.0 ). The samples were examined using a Philips 300 transmission electron microscOpe operating at 60 kV. 36 Negatively stained whole organiwms were examined by transmission electron microscapy. The micrographs taken of representive organisms are shown in Figures 2 and 4. We can see that they are all rod shape. The wild type organism is flagellated and has pili. Strains CL 3 and CL 17 have pili without flagella. Strain CL 5 was the same as wild type. Mutants in genes 222_A. £h§_§ both have flagella. The £12_A mutant do not have flagella while the £12_§ mutant has curly flagella. The m21_mutant was flagellated and the 22g mutant appeared nonflagellated. a a o o t a o s ' e b The total envelopes of a 1 liter cultured cells were resuspended in 1.5 ml of 10 mM HEPES ( pH 7.4 ). layered on discontinuous sucrose gradients and centrifuged to equilibr- ium. The profile of the various measurements -absorbance at 280 nm. succinate dehydrogenase activity and buoyant density - of a typical representive separation from wild type cells is shown in Figure 5. The profile of the absorbance at 280 nm indicates that the material resolved into two distinct bands. i.e.. the heavier fraction is enriched in outer membrane and the less-dense fraction enriched in cytoplasmic membrane. The profile of the gradients were similar among 37 Figure 4. Electron micrographs of 2. 2211 2h2 mutant ( A. X 10.000 )3 fla E mutant ( B. x 10.000 ) and mat mutant ( C.X6.900 ). 39 Figure 5. Sucrose gradient centrifugation of total membrane fractions from E. 2211 AW 405. Absorbance at 280 nm was measured across the gradient to locate the major bands. The activity of succinic dehydroge- nase(SDH) marks the location of cytOplasmic membrane. Two distinct bands are resolved. one enriched in outer membrane and the less dense fractions enriched in cytOplasmic membrane. 40 be we mu p m >~mo me new haemom\§s\sp . a ..»a H.“ .l l Heuo .. a: II; I. HeNu rm 1 / ....» z/ // xxx/Icesmwdw 1 “.mo 1 »o 0.0 l I/// I. Home.“ I. m - l I . I. fleHO 1.- e o. I u mu: xx: .sp.ou 1. m _ _ r. a .......... m 90 nu ma mm uo mosses guesses: zo. eon mwmcne u 41 various organisms. The buoyant densities of the outer and cytOplasmic membrane fractions as indicated in Table 1 did not show specific fluctuations and agree with the results of Schnaitman (4) except that the density of outer membrane from strain CL 3. CL 5 and CL 17 appeared somewhat higher. This may be due to the contamination with incompletely lysed cells. The profile of the succinate dehydrOgenase activity in these gradients indicated that most of the activity (>>70 % ) is found in the less-dense bands enriched in cytoplasmic membrane. The higher density bands also showed some succinate dehydrogenase activity. especially for gradients of membranes from £1222. £12_§. and 22g mutants. perhaps indicating contamination of cytoplasmic membrane in outer membrane fraction. Treatment of the outer membrane enriched fraction with Triton X-100 was carried out to remove contam- inating fragments of the cytOplasmic membrane. This prepar- ation was used to analyze outer membrane fractions. After wash ing each membrane fraction twice. the protein concentr- ation of each fraction was measured by the Lowry assay. In addition. KDO determinations were carried out on the hydrol- ysate by the thiobarbituric acid method in order to calculate lip0polysaccharide content. The data of protein and lip0polysaccharide analysis are listed in Table 2. It was found that significant amounts of LPS material was recovered in cytOplasmic membrane fractions. This may mean that the cytOplasmic membranes were contaminated by the 42 Table 1 Buoyant density of the outer and cytoplasmic membrane fractions from various organisms. Organism Membrane fraction Density (gm/Cc) 0.m. 1.210 I 0.004 w.t. I.M. 1.171 t 0.003 0.x. 1.244 1 0.005 CL 3 1.x. 1.175 t 0.005 0.m. 1.232 t 0.006 CL 5 + 1.x. 1.177 - 0.003 0.m. 1.236 t 0.004 CL 17 + 1.x. 1.180 - 0.003 0.M. 1.219 t 0.001 che A I.M. 1.170 t 0.005 0.x. 1.228 t 0.008 che B 1.x. 1.175 t 0.001 0.m. 1.211 t 0.005 fla A + 1.x. 1.183 - 0.002 0.m. 1.216 I 0.006 fla E + I.M. 1.168 - 0.003 0.M. 1.210 t 0.004 mot + 1.x. 1.178 - 0.002 0.M. 1.216 t 0.003 has I.M. 1.170 t 0.006 The values are the average density of the peak of each band from two separate gradients i standard deviation of the mean. Table 2 The result of protein and lip0polysaccharide assay of various organisms. ’43 ,‘ U2 8 E :10 (DN 00 “N NO\ Nd) COH coo mm 0" v4e\ .4U\ cvo~ .4a\ (0%) (3.4 tnra v4F\ «L: ‘38” H “V a. A o .... D. a); .4al r4V\ awn -#<> \Oaa cud) c>F\ cvo Wu: :3 '40: .491 c~c> (he: cac: cue) cart \na: OLH ca '4 «0 v4 01 8 V A 0 "1 p. 5 in \OO\ 50% 0) O\ «max “at NM 0000 Chm-3' OH O\ moan NOs H4? «duo MH H3 0 O O. O. O. O O. .0 .0 as no no no so co OH 00 NH HO \\ «a o H O s A m Sr! .39. gg 50 \OM mm 03%) 300 Nd’ NCO (Do 042' e. .0 o. a. e. e. .0 ea .0 $4 “\O 000 O\\O .70 00 on \Od’ O\CO "\d' Oat-g H N H H M H E a ...-:0 00 ee as ee e. ee .0 w as as as as as as as as as g OH OH OH OH OH OH OH OH OH U) 8 e m <: e +; “‘ ‘n o a m m ‘P . H H I: n H r-l O y 3 <3 <3 0 o 44 44 s .n 44 outer membranes. It is reasonable that the ratio of protein to lip0polysaccharide is higher for the cytOplasmic membrane fractions than for outer membrane fractions. because it is known that lip0polysaccharide exclusively exists in outer membrane while cytOplasmic membrane is composed of only protein and phospholipid. From the data obtained. it appears that the membranes of the :12_g mutant had less protein and a higher content of lip0polysaccharide while £122_J CL 5 and 222_§ mutants had a higher protein concentr- ations than that of the wild type cell. This may mean that these strains have a little different membrane composition or they have different sensitivity to alteration during treatment with EDTA and Triton X-100. E e t ha 3 s o b a 'on Membrane fractions from sucrose gradients were pooled. the outer membrane enriched fractions were treated with Triton X-100. then washed with 10 mM HEPES ( pH 7.4 ) and the pellet was resuspended in small volume of 0.0625 M Tris ( pH 6.8 ). As previously described. the fractions were mixed with SDS and mercaptoethanol. heated at 100°C for 2 min. then equal amounts of protein from each membrane fractions were applied to slab and tube SDS gels. Several samples of each fraction were run several times to assess the reproducibility of protein profiles. On the whole. there are less than 20 bands observed in the Triton x-100 45 treated outer membrane fractions. and more than 30 in cytOplasmic membrane fractions of which about 20 to 30 are very reproducible. The typical SDS electrOphoresis slab gels are shown in Figures 6 and 7. Figure 6 indicates the protein patterns of outer membrane fractions of AW 405. CL 3. CL 5. she... 9.11.8.5.- 1.11.1. 11.8.2. 1.1121- and has mutants together with six standard proteins to determine the molecular weight of the membrane proteins. Figure 7 indicates the typical gel pattern of cytOplasmic membrane fractions of CL 3. AW 405. CL 5. 222_A. 2n2__. i12_2, £12_E. 222. and 22g mutants. Figures 8 and 9 are typical tube gels and in the appendix are profiles of protein measured by absorbance scanning at 550 nm. Most bands visible to the eye were resolved on the scanning trace. either as separate peaks or as shoulders on other peaks. These scans enable the position of the peaks to be accurately located and their Rf values calculated for molecular weight determination. The relative quantities of the protein in each band could be approximately determined by comparing the heights of the peaks. The proteins from outer membrane fractions ranged from 12 K to 117 K in.the molecular weight. and the two major outer membrane bands - 27 to 39 K are dominated. The molecular weight of the major outer membrane protein bands is a little lower than that reported by Schnaitman. This may be due to differences in the conditions used for electr0phoresis. Several laboratories (7. 57. 58) have 46 .Haos some op mwflaaam mm; swepoaa no ma oma pson< .Anme cam mmmComoavhnov evapoma .Cwsspam>o .zuowH .mmdddpmo .sflssnam aspen osw>on mo mswopoaa camcsmpm Aoav ammm Amv .mma Amv «MIMHH Amv .d1mHM on .3 RV .INI: s 2: .m .8 :3 in .8 A3 Rs: 2.2 mass. can: 33.8 mcflopoaa sameness ampso an» no mammaocaoapoeao How evassamaomhaoa mam .o masmflm n N m" Q m _w n. w a 4? .Haos some as ceaaaam mm: saopoaa mo ma.onH psop< .Aume use ommsowouchnon ovmpoma use awssnam>o .xuuwH .ommaspso .sfisspam aspen esa>op mo mswovoam 388$ 33 Ems. RV same :3 filed at .3 3V alums A3 ......Iqfi 2: .m .3 9 33 3 e83 efi: as .n so A: :8 assesses assesses oasmmamo a one we mamoaosaoapooae How avasmahaomhfloa mam .m easmHm an .w in h 0 m .Q N N « 48 Figure 8. SDS polyacrylamide tube gel electr0phoresis of the outer membrane proteins. A: from left to right are: wild type AW 4053 CL 3: CL 53 CL 17: 2h2 A and standard proteins of bovine serum albumin. catalase. IgG-H. ovalbumin. lactate dehydrogenase and IgG-L. B: from left to ri ht are: che B; 21a A; 21a 2; 222; 22g_and also standard proteins. About 500‘pg of protein was applied to the gel. 50 Figure 9. SDS polyacrylamide tube gel electrOphoresis of the cytOplasmic membrane proteins. A: from left to right are: wild type AW 4053 CL 3; CL 5: CL 17; 2he A and standard proteins of bovine serum albumin. catalase. IgG-H. ovalbumin. lactate dehydrogenase and IgG-L. B: from left to right are: 2h2 5; 21a A; 21a E; 222) 22g and also 6 standard proteins. About 500 pg of protein was applied to the gel. 51 52 reported that some membrane proteins have different mobilit- ies on SDS acrylamide gel electr0phoresis depending on the temperature. heating time used prior to electrOphoresis as well as buffer systems used. Proteins from cytoplasmic membrane fractions ranged in molecular weight from 12 K to 125 K and were more numerous than that of the outer membrane. This is reasonable since it is known that there is a large diversity of proteins associated with inner membrane. It was seen that the major protein of the flagellum - flagellin is mainly associated with the outer membrane fractions and less of the protein fractionated with the cytoplasmic membrane. A band with the molecular weight of lysozyme ( 12 K to 14 K ) was present in all fractions. This apparently resulted from nonspecific adsorption of the basic protein to both outer and cytOplasmic membranes. ”‘93 :00 '9 O ‘ Hummij‘ ° ° 5 r 92'”; 01 0: 11:1 The proteins of the outer membrane fractions of E. 2211 AW 405 and the 9 mutant strains ( including :12. 225, 222, and 22g mutants ) were analyzed both by SDS polyacrylamide electrophoresis in tube and slab gels. The gel system we used is composed of 6 % polyacrylamide for the stacking gel and 12 % for the running gel. For each frcation. we apply the same concentration of protein to the gel. The concentr- ation of protein we applied was between 100 - 200 pg for each well on slab gels and abut 400 - 600 pg for tube gels. 53 The data are shown in Figures 6 and 8 and in the appendix. From the results obtained. it can be concluded that the protein composition of the outer membrane fractions does not appear to differ significantly among organisms studied. As shown in Figures 8 and 10 and in the appendix using the gel system I got basically the same profile of protein banding for each sample. There are about 10 bands that can be seen clearly. among them two major bands accounting for 70 % of outer membrane protein with molecular weight between 31K and 36 K. I named each visible band from the wild type cell as a standard and used it to compare with each mutant strain. The average molecular weight of each band is as follows: A-i. 95 K: A-2. 91 K: A-3. 69 K3 A-4. 50 K: A-6. 36 K: A-7. 34 K3 A-8. 31 K: A-9. 28.5 K: A-10. 28.2 K. The band A-4 corresponds to the main flagellar protein. flagellin. Inspection of strains CL 3. CL 17 and £12_2_ showed that the band A-4 was missing. This was expect because they are all flagella mutants. CL 3 also appeared to have a more intense A-10 band. CL 5 has less of the A-4 band. This may be due to its having fewer flagellar than its parent wild type. however. it has more A-9 band protein. CL 17 has less of the A-9 band protein. The £12_2_mutant has a more intense A-8 band. The molecular weight of the main bands of the £1222 mutant appeared a little higher. The percentage of main bands of 222 and 22g mutants seem to be a little higher than in the other strains. As can be 54 seen in Figure 6. the slab gel gave better resolution. more bands were visible. but the molecular weight calculated from these gels was lower. As for the tube gel system. I numbered the protein bands in a manner similar to that for the slab gel systems A-l. 85K3 A-Z. 83 K3 A-3. 53 K3 A-4. 46.6 K ( flagellin )3 A-5. 36.8 K3 A-6. 31 K ( main band )3 A-7. 29 K ( main band )3 A-8. 27 K3 A-9. 25 K3 A-10. 24 K3 A-ll. 19 K and A-12. 12.6 K ( lysozyme ). Again. strains CL 3. CL 17 and £12_A have no A-4 protein band. Strain CL 5 has reduced amounts of A-3 protein. The 222_A mutant has reduced amounts of proteins A-3. A-4 and more A-1O proteins. The 222_§_mutant is missing A-3 and A-5 proteins. The £12_A mutant lacked the A-8 protein and has reduced amounts of proteins A-1. A-2. A-3 and A-4. The f12_§ mutant again showed a different main protein pattern. Instead of molecu- lar weights of 29 K and 31 K. its main outer membrane proteins were 25 K and 31 K in molecular weight and the pattern of the minor bands also appeared different. The 223_mutant had a more intense A-9 band. The A-8 band of the 22g mutant appeared to be reduced in amount. In conclusion. for £12 mutants. no specific differences were found in outer membrane protein composition except the main flagellar protein. flagellin was missing. For 222 mutants. the 50 K and 69 K dalton proteins were found to be reduced in amount; these two proteins may be associated with the mechanism of chemotaxis. For mot and 22g mutants.the main band was found 55 to be more intense. This small change of main band protein may be related to the maintenance of structural stability. For the £12_§ mutant. the protein pattern differed signific- antly. These changes in the level of the different major proteins of the outer membrane may be involved in maintaining and regulating the structural stability. Although I treated each fraction with Triton x-100 to purify outer membrane fraction. it can be seen that each sample still appears to have some contamination from the cytOplasmic membrane. a so as ' embran at 'n o as o tw ' a d t st a' s As with outer membrane proteins. we analyzed the protein components of the cytOplasmic membrane. The results are shown in Figures 7 and 9 and in the appendix. From the tube gels of wild type cytoplasmic membrane there were 23 protein bands that could be resolved reproducibly: B-1. 125 K3 B-2. 94.5 K3 B-3. 90 K3 B-4. 84 K3 B-5. 83 K3 B-6. 81 K3 B-7. 76 K3 B-8. 72 K3 B-9. 70 K3 B-10. 68 K3 B-11. 65 K3 B-12. 60 K3 B-13. 55 K3 B-14. 54 K3 B-15. 51 K3 B-16. 49 K3 B-17. 44 K3 B-18. 38 K3 B-19. 36 K3 B-20. 35 K3 B-21. 32 K3 B-22. 31 K and B-23. 27 K. Among them. band B-14 is likely to be main flagellar protein. flagellin. and bands B-20 and B-21 are obviously major outer membrane protein. Their presence in these fractions means that cytOplasmic membrane fraction is contaminated with outer membrane in agreement with the results from the KDO determinations. From the banding 56 pattern of tube gels. it was shown that there appears to be some difference between wild type and mutants. Strain CL 3 lacks bands B-6. B-7. B-8 and B-15. Strain CL 5 appeared to be missing bands B-5. B-6. and B-7. Band B-2 can not be seen in cytOplasmic membranes from strain CL 17. The 22222 mutant lacks bands B-1. B-5. B-6. B-7. B-8. B-11 and B-16. Mutant 222_§ did not appear to contain protein bands B-3. B-4. B-6. B-7. B-8. B-9. B-12 and B-15. The {12_2 mutant lacks bands B-l. B-6. B-7. B-9. B-11. B-16. B-18 and B-19. Bands B-1. B-5. B-6. B-7. B-9. B-11 and B-18 were not found in the f12_§_mutant. The 221 mutant has a little different gel pattern for the higher molecular weight protein bands. Similar differences were seen in the 22g mutant. But. on the other hand. in the membrane isolation preparations visualized on slab gels. I did not see such drastic differe noes between the wild type and mutant strains. About 24 bands were visualized in the gel pattern. The molecular weight of the proteins were similar to those on the tube gels. Among the four most dense intense bands ( B-2. B-14. B-20 and B-21 ). band B-14 corresponds to flagellin. and B-20 and B-21 are the major outer membrane proteins. The differences that can be visualized clearly are as follows: Strain CL 3 lacks or had decreased amounts of bands of B-5. B-6. and B-7. Strain CL 5 seemed to have less of the band B-16 protein. The 222_2 mutant had nearly the same pattern as the wild type culture. The 222_§ mutant had decreased amounts of bands B-6. B-7. B-9. B-18 and B-19. The 31222 57 mutant had a little different gel pattern: the main outer membrane bands were not as intense as with the other organisms. The most intense band was band B-12 ( 61 K ) and the bands B-22. B-23. B-24 ( 26.6 K ). B-25 ( 24.5 K ) and B-26 ( 23.5 K ) were somewhat more intense than wild type. The 222 mutant had greater amounts of the second main band ( 32 K ) and B-16. The 22g mutant also had more of the B-16 and B-18 band proteins. In conclusion. for £12_mutants. the 81 K. 76 K. 70 K. 65 K and 38 K dalton proteins were found to be reduced in amount. Some of these proteins may be involved in the synthesis. assembly of flagella. For the 2112 mutants. the 90 K. 84 K. 81 K. 76 K. 72 K. 65 K and 51 K were found to be reduced in amount. Some of these proteins reduced in the mutant strain may be associated with the mechanism of chemotaxis. The 222 and nggrmutants.had more intense staining of protein bands of molecular weight 49 K. 38 K and 32 K. The increase in the amounts of these bands may reflect the need of proteins to maintain structural stability or for the motility process. seem .nu sweeps“ mmoH .3 58 wcwmmfls .x messes“ macs .+ 1 3 + m m.mm 0H3< + + x m.mm m3< + +. a an wu< + x em and s m.em sue x a o: mu< x x n x a on ens . x u u a me nu< : 2 am ~34 . a no Hu< passe; was m s e .s1m4w .MImmm .s1usm as so n so n so seasoeaos seem .mos as was» sass no sees» op madness mo mcoapomuw sameness umpso mo momma swoeoua mo somwnmasao n edema seem .nu mmsmpsw mmmH .3 59 mchmHs .x mmsmPsH macs .+ e um mmum a an mmum x mm «mum x mm om-m x mm maum + x x mm waum 2 es Baum + + x u a ms esum x x a pm 33m x em saum x mm msum x a so Naum a x x a we «Hum a me ceum x x x a om. mum a x x we ate x x x x x x x mm mnm x x x x x x 2 am mum x x a x x x mm mum 2 am sum x a om mum x a m.sm, mum x x x x a “NH Ham assess .mms was .mlmww m1mmw .mumqm .slmsm as so n so m so assesses: seem .nos as mass ease to emcee op messess we meowpomew meanness owsmmaaopho mo muses seepage mo somwamasoo e sense 60 Dis2ussion The protein assays on fractions from the gradients following the lysozyme - EDTA and Tris buffer treatment revealed four bands similar to what has been reported by others for E. 2211 and for 2. 3y221m22122 (7. 11). The four bands are : (i) the high density band which corresponds to the outer membrane fractionS3 (ii) the middle band which corresponds to total envelOpe material which had not separated well: (iii) and (iv) tow light bands corresponding to the inner membrane fractions. Although it is not known yet why the inner membrane fraction resolved into two bands. it was shown that those two bands have very similar chemical composition and bound enzyme activity. as well as almost identical electrophoretic protein patterns. The recovery of two bands may be due to different amounts of contamination with cell wall material. For simplification. we separated the membrane samples into two fractions - outer and cytOpla- smic membrane fractions by using HEPES buffer ( pH 7.4 ) instead of EDTA and Tris buffer. and a different system of discontinuous sucrose gradient containing 12 ml of 0.77 M. 16.8 ml of 1.44 M. 4.8 ml of 2.02 M sucrose in 10 mM HEPES ( pH 7.4 ). We recovered the two fractions from the envelOpes of every strain. The protein assay profiles of these gradients did not show specific differences in buoyant density between wild type and mutants except that the density of outer membrane from strain CL 3. CL 5 and CL 17 appeared 61 somewhat higher. This may be due to the contamination with incompletely lysed cells. From the succinate dehydrogenase activity assay. KDO determinations and electron microscopy. it was revealed that it is very likely that the outer membrane and cytoplasmic membrane fractions are contaminated with each other. This may be due to the complex structure of the envelOpe for Gram-negative bacteria. It may not be so easy to separate membrane layers. It was reported (53) that for better separation. the technique depends on various conditions such as the concentration of lysozyme. the time interval in which sucrose is diluted. the temperature and the buffer system. But the procedure for separation still could be improved. In order to gain more purified outer membrane fractions. we used the Triton x-100 treatment develOped by Schnaitman (10). This treatment improved the purity of the sample a little. but from our KDO measurement. we observed another problem. Triton X-100 treatment also removed KDO up to 50 %. Thus we can not be certain that we have the correct composition for protein and LPS in the treated membranes. For the KDO data. we know it is reasona- ble that most of the KDO is in outer membrane fractions. while the little amount KDO which is found in the cytOplasmic membrane fractions means contamination. For this study. we used KDO determinations to study weather the £12 mutant is really :12 mutant or if it is an LPS - deficient - deep rough strain. It appears that the £12 mutants we used are all £12 62 mutants not membrane mutants. We applied the outer membrane and inner membrane fractions to carbon-coated collodian covered grids and stained with 1 % PTA ( pH 7.0 ). then checked these fractions under electron microsc0py. Our observation were in agreement with what has been reported by others (60). The outer membrane fractions appeared as triple-layered vesicles. although they were contaminated with unlysed spher0plasts. Some flagella could be seen in wild type. ot.§12_§ and 222 mutants. 0n the other hand. the inner membrane fractions looked like open C-shaped vesicles. We could not observe any difference between the inner membranes of the wild type and the mutants. ‘ SDS polyacrylamide gel electr0phoresis has been one of the major popular techniques for examining the protein composition of membranes and this technique has been widely applied to the study of the outer and inner membrane proteins of Gram-negative bacteria. We compared the outer and inner membrane composition between wild type and mutants. Some differences were observed: but some of the proteins fluctua- ted from time to time and the gel preparation did not clearly resolved all the minor bands. In conclusion. for £12 mutants. no specific difference was found in outer membrane protein composition except the main flagellar protein. flagellim was missing while the 81 K. 76 K. 70 K. 65 K and 38 K dalton proteins of cytOplasmic membrane fractions were found to be reduced in amount. Some of these 63 proteins may be involved in the synthesis or assembly of flagella. For 222 mutants. the 50 K and 69 K dalton proteins of the outer membrane fractions were found to be reduced in amount. and the 90 K. 84 K. 81 K. 76 K. 65 K and 51 K dalton proteins of the cytOplasmic membrane fractions were found to be reduced in amount. Some of these reduced proteins may be associated with the mechanism of chemotaxis. For 222 and 22g mutants. the main outer membrane protein bands were found to be more intense. and the 49 K. 38 K dalton proteins of the cytoplasmic membrane fractions were also more intense. The increase in the amounts of these bands may reflect the need of these proteins to maintain structural stability or for the motility process. For the fi12_§_mutant. the protein pattern was quite different. the changes in the level of the different major proteins of the outer membrane may be involved in maintaining and regulating the structural stability. It is possible that during the purification procedure we may have removed some labile auxiliary structure. Also the polypeptides were separated on the basis of size and there may be proteins which were not resolved. Also. it is difficult for us to compare our results with others. We resolved two major proteins. which are smaller in size than reported by Schnaitman (4). He resolved four major protein bands which were larger in molecular weight. It was reported that this major protein complex may contain as many as 18 different proteins (62). The resolving power of the SDS 64 gel system depends on the conditions used and the treatment of the membrane preparation before applying it to the gel system. Thus. the protein banding pattern can easily be altered. It has been estimated that there are in the order of more than one hundred protein species associated with the inner membrane. including the many permease systems. DNA replication enzymes and the electron transport complex. So it is impossible to identify what the specific proteins we observed that were altered in the mutants. Up to now. only two proteins are identified: one is nitrate reductase. an enzyme which is induced anaerobically in the presence of nitrate and lacking in chlorate resistant mutants (18)3 another one is succinate dehydrogenase subunits. described by Spencer and Guest (11). In addition. many papers have reported that the cytoplasmic membrane protein composition can be altered in mutants or can be altered with changes in the growth conditions (11. 14. 16. 17. 18. 19. 21. 24. 44. 56). These fluctuating proteins however have not been identified yet. because of the complexity of inner membrane protein composition. So the observed difference must be considered tentative since the identification must await protein purification. Further genetic and chemical studies will be necessary to establish the maximum number of indivi- dual polypeptides which are present in membrane fractions. Also. Ames et al (61) have described a high resolution method for two-dimensional separation of membrane proteins. 65 It involves solubilizing membrane protein with SDS. then separation of protein components in the first dimension according to charge ( by isoelectric focusing ). For the second dimension. an SDS buffer is used to separate proteins on the basis of molecular weight. According to their report about 150 different proteins can be visualized from isolated E. 2211 envelOpes. According to Simon.et al (35). the basal structure of the flagellum controls both flagellar activity and assembly. They also found that 20 genes are required for the complete assembly and function of the organelle. Among these genes. perhaps not all of the £12 and 222 gene products are membrane proteins or flagellar structural proteins. Some of them may code for regulatory proteins which are still cytOplasmic components. Also. it was reported that purified basal regions were associated with cytoplasmic membrane fractions. McGroarty (40) reported that for flagella synthesis. not only RNA. protein synthesis are needed but also the presence of an intact cell envelOpe or concurrent synthesis of cell wall are required. From all these points. it is believed that some changes of membrane protein should be observed in :12. 221. 222_and 22g_mutants. In this study. we observed some differences. but in order to identify them, further study is needed. For further study. a two-dimensional gel system should be used. It would be more sensitive to allow observation of the differences among mutants. Also further 66 studies with more mutants should be undertaken to identify the protein composition of the motility system. Such studies should allow for a better understanding of the assembly. function. regulation and mechanism of the motility of bacteria. APPENDIX 67 Figures 10 - 14. Scans of SDS polyacrylamide tube gel electrOphoresis of membrane proteins shown in Figures 8 and 9. Scans a to j are from outer membrane fraction33 scans a' to j' are from cytOplasmic membrane fractions. The gels were scanned at 550 nm using a Gilford spectrOphotometer 240. £1 A 4 fla A fla E Figure 11 . l . x 1‘. ,1. ... t. . .r .l.‘1l |\. 1 . . . . I' A . y I . . . 1 t z _ 0 . . . 1 .. t ‘1‘ A! 0 I». v I: . a1‘ll . 6.1.. .07 .. 6“ .| I- 1 I I I 70 mot hag / bl a! AW 405 CL 3 Figure 12 5.." ... I a... la! a c ....l..- I ... ‘ A’ : .. t . . . . 1 t ’ 1... - - 3 3 I) - 1 . 1. - 1 t- x I... I .. ..I If . I/ \q \ . . u d . a .. - r. ’. l9 1.“. \.. “ Iii-wanna.” w\ .‘t K. . .11! . ..J 11. 1“ Q .91. u. .161- .- ..\ 1V. 1" ‘I‘ID- '0‘ ‘ II- I I. "llI-'|1- ‘3 0 1 ideas 1: .... .I :I ’v‘ I. ‘1! I‘ ... V J 3 I; u I I l ’1 .- aid-Inlet.) .l (J... z.u.w30».v.o- .....l :.-.Io \: III"... 3’. v -e \ (v A? BIBLIOGRAPHY 9. 10. 11. 12. 13. 14. 15. 16. BIBLIOGRAPHY Mitchell. P. Biological Structure and Function vol. 2. 581 (1961). Freer. J.H. and M.R.J. Salton The Anatomy and Chemist- ry of Gram-negative Cell Envelopes. 67-126 (1972). de Petris. S. J. Ultranstruct. Res. 12. 45-83 (1967). Schnaitman. C. J. Bacteriol. 122. 890-901 (1970). Oltmann. L.F. and A.H. Stouthamer Arch. Mikrobiol. .23. 311'325 (1973). Hasin. M.. S. Rottom and S. 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