JoURNAL OF BACTROLOGY, Feb. 1973, p. 937-945 Copyright @ 1973 American Society for Microbiology
Vol. 113, No. 2 Printed in U.SA.
Physiological Studies of Methane- and Methanol-Oxidizing Bacteria: Immunological Comparison of a Primary Alcohol Dehydrogenase from Methylococcus capsulatus and Pseudomonas sp. M27
R. N. PATEL,1 W. J. MANDY, AND D. S. HOARE2 Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712
Received for publication 27 September 1972
A primary alcohol dehydrogenase was purified from cell extracts of two apparently unrelated microorganisms, namely, Pseudomonas sp. M27 and Methylococcus capsulatus. Rabbit antiserum prepared against the purified enzyme from M. capsulatus revealed distinctive antigenic determinants by quantitative and gel precipitin reactions. Rabbit antiserum to M27 enzyme detected both distinctive and shared antigenic determinants. Certain methaneand methanol-oxidizing bacteria were grouped on the basis of serological cross-reacting enzyme specificities.
Pseudomonas sp. M27 and Methylococcus capsulatus are representative of two apparently unrelated groups of microorganisms (1, 18). Nevertheless, the two organisms are similar in certain physiological properties, particularly with reference to the oxidation of C-1 compounds. For example, cell suspensions of both organisms oxidize methane, methanol, formaldehyde, and formate to carbon dioxide (18). Recently, a nonspecific primary alcohol dehydrogenase (PAD) was purified from cell extracts of methanol-grown M. capsulatus and Pseudomonas M27 (2, 17). A comparison of the biochemical and physical properties of the PAD from the two organisms showed similarities in substrate specificity, requirement for ammonium ions, molecular weight, and subunit composition. Also, the purified enzymes had identical absorption properties, with optima occurring at 260 and 350 nm. It was suggested, therefore, that the enzymes operate by identical oxidative mechanisms, perhaps involving a heretofore unknown pteridine derivative (17). The purified enzymes, however, differ in their electrophoretic mobilities (17). Disc gel
electrophoresis experiments indicated that the M. capsulatus enzyme is more negatively charged. Also, preliminary amino acid analysis data show differences between the two enzymes in the content of acidic and basic amino acids. The present investigation was concerned with serological properties of the two enzymes. Antisera prepared against the purified PAD from M. capsulatus and Pseudomonas M27 were used in quantitative and agar gel precipitin reactions as well as enzyme inhibition experiments to show that certain antigenic similarities exist between the two enzymes. The morphological classification of certain methane-oxidizing bacteria (7, 22, 23) coincided with grouping on the basis of serological crossreactions with PAD from M. capsulatus and Pseudomonas M27.
MATERIALS AND METHODS Methane-oxidizing bacteria. M. capsulatus
(Texas) was reisolated from old contaminated cultures (8). M. capsulatus (Bath), Methylosinus sporium, Methylosinus trichosporium, Methylococcus minimus and cyst-forming culture (culture C.F.) were kindly provided by R. Whittenbury (Department of Microbiology, University of Edinburgh, Edinburgh, Scotland). The Texas strain of Pseudomonas methanica was provided by J. J. Perry (Department of Microbiology, North Carolina State University,
'Present address: Department of Biology, Yale University, New Haven, Conn 06520. 'Deceased, 16 May 1971. 937

PATEL, MANDY, AND HOARE

J. BACTOL
Raleigh). These microorganisms were maintained on minimal salts-agar plates in a desiccator under an atmosphere of methane and air (1:1) at 30 C, except for M. capsulatus (Texas and Bath), which was maintained at 37 C. Cell extracts were prepared from organisms grown at 30 C in 14-liter carboys containing methane (methane and air, 1:1) as sole carbon source (8). Methanol-oxidizing bacteria. Pseudomonas M27 was obtained from L. J. Zatman (Department of Microbiology, University of Reading, Reading, England). Hyphomicrobium B-522 was provided by P. Hirsch (Department of Microbiology, Michigan State University, East Lansing). The methanol-oxidizing "pink" and "white" organisms were obtained by enrichment culture techniques and were isolated from media containing methanol as sole carbon and energy source (R. J. Mehta and D. S. Hoare, unpublished data). These organisms were maintained at 30 C on basal medium agar plates containing 0.4% methanol. The methanol-oxidizing bacteria were grown at 30 C in 2-liter flasks containing 500 ml of basal medium (8) and methanol (0.4%). Cell extracts and purified PAD. Cells in 50 mM potassium phosphate buffer, pH 7.0, were disintegrated, and the cell extracts were obtained as described previously (18). The PAD from Pseudomonas M27 was purified as described by Anthony and Zatman (2). Essentially the same procedures were followed for the purification of PAD from M. capsulatus (17). Enzyme activity. A standard spectrophotometric assay was used to monitor PAD activity (2). Reaction mixtures (total volume of 3.0 ml) contained: 150 umoles of tris(hydroxymethyl)aminomethane (Tris)hydrochloride buffer, pH 9.0, 50 Amoles of ammonium chloride, 2 pmoles of phenazine methosulfate (PMS), 0.5 Mmole of 2,6-dichlorophenol-indophenol (DCPIP), 20 ;smoles of methanol, and cell extract or purified PAD. The rate of the reaction, measured as DCPIP reduction, was adjusted to give an absorbancy change of 0.3 per min per cm at 600 nm. The specific activities reported are expressed as nanomoles of DCPIP reduced per minute per milligram of

protein. A molar absorbancy for DCPIP of 1.9

per cm was

used (4). Protein concentrations were determined by the Folin-Ciocalteau method with bovine serum albumin as standard (14). Antisera. Antisera to crude or purified PAD from Pseudomonas M27 or M. capsulatus were prepared in random-bred New Zealand rabbits. Two injections of 2 mg each of the crude enzymes incorporated in complete Freunds adjuvant (Difco) were given subcutaneously at 2-week intervals. One week later and at weekly intervals thereafter, the animals were bled by marginal ear vein puncture and given booster injections of 1 mg of the antigen. Rabbits immunized with the purified PAD were given two successive injections of 1 mg of the antigen incorporated in complete Freunds' adjuvant at 1week intervals. Two weeks later, the rabbits were given four intraveneous injections of the soluble antigen (0.25 mg) at 2-day intervals. One week later and at weekly intervals thereafter, the animals were
bled by marginal ear vein puncture. The sera of successive bleedings which contained demonstrable antibody activity were pooled and stored at -20 C; no visible antigen-antibody reactions were produced with preimmune control sera. In preliminary experiments, it was found that preimmune rabbit control sera also exhibited some enzyme inhibition activity, presumably by binding of low-molecular-weight cofactors, such as PMS and DCPIP, with the nonspecific serum proteins. This nonspecific binding was eliminated by using globulin (IgG) fraction of immune and normal rabbit serum for all experiments. The IgG was obtained by the sequential procedures of sodium sulfate precipitation (11) and chromatography on diethylaminoethyl-cellulose (12). Protein concentrations were estimated from the absorbancy of the solution at 280 nm by use of a specific absorbancy of 15 per cm for a 1% immunoglobulin solution. Immunoelectrophoresis. Immunoelectrophoresis of the purified enzyme preparations in 50 mM barbital buffer, pH 8.6, was carried out by the method of Scheidegger (19). For these experiments, the precipitin bands were developed with the IgG fraction of antiserum to the crude enzyme preparations. Immunodiffusion. The Ouchterlony double-diffusion reactions were carried out in 1% Oxoid agar gels made in saline-borate buffer, pH 8.0 (21). The IgG fraction of rabbit anti-PAD was used at a concentration of 10 mg per ml. The dilution of antigen, i.e., purified enzyme or cell extract, which gave the sharpest band with immune serum was determined in preliminary tests. All diffusion reactions were evaluated after incubation for 30 hr in a humid atmosphere at room temperature. Inhibition studies. Various amounts of the IgG fraction of anti-PAD antisera were incubated with the purified enzyme for 20 min at room temperature in a reaction mixture (3.0 ml) containing the neces. sary reagents for enzyme assay. Methanol (20 Asmoles) was added after the incubation period. As a control, the enzyme was preincubated with equivalent amounts of normal rabbit IgG. The percent inhibition was estimated from the amount of DCPIP reduced in the presence of antienzyme antibody as compared with the amount reduced in the control experiments. Quantitative precipitin tests. The amount of antibody precipitated by the various antigens was determined by the standard quantitative precipitin reaction (15). Volumes of 0.2 ml of various dilutions of the purified or crude enzyme preparations were added to equal volumes of the IgG fraction of rabbit anti-PAD (10 mg/ml). The precipitating mixtures (0.4 ml) were incubated at 37 C for 1 hr and ovemight at 4 C. The immune precipitate which formed was then centrifuged, washed three times with physiological saline, and dissolved in 1.0 ml of 0.02 N NaOH. The amount of protein recovered as the enzyme-antienzyme precipitate was determined from the absorption at 280 nm. Ultracentrifugation. Sedimentation velocities of protein solutions were measured at 20 C in a 40,

VOL. 113, 1973

SEROLOGY OF A PRIMARY ALCOHOL DEHYDROGENASE
12-mm cell in a Spinco model E ultracentrifuge at 59,780 rev/min. Acrylamide gel electrophoresis. Disc gel electrophoresis was performed as described by Hay et al (9). Untreated enzyme preparations were subjected to electrophoresis on 7% gels with 0.1 M Tris-glycine buffer, pH 8.9, for 2 hr at 4 ma/gel. The gels were stained with aniline blue black (1%, w/v, in 7% acetic

acid).

RESULTS Throughout these studies, PAD was purified from cell extracts of several batches of methanol-grown M. capsulatus and Pseudomonas M27. The purity of the enzyme preparations was assessed by the following criteria: ultracentrifugation analyses showing a single symmetrical peak with S2:0, values of approximately 7.7, acrylamide gel electrophoresis of the enzyme showing a single band stained for protein (Fig. 1), and immunoelectrophoresis of the enzyme revealing a single precipitin arc with antiserum to the crude enzyme extract containing the homologous antigen (Fig. 2). Figures 1 and 2 also show differences between the two enzymes in their electrophoretic mobilities. Immunoelectrophoresis of a mixture of the two enzymes gave overlapping arcs and a spur (Fig. 2C). These findings suggested that the two enzymes have distinctive as well as similar antigenic determinants. Serological comparison of PAD from M. capsulatus and Pseudomonas M27. Figure 3 illustrates a qualitative analysis of the reaction between specific antiserum and PAD. The gel diffusion patterns show that the antiserum directed against the M. capsulatus enzyme was specific and did not react with the M27 enzyme (Fig. 3B). The antiserum directed against the Pseudomonas M27 PAD, however, reacted with both enzymes (Fig 3A). The spur formed by reaction of the antiserum with the M. capsulatus PAD and the M27 PAD in adjacent FIG. 1. Disc gel electrophoresis of purified priwells reveals that M27 PAD possessed antigenic determinants not found on M. capsulatus mary alcohol dehydrogenase from Pseudomonas M27 (left) and M. capsulatus (right). Each enzyme prepaPAD (Fig. 3A). ration The serological specificity of the antisera was hr at 4(75 ug) was subjected to electrophoresis for 2 ma/gel in 0.1 buffer, also demonstrated by quantitative precipitin The protein migrated M Tris-glycine(top) to pH 8.9. from cathode anode. analyses. For example, antibody to the M. capsulatus enzyme was precipitated only by the homologous antigen (Fig. 4), whereas anti- group of antigenic determinants. body prepared against the PAD from Figures 6 and 7 illustrate the capacity of Pseudomonas M27 was precipitated by both antibody to inhibit PAD activity. For anenzymes (Fig. 5). A comparison of the preciptin ti-M27, the inhibition was linear within the curves obtained with homologous antigen-anti- range of 0 to 0.75 mg of the immunoglobulin body systems shows that the anti-M. fraction (Fig. 6A). It is of interest to note that, capsulatus serum was more homogeneous and although the inhibitory activities of the antiapparently was directed to a more restricted sera for each enzyme were comparable, the

J. BACTMOL.

20 ENZYME (pg)
FIG. 2. Immunoelectrophoresis of purified primary alcohol dehydrogenase from: (A) Pseudomonas M27, (B) M. capsulatus, or (C), a mixture of each purified enzyme preparation. The precipitin bands were developed with antiserum to a crude enzyme preparation. Pattern C was developed with a mixture of antiserum to each enzyme preparation.
FIG. 4. Quantitative precipitin curves obtained with anti-M. capsulatus primary alcohol dehydrogenase tested against various amounts of the purified primary alcohol dehydrogenases of M. capsulatus (0) or Pseudomonas M27 (A). Precipitin reactions were carried out in saline-borate buffer, pH 8.0, ionic strength 0.16. Total volume of each reaction mixture was 0.4 ml; 2.0 mg of antibody protein was used in each test.
FIG. 3. Double gel diffusion precipitin patterns of purified primary alcohol dehydrogenase of Pseudomonas M27 (well 1) and M. capsulatus (well 2). The center wells, A and B, contained antiserum prepared against the purified enzyme preparations of M27 and M. capsulatus, respectively.
amount of antibody precipitated by the homologous antigen (i.e., M27) was approximately fivefold greater than that precipitated by the M. capsulatus enzyme (Fig. 5). Enzyme activity was also inhibited by the anti-M. capsulatus antiserum, but to a lesser extent (Fig. 6B). The inhibition provided by the antiserum to the M. capsulatus enzyme was also examined
(jYg) FIG. 5. Quantitative precipitin curves obtained with anti-M27 primary alcohol dehydrogenase tested against various amounts of the purified primary alcohol dehydrogenase of Pseudomonas M27 (A) or M. capsulatus (0). Precipitin reactions were carried out in saline-borate buffer, pH 8.0, ionic strength 0.16. Total volume of each reaction mixture was 0.4 ml; 2.0 mg of antibody protein was used in each test.
at various substrate concentrations. According to the Lineweaver-Burk plots of the data, the inhibition of Pseudomonas M27 PAD was simple noncompetitive. With M. capsulatus PAD, the inhibition was complex noncompetitive, probably as a result of a combination of mech-

20 ENZYME

0.50 0.75 1.00 IMMUNOGLOBULIN

05 (mg/ASSAY) 15

FIG. 6. Inhibition of primary alcohol dehydrogenase (PAD) activity by the IgG fraction of (A) anti-M27 primary alcohol dehydrogenase and (B) anti-M. capsulatus PAD. Details for the inhibition tests are given in the text. All values were corrected for the amount of inhibition obtained by equivalent amounts of normal rabbit immunoglobulin. Purified M. capsulatus PAD (0); purified M27 PAD (A).

anisms involving noncompetitive and uncompetitive types of inhibition. Serological comparison of PAD from methane- and methanol-oxidizing bacteria. Each antiserum used in this study was specific for the purified enzyme protein. This was demonstrated by the development of a single precipitin band when the antisera were tested against crude enzyme preparations after immunoelectrophoresis. Therefore, the PAD enzyme in cell extracts of various methane- and methanol-oxidizing bacteria was amenable to a comparative immunological study without further purification. The results of a quantitative precipitin analysis obtained with antisera and the cell extracts of a number of different microorganisms are given in Table 1. The values reported are the amount of protein precipitated at the optimal antigen concentration. According to the results, the microorganisms listed can be categorized into three or five groups on the basis of protein precipitated with anti-M. capsulatus or anti-M27 PAD, respectively. There was some question, however, as to whether M. minimus belongs with M. capsulatus or with P. methanica. The serological cross-reactions of the crude extracts were also tested by qualitative gel-diffusion reactions (Fig. 7). As shown in Fig. 7A, anti-M. capsulatus gave precipitin bands with the homologous antigen and with M. capsulatus (Bath), M. minimus, P. methanica, and culture C.F. The fusion of precipitin arcs between the Texas and Bath strains shows reactions of complete identity. Likewise, the
reactions with M. minimus, P. methanica, and culture C.F. show complete identity. These, however, gave only partial identity with the homologous antigen as evidenced by the spurs. No precipitin bands were developed with antiM. capsulatus and the cell extracts of M. sporium, M. trichosporium, or the methanoloxidizing bacteria listed in Table 1. With the exception of the "white" organism, cell extracts of the methane- and methanol-oxidizing bacteria reacted in gel-diffusion tests with anti-M27 PAD (Fig. 7B and C). The methane-oxidizing bacteria M. capsulatus (Texas and Bath), M. minimus, P. methanica, and culture C.F. gave reactions of complete identity with one another, but gave reactions of partial identity with Pseudomonas M27 (Fig. 7C). Pseudomonas M27 and "pink" organisms gave reactions of complete identity (Fig. 7B). Methylosinus sporium, M. trichosporium, and Hyphomicrobium B-522 gave reactions of complete identity with each other (Fig. 7B), but gave reactions of partial identity with the homologous antigen (Fig. 7D). By comparing M. sporium, 'as the prototype, with M. capsulatus, P. methanica, and culture C.F., partial antigenic identity was observed also with M. trichosporium and Hyphomicrobium B-522 (Fig. 7E). These results, too, are summarized in Table 1. The organisms are grouped according to those comparisons showing complete antigenic identity. DISCUSSION The growth of M. capsulatus and of Pseudomonas sp. M27 in media containing

J. BACTFRIOL.

FIG. 7. Immunodiffusion studies showing cross-reactions between the primary alcohol dehydrogenase (PAD) of various methane- and methanol-oxidizing bacteria. (A) Center well contained antiserum prepared against the purified PAD preparation from M. capsulatus. (B-E) Center wells contained antiserum prepared against the purified PAD of Pseudomonas M27. Sample wells contained cell extracts from: (1) M. capsulatus (Texas), (2) M. capsulatus (Bath), (3) M. minimus, (4) P. methanica, (5) culture C.F., (6) M. sporium, (7) M. trichosporium, (8) Hyphomicrobium B-522, (9) Pseudomonas M27, and (1O)"pink" organism. The amount of cell extract placed in sample wells was the amount giving the best reaction as determined by preliminary gel diffusion studies.
methanol as a sole carbon source appears to be
dependent upon the catalytic activity of a PAD. The purification of PAD from each microorganism has been reported (2, 17). The enzymes are identical in several respects but differ in electrophoretic mobility; the enzyme from M. capsulatus is the more negatively
charged. This was confirmed by amino acid analyses, which showed differences in the content of acidic and basic amino acids (Patel and Mandy, unpublished data). The results reported herein clearly show that each enzyme has distinctive antigenic determinants, indicating significant conformational
TABLE 1. Immunological comparison of pri'mary alcohol dehydrogenase from various methane- and methanol-oxidizing bacteria

Groups

Cell extracts of

precipitateda Anti-M.

capsulatus

Gel diffusion

Anti-M. capsulatus

reactionsb

Anti-M27

AM27n M2

Methane-oxidizing bacteria
M. capsulatus (Texas) M. capsulatus (Bath) M. minimus

74.0 72.8 52.5

40.7 38.3 0.0 0.0
31.5 Confluent precipitin Confluent precipitin lines 33.3 lines 37.7 18.5 Confluent precipitin 20.4 lines
77.8 No lines 71.6 Confluent precipitin
P. methanica Culture C.F.
M. sporium M. trichosporium
lines Confluent precipitin lines
Methanol-oxidizing bacteria

Hyphomicrobium B-522

Pseudomonas M27 "Pink organism"
"White organism"

0.0 0.0

154.3 145.7 0.0

No lines

The values listed are micrograms of antibody precipitated at optimal antigen concentration (see Fig. 4 and
5). b These microorganisms are grouped according to the comparative gel diffusion reactions depicted in Fig. 7.
differences which presumably reflect variation in primary structures that can be detected by specific antibody. The enzymes also have certain antigenic determinants in common, but the detection of these structures was dependent upon the choice of antisera used in the tests. These studies, however, do not provide precise estimates as to the extent of structural similarities or differences between the enzymes. It may be that the antigenic portions(s) of each enzyme only represents a small fraction of the entire molecule. Indeed, an analysis of the tryptic peptides, separated by two-dimensional electrophoresis and partition chromatography, revealed extensive identity between the two proteins (Aggarwal and Mandy, unpublished data). A more accurate quantitative assessment of the degree of homology would require a comparison of their amino acid sequence. This investigation also touches upon one of the major difficulties encountered in comparative immunological studies, namely, the reliance upon a single antiserum prepared against one of the antigens to be compared. For example, if we considered only the reactions with anti-M. capsulatus serum, our findings would have revealed complete nonidentity between the antigens of M. capsulatus and Pseudomonas M27 enzymes. This observation would be difficult to reconcile in light of their

functional biochemical similarities (17). In other words, structural homologies are expected between proteins which have identical substrate and cofactor requirement for catalytic activity. It was important, therefore, to consider also the M27 enzyme as an antigen. Thus, we showed that the anti-M27 serum revealed not only the distinctive determinants but also the common antigens. Despite the lack of conclusive evidence, it is tempting to postulate that the antigenic determinants which are common to each enzyme represent part of the molecular conformations necessary for catalytic activity. Such homologous regions could include the noncovalent attachment sites of a pteridine cofactor which presumably participates in the oxidation of primary alcohol substrates (3, 17). The antisera were effective inhibitors of enzymatic activity. The inhibition exhibited by anti-M. capsulatus was mediated by mechanisms other than substrate competition. The anti-M. capsulatus serum which did not give precipitin reactions with M27 was effective in enzyme inhibition experiments. Although the inhibitory activity of anti-M. capsulatus for each enzyme was comparable, the amount of antibody (as the IgG fraction) was greater than that expected for such a specific reagent. Apparently, most of the antienzyme antibody

J. BACTROL.

TABLrE 2. Properties of methane-oxidizing bacteriaa

Organism

Resting stage
Immature azotobactertype cyst Immature azotobactertype cyst

Membrane

Morphology
Coccus Rod Rod Rod or pear

fRorsmette

Cloua grolupal ru
Methylococcus M. capsulatus M. minimus Methylomonas P. methanica

I I I U

Rod/coccoid group
Methylobacter Culture C.F. Methylosinus M. sporium (5)

M. trichosporium (PG)

a From
Azotobacter-type cyst Exospore

shaped

Whittenbury et al. (7, 22, 23).

Vibrioid group

does not interefere with enzymatic activity. This suggests that the enzyme molecule assumes a rather rigid configuration and is thus able to withstand any allosteric pressures brought on by the interaction of antibody with the enzyme. Our attempts to categorize other methaneand methanol-oxidizing bacteria by using immunological criteria were satisfying (Table 1). The two strains of M. capsulatus appear to be identical by both quantitative and qualitative tests. M. minimus, though a member of the Methylococcus group, shows greater identity with P. methanica and culture C.F. These, however, have been categorized as a Methylomonas and a Methylobacter, respectively (Table 2). The representatives of Methylosinus (i.e., M. sporium and M. tricosporium) apparently have PAD enzymes more closely related to the methanol-oxidizing bacteria. Hyphomicrobium B-522, on the other hand, shows serological identity with two members of the Methylosinus group, even though this organism cannot grow at the expense of methane (10). In another respect as well, namely, the possession of a peripheral membrane system (5, 7), the placement of Hyphomicrobium B-522 and Methylosinus in the same group seems appropriate. The enzyme from Pseudomonas M27 is particularly interesting, for it apparently has antigenic determinants in common with a number of other methane- or methanol-oxidizing bacteria. The "pink" isolate obtained locally from enrichment culture techniques is serologically identical with M27, but differs in its ability to grow on N-propanol and in its inability to grow on formate (R. J. Mehta, M.S. thesis). The "white" isolate (WI), a new obligate methylotroph, utilizes methanol or methylamine as a sole carbon source (6), but apparently possesses a PAD

enzyme serologically quite different from either M. capsulatus or Pseudomonas M27. (The
biochemical and physical properties of this enzyme have not yet been elucidated.) As has been illustrated with other distinct microbial enzyme systems (13, 16, 20), the serological cross-reactions between the PAD of related methane- and methanol-oxidizing bacteria may be advantageous as a taxonomic aid.

ACKNOWLEDGMENTS

This work was supported by Public Health Service research grant AI-07184 from the National Institute of Allergy and Infectious Disease, by National Science Foundation grant GB-20662, and by Robert A. Welch Foundation grant F-209. Acknowledgement is also made to the donors of the Petroleum Research Fund administered by the American Chemical Society. W. J. Mandy is supported by a Public Health Service Career Development Award.
LITERATURE CITED 1. Anthony, C., and L. J. Zatman. 1964. Isolation and properties of Pseudomonas sp. M27. Biochem. J.

92:609-614.

2. Anthony, C., and L. J. Zatman. 1967. The microbial oxidation of methanol: purification and properties of the alcohol dehydrogenase of Pseudomonas sp. M27. Biochem. J. 104:953-955. 3. Anthony, C., and L. J. Zatman. 1967. The microbial oxidation of methanol: the prosthetic group of the alcohol dehydrogenase of Pseudomonas sp. M27. Biochem. J. 104:960-969. 4. Basford, R. E., and F. M. Huennkeus. 1955. Oxidation of thio groups by 2,6-dichlorophenol-indophenol. J. Amer. Chem. Soc. 77:3872-3877. 5. Conti, S. F., and P. Hirsch. 1965. Biology of budding bacteria. m. Fine structure of Rhodomicrobium and Hyphomicrobium spp. J. Bacteriol. 89:503-512. 6. Dahl, J. S., R. J. Mehta, and D. S. Hoare. 1972. New obligate methylotroph. J. Bacteriol. 109:916-921. 7. Davies, S. L., and R. Whittenbury. 1970. Fine structure of methane and other hydrocarbon utilizing bacteria. J. Gen. Microbiol. 61:227-232. 8. Foster, J. W., and R. H. Davis. 1966. A methane-dependent coccus, with notes on classification and nomenclature of obligate methane-utilizing bacteria. J. Bacteriol. 91:1924-1931. 9. Hay, A. J., J. J. Skehel, and D. C. Burke. 1968. Proteins
SEROLOGY OF A PRIMARY AL COHOL DEHYDROGENASE

14. 15.

synthesized in chick cells following injection with Semliki Forest virus. J. Gen. Virol. 3:175-183. Hirsch, P., and S. F. Conti. 1964. Biology of budding bacteria. I. Enrichment, isolation and morphology of Hyphomicrobium spp. Arch. Mikrobiol. 48:339-357. Kekwick, R. A. 1940. The serum proteins in multiple myelomatosis. Biochem. J. 34:1248-1257. Levy, H. B., and H. A. Sober. 1960. A simple chromographic method for preparation of gamma globulin. Proc. Soc. Exp. Biol. Med. 103:250-259. London, J., E. Y. Meyer, and S. Kulczyk. 1971. Comparative biochemical and immunological study of malic enzyme from two species of lactic acid bacteria. evolutionary implications. J. Bacteriol. 106:126-137. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. Maurer, P. H. 1971. The quantitative precipitin reaction, p. 1-58. In C. A. Williams and M. W. Chase (ed.), Methods in immunology and immunochemistry, vol. 3. Academic Press Inc., New York. Murphy, T. M., and S. E. Mills. 1969. Immunochemical and enzymatic comparisons of the tryptophan synthase a subunits from five species of enterobacteriaceae. J. Bacteriol. 97:1310-1320. Patel, R. N., H. R. Bose, W. J. Mandy, and D. S. Hoare.

1972. Physiological studies of methane- and methanoloxidizing bacteria: comparison of a primary alcohol dehydrogenase from Methylococcus capsulatus (Texas strain) and Pseudomonas sp. M27. J. Bacteriol. 110:570-577. 18. Patel, R. N., and D. S. Hoare. 1971. Physiological studies of methane and methanol-oxidizing bacteria: oxidation of C-1 compounds by Methylococcus capsulatus. J. Bacteriol. 107:187-192. 19. Scheidegger, J. J. 1955. Une Micro-methode de l'immunoelectrophorese. Int. Arch. Allergy Appl. Immu-
nol. 7:103-110. 20. Stanier, R. Y., D. Wachter, C. Gasser, and A. C. Wilson. 1970. Comparative immunological studies of two Pseudomonas enzymes. J. Bacteriol. 102:351-362. 21. Stollar, D., and L. Levine. 1963. Two-dimensional immunodiffusion, p. 848-854. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 6. Academic Press Inc., New York. 22. Whittenbury, R., S. L. Davies, and J. F. Davey. 1970. Exospore and cyst formed by methane utilizing bacteria. J. Gen. Microbiol. 61:219-226. 23. Whittenbury, R., K. C. Phillips, and J. F. Wilkinson. 1970. Enrichment, isolation and some properties of methane utilizing bacteria. J. Gen. Microbiol. 61:205-218.

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ROUTING Data Center MX960 MX480 MX240 MX80 M320 M120 M10i SWITCHING EX8200 Line EX4500 Line EX4200 Line QFX3500 SECURITY/VPN SRX5800 SRX5600 SRX3600 SRX3400 SRX1400 ISG20001 ISG10001 NetScreen-5400 NetScreen-5200 SRX3600 SRX3400 SRX1400 ISG20001 ISG10001 NetScreen-5400 NetScreen-5200 WIRELESS WLC2800 ACCESS CONTROL SA6500 FIPS SA6500 IC6500 FIPS IC6500 SBR Enterprise Series WAN OPTIMIZATION WXC3400 POLICY MANAGEMENT IC6500 FIPS IC6500 SBR Enterprise Series
Performance-Enabling Services and Support
Juniper Networks Service Portfolio offers performanceenabling services designed around a time-to-value experience that accelerates, extends, and optimizes the value of high-performance networking.

Technical Services

Protect your high-performance business investment through operational assistance

Education Services

Improve the productivity and self-sufficiency of your technical staff

Consulting Services

Accelerate your networks value with expert assistance
Installation and Conguration Services

Campus

MX960 MX480 MX240 MX80 M10i M7i
EX8200 Line EX4500 Line EX4200 Line EX3200 Line

WLC2800 WLC800 WLC200 WLA432 WLA522 WLA632
SA6500 SA6500 FIPS SA4500 SA4500 FIPS IC6500 IC6500 FIPS IC4500 OAC SBR Enterprise Series Enterprise Guest Access

WXC2600

IC6500 FIPS IC6500 IC4500 SBR Enterprise Series C4000 C2000
Start your high-performance, high-value network rapidly, condently Juniper Care 24x7 JTAC access Software releases CSC online eSupport Junos Space Service Now eLearning HW replacement options Juniper Care Plus Training Credits Consulting Credits Service Manager Junos Space Service Insight Expert to Expert Access Complementary Options Resident Engineer Resident Consultant Focused Technical Support Installation and Conguration Services Basic installation Site survey Engineer, furnish, and installation Implementation consulting SSL quick start services Firewall branch office quick start services WXC Series platform quick start services Vendor Introduction Program (VIP) Juniper Enterprise Transition (JET) UAC quick start service Conguration service Firewall migrations Router migrations Education Services Juniper Networks Certication Program Juniper Networks Authorized Education Partners Juniper Networks Academic Alliance Technical training Prescriptive training Consulting Services Security assessment and risk mitigation Routing policy optimization Security policy optimization Assessment services High-level design review High-level design Low-level design review Low-level design Low-level design validation and testing Implementation: planning and plan review Strategic network consulting Proof-of-concept testing Product issue impact review Design resident engineer

Branch/Regional Office

J63502 J43502 J23502 J23202 SRX650 SRX240 SRX210 SRX100
EX4200 Line EX3200 Line EX2200 Line
SSG550M SSG520M SSG350M SSG320M SSG140 SSG203 SSG53 SRX650 SRX240 SRX210 SRX100
AX411 SA2500 WXC1800 SBR Enterprise Series CX111 SA700 J Series (ISM200) SRC Series SSG54 IC4500 IC4500 SSG204 OAC C4000 3G ExpressCard3 SBR Enterprise Series C2000 WLC200 Enterprise Guest Access WLC8 WLC2 WLA432 WLA522 WLA632
Extended Enterprise Mobile Worker, Teleworker Network Management and Automation

Secure remote and mobile remote access with Junos Pulse via SA Series SSL VPN; Comprehensive mobile device antimalware, loss and theft protection, monitoring, and control with Junos Pulse Mobile Security Suite; Secure clientless access via SA Series SSL VPN OAC; Secure remote and mobile remote access with Junos Pulse via SA Series SSL VPN; Comprehensive mobile device antimalware, loss and theft protection, monitoring, and control with Junos Pulse Mobile Security Suite; Secure clientless access via SA Series SSL VPN Ethernet Design, Network Activate, QoS Design, Route Insight, Security Design, Service Insight, Service Now, Virtual Control, SBR Service Provider Series, SRC Series, STRM Series, J-Web, NSM and CMS Junos SDK, Junos Space SDK
Junos Development Platform
Optional integrated IPS Optional integrated Avaya IG550 Media Gateway and Modules
Optional integrated on SRX210 Optional integrated wireless LAN access point
Corporate and Sales Headquarters Juniper Networks, Inc. 1194 North Mathilda Avenue Sunnyvale, CA 94089 USA Phone: 888.JUNIPER (888.586.4737)or 408.745.2000 Fax: 408.745.2100 www.juniper.net
APAC Headquarters Juniper Networks (Hong Kong) 26/F, Cityplaza One 1111 Kings Road Taikoo Shing, Hong Kong Phone: 852.2332.3636 Fax: 852.2574.7803
EMEA Headquarters Juniper Networks Ireland Airside Business Park Swords, County Dublin, Ireland Phone: 35.31.8903.600 EMEA Sales: 00800.4586.4737 Fax: 35.31.8903.601
Copyright 2011 Juniper Networks, Inc. All rights reserved. Juniper Networks, the Juniper Networks logo, Junos, NetScreen, and ScreenOS are registered trademarks of Juniper Networks, Inc. in the United States and other countries. All other trademarks, service marks, registered marks, or registered service marks are the property of their respective owners. Juniper Networks assumes no responsibility for any inaccuracies in this document. Juniper Networks reserves the right to change, modify, transfer, or otherwise revise this publication without notice. 3010015-006-EN Feb 2011