Vol. 134, No. 2

JOURNAL OF BACTERIOLOGY, May 1978, p. 674-676 0021-9193/78/00134-0674$02.00/0 Copyright X 1978 American Society for Microbiology

Printed in U.S.A.

Lipoprotein from Proteus,inirabilis ELEANORE KATZ, DENISE LORING, SUMIKO INOUYE, AND MASAYORI INOUYE* Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 Received for publication 5 December 1977

The biosynthesis of a Proteus mirabilis outer membrane protein of molecular weight of approximately 7,000 was found to be relatively resistant to puromycin and rifampin, as is the case for the Escherichia coli lipoprotein. Furthermore, the existence of the lipoprotein in P. mirabilis was indicated by a comparison of the amino acid compositions of the purified free and bound forms of this protein with those of the E. coli free and bound lipoproteins.

Unique features of the Escherichia coli lipoprotein, an outer membrane protein, have been well documented (for reviews, see references 1 and 9). The existence of the lipoprotein in other gram-negative bacteria has been examined for the bound form (2) as well as for the free form (4). From analysis of the Proteus mirabilis peptidoglycan, Braun et al. (2) concluded that P. mirabilis has no lipoprotein. We have also shown that P. mirabilis does not contain a protein that is cross-reactive against the anti-E. coli lipoprotein serum (4). Furthermore, the mRNA for the E. coli lipoprotein does not hybridize with P. mirabilis DNA (K. Nakamura and M. Inouye, manuscript in preparation). Recently Gruss et al. (3) suggested the existence of the bound form of the lipoprotein in P. mirabilis. Thus, in the present communication, we first examined the existence of the lipoprotein in P. mirabilis from the biosynthetic point of view. The biosynthesis of the E. coli lipoprotein has been shown to be unusually resistant to puromycin and rifampin (5-7, 9). Second, we attempted to purify the free and bound forms of the lipoprotein and to determine their amino acid compositions. Effect of puromycin. Figure 1 shows the effect of puromycin on the biosynthesis of total membrane proteins of P. mirabilis. The biosynthesis of all membrane proteins was drastically inhibited except for the peak I and II proteins. Apparent molecular weights of peak I and II proteins were estimated to be approximately 7,000 and 17,000, respectively. Peak I protein is almost the same size as the E. coli lipoprotein, and its biosynthesis showed clear resistance to puromycin, as is the case for the E. coli lipoprotein (5-7). Effect of rifampin. When rifampin was added to the culture, it was again found that the biosynthetic mechanisms of peak I and II proteins were relatively resistant to the antibiotic

(Fig. 2). Under the culture conditions used in the present study, the stability of the mRNA's for peak I and II proteins seemed to be much less than that of the E. coli lipoprotein mRNA. However, the half-lives of the mRNA's for peak I and II proteins were found to be two to three times longer than those of the mRNA's of the other membrane proteins (data not shown). Therefore, the mRNA's for peak I and II proteins are still more stable than other membrane protein mRNA's, as is found for the mRNA coding for lipoprotein in E. coli. Amino acid composition of peak I protein. The results described above show that peak I protein in particular is similar in size and relative mRNA stability to the E. coli lipoprotein. Furthermore, we found that this protein is located primarily in the outer membrane fraction (data not shown). Therefore, to identify peak I protein as the P. mirabilis lipoprotein, we attempted to purify the protein according to the purification method for the E. coli lipoprotein (11), in conjunction with filtration on Sephadex G-200 in 0.5% sodium dodecyl sulfate (SDS). The final preparation gave a single band in SDS-polyacrylamide gel electrophoresis (data not shown). The amino acid composition of the purified peak I protein is shown in Table 1. When the amount of each amino acid was calculated, assuming that the protein contains 11 aspartic acid residues, the total number of amino acid residues in peak I protein was estimated to be 57 to 59. This value is in very good agreement with the total of 58 amino acid residues in the E. coli lipoprotein (see Table 1). Gruss et al. also performed a tentative amino acid analysis, and their findings were not substantially different (3). Most importantly, peak I protein contains glycerylcysteine, which is so far known to exist only in the lipoprotein (1). Furthermore, peak I protein has no histidine, proline, or phenylalanine, and these amino acids are also absent in 674

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VOL. 134, 1978

mirabilis lipoprotein. To examine whether there is a bound form of the lipoprotein, the peptidoglycan was purified 6 by extensive extraction of the envelope fraction _ with 2% SDS at 100°C (15 times). The amino acid composition of the purified peptidoglycan I n 4 is shown in Table 1. The amino acid composition v t of the protein component attached to the pepwas almost exactly the same as that tidoglycan 11 n 21 E 2 of the free form except for glutamic acid and 1l_ 1l A k \k1 ffi alanine, which are the components of the pep,tidoglycan. From the content of glutamic acid, ___________________________ > (x lop) one can calculate the number of peptidoglycan l d b units per bound form of the lipoprotein to be I ;; $C 0 66.4 (= 76.4 - 10.0). This number indicates that the P. mirabilis peptidoglycan contains five to ^ _ o _6 six times less bound-form lipoprotein than does v A the E. coli peptidoglycan (1). Il _ 4_ The results clearly show the existence of the free form of the lipoprotein in P. mirabilis and also confirm the existence of the bound form 2 discovered by Gruss et al. (3). Braun et al. (2) could not detect the existence of the bound form, I possibly because of its extremely low content in (x 103) A A a

b

c

d

a

f

-

-

0

20

60 40 SLICE NO.

80

100

FIG. 1. Effect of puromycin on the biosynthesis of membrane proteins of P. mirabilis. P. mirabilis (obtained from G. Tortora, Stony Brook) was routinely grown at 37°C in tryptone broth (Difco) and 0.5% NaCI. A 30-ml culture of exponentially growing cells (about 2 x 108 cells/ml) was subdivided into two 15ml cultures. Thirty milligrams of puromycin (Nutritional Biochemicals Corp.) was added to one culture. Both cultures were incubated for 2 min at 37°C and pulse-labeled with 150 iaCi ofl3HJarginine (New England Nuclear Corp.; 230 Ci/mmol) for 5min. Growth was terminated with addition of 0.1 ml of 40% formaldehyde and 2 ml of 2-mg/ml nonradioactive arginine. The cell envelope fraction was prepared, solubilized in 0.2 ml of solubilizing solution, and subjected to SDS-polyacrylamide gel electrophoresis with use of 7.5% gels as described previously (16). After electrophoresis, the gels were sliced with razor blades and the radioactivity in each slice was measured as described previously (10). (A) Untreated control cells; (B) cells treated with puromycin. Small arrows with letters indicate the positions of the internal molecular-weight standard (8): (a) dimer; (b) monomer ofDANS-bovine serum albumin; (c) dimer; (d) monomer of DANS-hen egg white lysozyme; (e) cytochrome c; (f) DANS-insulin. (DANS indicates 1-

dimethylaminonaphthalene-5-sulfonyl).

x13 A a 40

b

c 4

b

c 4

e

d

f

32

24 16

E 8>_ >

B

FP

Xx 102 o 4i

d

4

e 4

f

8_ 6 4 2

2

0

80 60 40 SLICE NO. FIG. 2. Effect of rifampin on the biosynthesis of membrane proteins ofP. mirabilis. Experiments were carried out as in Fig. 1 except that 300(lAg of rifampin ofpuromycin, was added (Calbiochem) perml addition of after the 10 mininstead was added 20

the E. coli lipoprotein. Peak I protein may contain one glycine residue; glycine is not found in the E. coli lipoprotein. It should also be noted tmH]arginine peak I protein lacks methionine, in contrast the drug, and the mixture was incubated for only 2 that to two methionine residues present in the E. coli min. (A) Untreated control cells; (B) cells treated with lipoprotein (11). These results clearly indicate rifampin. Assignments for internal molecular-weight that peak I protein is the free form of the P. standards are described in Fig. 1.

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TABLE 1. Amino acid composition of the free and bound forms of the lipoprotein of P. mirabilis Ratio'

Integer value for lipoprotein from:

Amino acid

Free form

Lysine

2.9 Histidine ....... 0 Arginine ........ 2.9 Aspartic acid .... 11.0 Threonine ...... 2.7 Serine .......... 6.9 Glutamic acid ... 10.0 Proline ......... 0 Glycine ......... 0.8 Alanine ......... 8.5 0 Half-cysteine Valine .......... 3.8 Methionine ..... 0 Isoleucine 0.8 Leucine 4.2 Tyrosine ........ 1.6 0 Phenylalanine Glycerolcysteinef 0.2 Tryptophan ..... ND

Bound form

P mi-

E. Col

2.9 0 2.9 11.0 3.0 6.7 76.4 0 1.5 118.0 0 3.6

3 0 3 11 3 7 10 0 0-1 8-9 0 4 0 1 4 2 0 1

5 0 4 14 2 6 5 0 0 9 0 4 2 1 4 1 0 1 0

NDd 0.9 NDe 1.7 0 ND ND

rabi-

Total 57-59 58 aAll values are calculated assuming that there are 11 aspartic acid residues per molecule of the lipoprotein. Hydrolysis was carried out in 6 N HCl at 105°C for 24 h as described previously (11). 'Obtained on the basis of the amino acid composition of the free form. 'From the amino acid composition from the E. coli lipoprotein (11). d ND, Not determined. e Not determined because of glucosamine- that was eluted very close to the leucine peak. f Obtained as an oxidized form of glycerylcysteine as described previously (11). In case of the E. coli lipoprotein, values of 0.2 to 0.4 were obtained.

the peptidoglycan fraction. Recently we have found that Pseudomonas aeruginosa also contains "lipoprotein-like protein" (J. DiRienzo, M. Yasumura, and M. Inouye, manuscript in prep-

aration). These results, together with the fact that Salmonella and Serratia also contain the lipoprotein (2, 4), indicate widespread existence of the lipoprotein in gram-negative bacteria. We thank J. DiRienzo for critical reading of this manuscript. This research was supported by Public Health Service grant GM 19043 from the National Institute of General Medical Sciences and by the American Cancer Society grant BC67.

LITERATURE CITED 1. Braun, V. 1975. Covalent lipoprotein from the outer membrane of Escherichia coli. Biochim. Biophys. Acta 415:335-377. 2. Braun, V., K. Rehn, and H. Wolff. 1970. Supermolecular structure of the rigid layer of the cell wall of a number of lipoprotein molecules in a membrane layer (Salmonella, Serratia, Proteus, and Pseudomonas fluorescens). Biochemistry 9:5041-5049. 3. Gruss, P., J. Gmeiner, and H. H. Martin. 1975. Amino acid composition of the covalent rigid-layer lipoprotein in cell walls of Proteus mirabilis. Eur. J. Biochem. 57:411-414. 4. Halegoua, S., A. Hirashima, and M. Inouye. 1974. Existence of a free form of a specific membrane lipoprotein in gram-negative bacteria. J. Bacteriol. 120:1204-1208. 5. Halegoua, S., A. Hirashima, and M. Inouye. 1976. Puromycin-resistant biosynthesis of a specific membrane lipoprotein of Escherichia coli. J. Bacteriol. 126:183-191. 6. Hirashima, A., G. Childs, and M. Inouye. 1973. Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli. J. Mol. Biol. 79:373-389. 7. Hirashima, A., and M. Inouye. 1973. Specific biosynthesis of an envelope protein of Escherichia coli. Nature (London) 242:405-407. 8. Inouye, M. 1971. Internal standards for molecular weight determinations of proteins by polyacrylamide gel electrophoresis. J. Biol. Chem. 246:4834-4838. 9. Inouye, M. 1975. Biosynthesis and assembly of the outer membrane proteins of Escherichia coli, p. 351-391. In A. Tsagaloff (ed.), Membrane biogenesis. Plenum Publishing Corp., New York. 10. Inouye, M., and A. B. Pardee. 1970. Changes of membrane proteins and their relation to DNA synthesis and cell division. J. Biol. Chem. 245:5813-5819. 11. Inouye, S., K. Takeishi, N. Lee, M. DeMartini, A. Hirashima, and M. Inouye. 1976. Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form. J. Bacteriol. 127:555-563.