Vol. 136, No. 2

JOURNAL oF BACTERIOLOGY, Nov. 1978, p). 812-814 () Copcright C 1978X American Society for 'Microbiology

()021-919:3/78,'( 136-08128$02.()

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Changes in Composition of Envelope Proteins in Adenylate Cyclase- or Cyclic AMP Receptor Protein-Deficient Mutants of Escherichia coli RIKIZO AONOfr MIAKARI YNAMASAKI, AND GAKTZ() TAMUTRA Departm entIof Agrico/ltora l Che/mistrv, Facu/ltv o/Agriculture, The niluers.itv of Toko, I3Bunko-ku, Tokyo 11 3 Jap a n R{eceivecflfor I)Ublication1:0 3 uly 1978

Synthesis of several envelope proteins in Escherichia coli K-12 is regulated by cyclic AMI and cyclic AMP receptor protein. phate buffer system (0.1 M NaH,PO4, pH 7.1; 0.5c'% SDS), the gel was stained with Coomassie brilliant blue R-250 bv the method of Fairbanks et al. (3). Densitometrv tracing of the stained gel was carried out with a Gilford multiple photom-

Adeinvlate c\vclase-(efticient ( (C X I ilut antats of' ES(hci'hia ('c/li K- 12 grown in the absence of cY lic AMI) (cAMP) show spherical or short-r-od morphology (5, 10, 14). This shape is restored to a nornmal rod by the addition of cAMP to the mlediuimi. The cAMP receptor pIrotein-deficient (cqp) mutants hav e the same morphology, which cannot be restored by cAMP. It has been pro-

eter equipped with a linear transport mechanism.

Analvsis of envelope proteins by SDS-polyacrvlamide gel electrophoresis revealed that the protein conmposition of the envelope of CA7902 (cya [1.3]) was greatly influenced by exogenous cAMI' (Fig. 1). Peaks Cl, C2, C3, C4, and C5, which were tentatively numbered, increased in the envelope fraction prepared from CA7902 grown in the presence of cAMP. These peaks, however, were larger than in revertants (cya+) derived from CA7902 (data not shown). Peaks C6 and C7 decreased in the same fraction. This result suggests that cAMP should regulate the synthesis or the integration of proteins from Cl to C7. The other peaks were not included in our study because the amount of these peaks was not so large, the changes in their amounts were not remarkable, or because we obtained differing amounts under the same conditions. The changes in amount of the peaks from Cl to C7 were remarkable in each of several preparations. The protein composition of the envelope fraction prepared from 5333 (Cl'p [1]) was not affected by exogenous cAMP at all (Fig. 2). The composition was similar to that of CA7902 grown in the absence of cAMP. In the case of a spontaneous revertant (5333R1) derived from 5333, peaks Cl, C2, C3, C4, and C5 increased, and peak C7 decreased as compared with the respective peaks of 5333. This result indicates that protein Cl, C2, C3, C4, C5, and C7 are also under the regulation of cAMP receptor protein. Therefore, we could conclude that cAMP does not regulate integration of these proteins into the envelope, but rather the synthesis of these pro-

p)osed

that certain outer miembrane p1rotein(s) would first form a rod frame, ancl then a peptidoglvcan layer would be constituted on the rod frame and maintain the rod morphology of the cells (4, 6-8). We assumed that cAMP and cAMP receptor protein should regulate the svnthesis of the protein(s) deteriimining the cell shape of E. coli K-12. In this paper, we analyze the comnposition of envelope proteins in cva or ct-p strains grown in the presence or absence of

cAM1P b)v sorlium dlodecvl sulf'ate (SI)S)-polvacrvlamirle gel elect r)phoresis in relation to their mnorphological c hanges. All the strains were cultured in nutrient broth supplemented with or without 1 mM cAMP with shaking at 3 7C, as previously described (14). Nutrient broth contained 10 g of peptone, 3 g of meat extract, and 5 g of NaCl per liter of distilled water (pH 7.0). Cells in mid-logarithmic phase of growth were harvested and broken by ultrasonic treatnment as previously described (15). An env,elope fraction was prepared as a 100,000 x g (30 min) precipitate of this lysate and solubilized in 10 mM phosphate buffer (pH 7.1) containing 1' SDS, 1'. /-mercaptoethanol, and 10('-C glvcerol at 30°C overnight. A sample containing ca. 30 ,ug of proteins was loaded on an SDS-polvacrvlamide gel, which was composed of 7.5'%, acrvlamide, 0.2(' methvlenebisacrvlamide, 0.5', SDS, and 0.1 M sodiunm phosphate buffer (pH 7.1) together with Pvronin G as a rmarker dve. After electrophoresis, using a phost Present address: The Institute of Physical and Chemical

teins.

These results are summarized in Table 1. The

Research, Wako-shi, Saitama-ken 351 Ja)an. 812

NOTES

VOL. 136, 1978

813

C5

I 1I

C6

II

II II

I'

II

C31

I,li

C2$

I

C'

C7

A II I

r\

I.

I/ \

\J

C FIG. 1. Electrophoretic patterns of envelope proteins from CA7902. The envelope fraction was prepared from CA7902 grown in the presence (A) or absence (B) of 1 mM cAMP and solubilized with SDS. Arrows indicate peaks Cl, C2, C3, C4, C5, C6, and C7, which were changeable in amount by the addition of cAMP. The direction of migration by electrophoresis is from the left to the right of the figure. amount of proteins Cl, C2, C3, C4, and C5

was recep-

positively regulated by cAMP and cAMP tor protein. Among these proteins, we could conclude on several grounds that C2 protein is flagellin (unpublished data). The formation of flagella is known to be regulated by cAMP (16). So far, the function of other proteins has not been identified. Proteins C5 and C6 may correspond to proteins 4 and 7 of Inouye et al., re-

FIG. 2. Electrophoretic patterns of envelope proteins from 5333 and 5333R1. The envelope fraction was prepared from 5333R1 grown in the absence of cAMP (A). The fraction was prepared from 5333 grown in the presence (B) or absence (C) of 1 mM cAMP. They were solubilized with SDS. Arrows indicate peaks Cl, C2, C3, C4, C5, and C7, which were changeable in amount between 5333 and 5333R1. The direction of migration by electrophoresis is from the left to the right of the figure.

spectively (9). The amount of protein C7 is repressed or negatively regulated by cAMP and AMP receptor protein. Other examples of negatively controlled proteins in the cAMP-cAMP receptor protein system are glutamate synthetase and glutaminase A, but these are cytoplasmic (12). The intracellular concentration of cAMP in E. coli is lowered by the addition of glucose to the medium (2, 11, 13). The amount of protein

814

J. BACTERIOL.

NOTES

TABLE 1. Changes in the amount of envelope proteins Protein' Peak no.

Apparent mol -cAMP wtb

-CRPd

+Glucose'

C1 65,000 C2 60,000 C3 55,000 C4 50,000 C5 45,000 + C6 30,000 + + + C7 18,000 in Increase the amount of protein; -, decrease a+, in the amount of protein. bThe apparent molecular weight of each protein was estimated from its mobility in comparison with the mobilities of standard proteins such as egg white lysozyme, chymotrypsinogen A, egg albumin, and bovine serum albumin. 'Envelope proteins of CA7902 grown at 37°C in nutrient broth in the presence and absence of 1 mM cAMP were compared. d CRP, cAMP receptor protein. Envelope proteins of 5333 and its revertants grown at 37°C in nutrient broth in the absence of cAMP were compared. e Envelope proteins of a revertant of CA7902 grown at 37°C in nutrient broth supplemented with or without 0.5% glucose were compared.

Cl, C2, C3, C4, and C5 decreased, and that of C7 increased by the addition of 0.5% glucose to a wild (cya+, crp+) strain (Table 1). The cell had a normal rod shape under these conditions. It seems, therefore, that these proteins are not related to the determination of cell shape. Our results, however, clearly show that the amount of some envelope proteins is regulated by a cAMP and cAMP receptor protein system. We thank T. Yokota and K. Nakazawa for kindly supplying the bacterial strains, and Yamasa .Shoyu Co. for supplying the cAMP. This work was supported by a grant from the Ministry of Education, Japan.

LITERATURE CITED B. de Crombrugghe, I. Pastan, and R. 1. Emmer, O., Perlman. 1970. Cyclic AMP receptor protein of E. coli:

its role in the synthesis of inducible enzymes. Proc. Natl. Acad. Sci. U.S.A. 66:480-487. 2. Epstein, W., L. B. Rothman-Denes, and J. Hesse. 1975. Adenosine 3',5'-cyclic monophosphate as mediator of catabolite repression in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:2300-2304. 3. Fairbanks, G., T. L. Stack, and D. F. H. Wallach. 1971. Electric analysis of the major polypeptide of the human erythrocyte membrane. Biochemistry 10: 2606-2617. 4. Haller, I., and U. Henning. 1974. Cell envelope and shape of Escherichia coli K-12. Crosslinking with dimethyl imido esters of the whole cell wall. Proc. Natl. Acad. Sci. U.S.A. 71:2018-2021. 5. Harwood, C. R., and E. Meynell. 1975. Cyclic AMP and the production of sex pili by E. coli K-12 carrying derepressed sex factors. Nature (London) 254:628-630. 6. Henning, U., and I. Haller. 1975. Mutants of Escherichia coli K-12 lacking all major proteins of the outer cell envelope membrane. FEBS Lett. 55:161-164. 7. Henning, U., B. Hohn., and I. Sontag. 1973. Cell envelope and shape of Escherichia coli K-12: the ghost membrane. Eur. J. Biochem. 39:27-36. 8. Henning, U., K. Rehn, and B. Hohn. 1973. Cell envelope and shape of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 70:2033-2036. 9. Inouye, M., and J. P. Guthrie. 1969. A mutation which changes a membrane protein of E. coli. Proc. Natl. Acad. Sci. U.S.A. 64:957-961. 10. Kumar, S. 1976. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli. J. Bacteriol. 125: 545-555. 11. Makman, R. S., and E. W. Sutherland. 1965. Adenosine 3',5'-phosphate in Escherichia coli. J. Biol. Chem. 240:1309-1314. 12. Prusiner, S., R. E. Miller, and R. C. Valentein. 1972. Adenosine 3',5'-cyclic monophosphate control of the enzyme of glutamine metabolism in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:2922-2926. 13. Schwartz, D., and J. R. Beckwith. 197t). Mutants missing a factor necessary for the expression of catabolitesensitive operons in E. coli, p. 417. In J. R. Beckwith and D. Zipper (ed.), The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 14. Yamasaki, M., R. Aono, and G. Tamura. 1976. FL1060 binding protein of Escherichia coli is probably under the control of adenosine 3',5-cyclic monophosphate. Agric. Biol. Chem. 40:1665-1667. 15. Yoda, K., K. Nagai, and G. Tamura. 1977. Properties of proteins produced after damage to deoxyribonucleic acid of Escherichia coli. J. Biochem. 81:1357-1365. 16. Yokota, T., and J. S. Gots. 1970. Requirement of adenosine 3',5'-cyclic phosphate for flagella formation in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 103:513-516.

Changes in composition of envelope proteins in adenylate cyclase- or cyclic AMP receptor protein-deficient mutants of Escherichia coli.

Vol. 136, No. 2 JOURNAL oF BACTERIOLOGY, Nov. 1978, p). 812-814 () Copcright C 1978X American Society for 'Microbiology ()021-919:3/78,'( 136-08128$...
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