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The EMBO Journal Vol.2 No.8 pp.l275-1279, 1983 The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence Jan Tommassen*, Huub van Tol and Ben Lugtenberg Institute for Molecular Biology and Department of Molecular Cell Biology, Section of Microbiology, State University, Transitorium 3, Padualaan 8, 3584 CH Utrecht, The Netherlands Commitnunicated by M. Hofnung Received on 20 April 1983 To study the role of the signal sequences in the biogenesis of outer membrane proteins, we have constructed two hybrid genes: a phoE-ompF hybrid gene, which encodes the signal sequence of outer membrane PhoE protein and the structural sequence of outer membrane OmpF protein, and a bla-phoE hybrid gene which encodes the signal sequence as well as 158 amino acids of the structural sequence of the periplasmic enzyme 1-lactamase and the complete structural sequence of PhoE protein. The products of these genes are normally transported to and assembled into the outer membrane. These results show: (i) that signal sequences of exported proteins are export signals which function independently of the structural sequence, and (ii) that the information which determines the ultimate location of an outer membrane protein is located in the structural sequence of this protein, and not in the signal sequence. Key words: biogenesis/hybrid genes/outer membrane proteins/signal sequence Introduction The cell envelope of Escherichia coli K- 12 contains two membranes, i.e., the cytoplasmic membrane and the outer membrane, which are separated by the peptidoglycan layer and the periplasmic space. Since protein synthesis occurs in the cytoplasm, both periplasmic and outer membrane proteins have to pass the cytoplasmic membrane before reaching their ultimate location. Outer membrane proteins face an additional problem since they have to be assembled into the outer membrane. Periplasmic and outer membrane proteins are initially synthesized as larger precursor molecules, with an amino-terminal peptide extension of 15 30 amino acids, the signal sequence (for a review, see Halegoua and Inouye, 1979). This sequence is essential for transport of these proteins through the cytoplasmic membrane (Bedouelle et al., 1980; Emr et al., 1980) and it is cleaved off during or after this process. It is not clear yet whether the signal sequence also determines the ultimate location of an exported protein (i.e., the periplasmic space or the outer membrane). The amino acid sequences of the signal peptides thus far determined are very heterogeneous (Halegoua and Inouye, 1979) and no significant differences between the signal peptides of the periplasmic proteins on the one hand and those of outer membrane proteins on the other have been detected. The outer membrane contains large quantities of relatively few protein species, of which OmpC protein and OmpF protein are involved in the formation of pores through which small hydrophilic solutes can pass (for a review, see Nikaido, - *To whom reprint requests should be sent. O IRL Press Limited, Oxford, England. 1979). Expression of the genes ompC and ompF is dependent on the ompR gene product (Hall and Silhavy, 1981). Growth of cells under phosphate limitation (Overbeeke and Lugtenberg, 1980a), or a mutation in one of the genes phoR, phoS, phoT or pst (Tommassen and Lugtenberg, 1980) results in the synthesis of another pore protein, designated PhoE protein (Tommassen and Lugtenberg, 1981), which has similar properties (Lugtenberg et al., 1978; Pugsley and Schnaitman, 1978; van Alphen et al., 1978). Since these abundant pore proteins have been studied extensively, they are very suitable for investigations of the biogenesis of outer membrane proteins. We have recently cloned the structural genes for two of these proteins, phoE (Tommassen et al., 1982a) and ompF (Tommassen et al., 1982b) and from heteroduplex analysis it was concluded that these genes are very homologous (Tommassen et al., 1982b). Comparison of the primary structures of these proteins showed that almost 70% of their amino acid residues are identical. In contrast, their signal sequences are very different (Overbeeke et al., 1983). The use of hybrid genes, encoding part of an exported protein as well as the nearly complete sequence of the cytoplasmic enzyme 3-galactosidase, has been very fruitful in studies of the initial steps of the secretion process (for a review, see Silhavy et al., 1979). To study the degree of specificity of signal peptides of exported proteins, and to obtain more insight into the factors which determine the ultimate location of an exported protein in the cell envelope, we have constructed two new hybrid genes. One hybrid gene encodes the signal sequence of PhoE protein and the structural sequence of OmpF protein, whereas the other one encodes the signal sequence as well as part of the structural sequence of the periplasmic enzyme 3-lactamase and the structural sequence of PhoE protein. The cellular location of the hybrid gene products was determined. Results Can the signal peptide of PhoE protein replace that of OmpF protein? OmpF protein and PhoE protein are very homologous (Tommassen et al., 1982b; Overbeeke et al., 1983) but the primary structures of their signal peptides are completely different (Figure 1). To determine whether the signal sequences of these proteins can be exchanged, we wanted to construct a hybrid gene which encodes the signal sequence of PhoE protein and the structural sequence of OmpF protein. Both genes, phoE and ompF, contain a PstI site in the same reading frame, very close to the cleavage sites of the signal peptidase(s) in the precursor proteins (Figure 1). Thus, cloning of the Pstl-3 to PstI-4 restriction fragment of the ompFcontaining plasmid pJP33 (Figure 2) into the Pstl site of the PhoE gene on pJP 12 in the proper orientation would result in the desired phoE-ompF hybrid gene. Plasmid pJP38 (Figure 2), constructed as described in Materials and methods, contains this hybrid gene. To study the expression of the hybrid gene, plasmid pJP38 1275 J.Tommassen, H.van Tol and B.Lugtenberg phoE 4 ATGAAAAAGAGCACTCTGGCATTAGTGGTGATGGG,CATTGTGGCATCTGCATCTGTACAGGCTGCAGAAATAT MetLysLysSerThrLeuA laLeuVa ZVaZMetGZyIZeVaZA ZaSerA ZaSerVaZGZnA ZaA ZaG ZuIZeTyr -21 -1 A 1+ ompF ATGATGAAGCGCAATATTCTGGCAGTGATCGTCCCTGCTCTGTTAGTAGCAGGTACTGCAAACGCTGCAGAAATCTAT MetMetLysArgAsnIZeLeuA ZaVaZlZeVaZProAZaLeuLeuValA ZaGZuThrA ZaAsnA ZaA ZaGZuIZeTyr -22 -1 ib+iL+4 ACCACGATGCCTGCAGCAATGGCA ThrThrMe tProA ZaA ZaMe tA la 155 162 Fig. 1. DNA sequence and deduced amino acid sequence of the signal sequences of phoE (Overbeeke et al., 1983) and ompF (Mutoh et al., 1982) and of the DNA sequence and the deduced amino acid sequence around the PstI site within the bla gene of pBR322 (Sutcliffe, 1978). The numbers correspond to the amino acids of the mature proteins. The Pstl sites are indicated by arrows. The cleavage sites of the signal peptidase(s) are indicated by a triangle. | 'l pJP12 6kb pJP14 pJP33 Iz MS 0kb 8.9 kb phoE t=-i cm 1Q1 v 1~ M I pBR322 -----4 6kb cm phoE 8.9 kb m0_5m - cm Fig. 2. Construction of a phoE-ompF hybrid gene. Plasmid pJPI2 consists of the multicopy plasmid vector pACYC184 (Chang and Cohen, 1978), indicated by the thick line, and a 4.9-kb DNA fragment, containing the phoE gene, cloned in the Sall site of pACYC184 (Tommassen et al., 1982a). The vector part of the DNA contains an intact chloramphenicol resistance gene (cm). The direction of transcription of the phoE gene is indicated by an arrow (Overbeeke et al., 1983). Plasmid pJP33 consists of the same vector and a cloned 7.1-kb DNA fragment containing ompF (Tommassen et al., 1982b). Plasmid pJP38 contains a phoE-ompF hybrid gene and was constructed by cloning the PstI-3 to Pstl-4 DNA fragment of pJP33 into the Pstl site of pJP12. _ _Qn p LJ Fig. 3. SDS-polyacrylamide gel electrophoresis patterns of the cell envelope proteins of ompF strain K1O (a), its phoR derivative C9 (c), ompR phoE strain CE1224 (e), its phoS derivative CE1222 (g) and of pJP38-containing derivatives of strains KIO (b), C9 (d), CE1224 (f) and CE1222 (h). Only the relevant part of the gel, containing the proteins with apparent mol. wts. between 40 000 and 35 000 is shown. was transformed into ompR + ompF strain K 10 and its phoR derivative C9, and into ompR strain CE1224 and its phoS derivative CE1222. Analyses of the cell envelope protein patterns of the transformants (Figure 3) showed that large amounts of OmpF protein are produced only in transformants of the strains containing a phoR or phoS mutation (Figure 3, lanes d and h). This synthesis of OmpF protein is independent of the presence of an ompR + gene product. In addition, only these strains were found to be sensitive to the OmpF protein-specific phage Tula in contrast to the plasmidless strains and the pJP38-containing derivatives of strains KlO and CE1224. Thus, in cells containing the hybrid bla ~ ~ 4.3 kb H n PJ.4J3 kt, ~~~~~~~~10.5 _i_ _0kb 10.6kb omccpf pJP38 1276 L- c,- i Okb Cl (2 cm T _ -0 Z I I 6kb cm xl 8.6 kb Fig. 4. Construction of a bla-phoE hybrid gene. Plasmid pJP14 is identical to pJP12 (Figure 2), except that the cloned 4.9-kb DNA fragment containing phoE is located in the opposite orientation, relative to the pACYC184 sequence (Tommassen et al., 1982a). Plasmid pBR322 is a multicopy cloning vector, containing a bla gene which encodes 3-lactamase (Bolivar et al., 1977). pJP43 contains a bla-phoE hybrid gene and was constructed by replacing the Hindlll-Pstl fragment of pJP14 by the Hindlll-Pstl fragment of pBR322. gene, OmpF protein is produced under control of the pho regulon. It, therefore, can be concluded that the presence of the signal sequence of PhoE protein instead of that of OmpF protein, still allows a proper translocation to and assembly into the outer membrane. Does the signal peptide determine the ultimate location of an outer membrane protein? To determine whether translocation and assembly are influenced when the signal sequence of an outer membrane protein is replaced by that of a periplasmic protein, we wanted to construct a hybrid gene which encodes the signal sequence (and part of the structural sequence) of the periplasmic enzyme f3-lactamase as well as the structural sequence of PhoE protein. Plasmid pBR322 contains a unique PstI site in the bla gene, which is the structural gene for f3-lactamase (Bolivar et al., 1977). Comparison of the DNA sequences of phoE and bla (Figure 1) shows that cloning of the 0.75-kb HindIII to PstI DNA fragment of pBR322 (see Figure 4) in the PstI site of phoE would result in fusion of phoE to a large part of the bla gene in the proper reading frame. Plasmid pJP43 (Figure 4), constructed as described in Materials and methods, contains the desired bla-phoE hybrid gene. To study the expression of the hybrid gene and the location of its product in the cell, plasmid pJP43 was transformed into strain CE 1222 and the protein patterns of total cells, periplasm and cell envelopes of transformants and of the parental strain were analyzed on SDS-polyacrylamide gels (Figure 5). Biogenesis of outer membrane proteins A B C a' ,,W .. _-- Epmw40411:< ip* - AI P~~I __,40 _.4" 3VW-q q ... .4q. a a b c d e f gh i Fig. 5. SDS-polyacrylamide gel electrophoresis patterns of proteins of omnpR, phoE, phoS strain CE1222 (a, c, e, g and h) and of a representative pJP43 containing derivative of this strain (b, d, f, i and j). The lanes show total cell proteins (a and b), periplasmic proteins (c and d), cell envelope proteins (e and f), Triton X-100-insoluble fractions (g and i) and Triton X-100-soluble fractions (h and j). The 60-K protein is indicated by an arrow. In the cell envelope fraction of the transformants (Figure 5, lane f) an additional protein with an apparent mol. wt. of 60 000 (60 K) was observed, which was not found in the plasmidless parental strain (lane e). This mol. wt. is in good agreement with the expected mol. wt. of the hybrid gene product, i.e., an apparent mol. wt. of 40 000 for the complete PhoE protein plus a calculated mol. wt. of 18 028 for the amino-terminal fragment of the mature 3-lactamase up to the PstI site in the gene. The gel radioimmunoassay technique was used to determine whether this 60 K protein is indeed the expected hybrid protein. Cell envelope proteins of strain CE 1222 and a pJP43-containing derivative were separated on SDS-polyacrylamide gels and longitudinal gel slices were incubated with antisera. The results show that both the anti-PhoE protein serum and the anti-o-lactamase serum indeed react with a 60-K protein, which is not present in the plasmidless strain (Figure 6). No bands reacting with anti-PhoE protein serum or anti-o-lactamase serum were observed in a gel containing the periplasmic protein fraction (not shown). In conclusion, the 60-K protein found in the cell envelope fraction is the expected hybrid protein. For a more precise localization of the 60-K protein, the cell envelopes were extracted with Triton X- 100 in the presence of Mg2+ ions. The 60-K protein was found in the Tritoninsoluble fraction (Figure 5, lane i), suggesting a location in the outer membrane. This notion was supported by the observation that the 60-K protein, like PhoE protein (Lugtenberg et al., 1978), could be isolated associated with the peptidoglycan fraction after extraction of the cell envelopes with 2tVo SDS at 60°C (not shown). To determine whether the PhoE moiety of the hybrid protein is normally incorporated into the outer membrane, cells containing pJP43 were tested for sensitivity to the PhoE b a b a b Fig. 6. SDS-polyacrylamide gel electrophoresis protein patterns and gel radioimmunoassays of the cell envelope proteins of strain CE1222 (a) and a pJP43-containing derivative of this strain (b). After slicing of the gel, one slice was stained (A), whereas others were incubated with anti-3-lactamase serum (B) or anti-PhoE protein serum (C) and subsequently incubated with 1251-labeled Protein A and autoradiographed. protein-specific phage TC45. The plasmid was found to render cells of strain CE1222 sensitive to this phage. In conclusion, PhoE protein is normally translocated to the outer membrane with the signal sequence of the periplasmic enzyme 3-lactamase instead of its own signal sequence and assembly into this membrane is not influenced by the presence of the large f-lactamase fragment, since the hybrid protein can be isolated associated with the peptidoglycan and functions as the receptor for TC45. Discussion We have constructed hybrid genes to study the role of the signal sequence in the determination of the localization of outer membrane pore proteins. Although the signal sequences of the outer membrane proteins OmpF and PhoE differ considerably (see Figure 1), replacement of the OmpF signal sequence by the PhoE signal sequence results in normal transport of OmpF protein to and assembly into the outer membrane. Thus, the unrelated signal sequences of these related proteins are not gene-specific and are exchangeable. Replacement of the signal sequence of PhoE protein by the signal sequence and a large part of the periplasmic enzyme lactamase results in the production of a hybrid protein with an apparent mol. wt. of 60 000. This protein is transported to the outer membrane and apparently is assembled in this membrane essentially in the same way as wild-type PhoE protein, since it can be isolated associated with the peptidoglycan and can serve as the receptor for phage TC45. From these results we conclude: (i) that signal sequences are general, nonspecific, export units which can probably lead any protein, which is meant to be exported, out of the cytoplasm, and (ii) that the ultimate location of an exported protein, i.e., the periplasmic space or the outer membrane, is not determined by the signal sequence. Thus, the information which determines the outer membrane location of PhoE protein must be located in the structural sequence of this protein itself. It has already been suggested that some instructions needed 3- 1277 J.Tommassen, H.van Tol and B.Lugtenberg Table I. Bacterial strains Strain Characteristicsa Sourceb, reference K1O C9 AB1 157 Hfr Cav, re/AI tonA22 pit-0 spoTI ompF phoR18 derivative of KIO F-, thr leu proA2 (del proA-phoE-gpt) his thi argE lacY galK xyl rpsL ompR derivative of AB1 157 phoS21 derivative of ABI 157 CGSC CGSC Adelberg CE1224 CE1 194 CE1223 CE1222 CE1265 PC1505 recA56 his' derivative of CEI 194 ompR derivative of CE1223 F-, thi pyrF thy ilvA argG rpsL cod dra vtr g/pR ompR471 phoR18 recA56 del phoE-proA,B Hfr KL16, thr ilv recA56 This study Tommassen et al. (1982a) This study This study Overbeeke PC aGenotype descriptions follow the recommendations of Bachmann and Low (1980), except for phoE, which is the structural gene for PhoE protein (Tommassen and Lugtenberg, 1981), and ompR, which is a regulatory gene for OmpF protein and OmpC protein (Hall and Silhavy, 1981). bCGSC, E. coli Genetic Stock Center, Department of Human Genetics, Yale University School of Medicine, New Haven, CT (B.J.Bachmann, Curator). PC, Phabagen Collection, Department of Molecular Cell Biology, Section Microbiology, State University of Utrecht, Utrecht, The Netherlands. specifically for transfer to the outer membrane are contained in the sequence of the mature LamB protein (Silhavy et al., 1979). We demonstrate here that this is the case for all of these instructions for PhoE protein. Recently, it has been suggested that the first amino acid residue of the structural sequence is involved in this process, as this residue is very hydrophilic for all periplasmic proteins, whereas it is hydrophobic for all of the outer membrane proteins (Mutoh et al., 1982). However, since the first residue of the structural sequence of the 3-lactamase-PhoE hybrid protein is histidine, as in 3-lactamase, such a role for the first residue seems to be unlikely. The hybrid genes described here, can be very useful instruments for further studies on the assembly of pore proteins into the outer membrane. Whereas fusions of lacZ to the promoters of outer membrane protein genes allow us to detect whether mutations or cultural conditions affect the transcription of these genes, the hybrid genes we described, allow us to detect whether a step after initiation of translation and translocation is affected. Materials and methods Strains, phages and growth conditions All bacterial strains used are derivatives of E. coli K-12. The sources and relevant characteristics of the strains are listed in Table 1. recA56 derivatives of strains were obtained by crosses with Hfr strain PCI505, selecting for his' rpsL transconjugants. The recA56 derivatives were selected as u.v. lightsensitive transconjugants. ompR derivatives of strains were obtained by selecting simultaneously for resistance to the OmpC protein-specific phage Mel (Verhoef et al., 1977) and the OmpF protein-specific phage Tula (Datta et al., 1977). Phage TC45 uses PhoE protein as part of its receptor (Chai and Foulds, 1978). Cells were grown overnight at 37°C under aeration in L-broth, which contains 107% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.002% thymine, pH 7.0. For growth of cells containing plasmid derivatives of pACYC184, the medium was supplemented with chloramphenicol (25 ,ug/ml). Genetic techniques Transformation was carried out as described by Brown et al. (1979). Sensitivity to bacteriophages was determined by cross-streaking. 1278 DNA techniques and plasmids The conditions for restriction endonuclease reactions were those proposed by the manufacturers. Plasmid DNA was isolated by the cleared lysate technique of Clewell and Helinski (1969), followed by CsCl-ethidium bromide isopycnic centrifugation. Plasmids used in this study and their relevant genes and restriction sites are shown in Figures 2 and 4. Plasmid pJP38 was constructed by cloning the 1.6-kb PstI-3 to Pstlf4 DNA fragment of pJP33 into the Pstl site in the phoE gene of pJP12 as follows. pJP33 was digested with Pstl and, to prevent re-circulation, with Sall and EcoRI; the resulting solution was mixed with PstI-digested pJP12. After ligation with T4 DNA ligase (Tanaku and Weisblum, 1975), the DNA mixture was used to transform strain CE1265, selecting for chloramphenicol-resistant colonies. Plasmid DNA was isolated (Birnboim and Doly, 1979) from transformants which were resistant to the PhoE protein-specific phage TC45, indicating insertional inactivation of the phoE gene, and the DNA was analyzed on agarose gels (Van den Hondel et a/., 1979) after digestion with Pstl to determine whether the right fragment had been cloned, and with Bg/ll to determine the orientation of the cloned fragment. pJP38 is one of the recombinant plasmids and is depicted in Figure 2. In plasmid pJP43 the 1.1-kb Hindlll to Pstl DNA fragment of pJPl4 is exchanged for the 0.75-kb HindIll to Pstl DNA fragment of pBR322 as follows. Plasmids pJPl4 and pBR322 were mixed and digested with restriction enzymes HindllI and Pstl. After subsequent ligation, the DNA mixture was used to transform strain CE 1194, selecting for chloramphenicol-resistant colonies. Plasmid DNA was extracted from the transformants and analyzed on agarose gels after digestion with HindIll and Pstl. pJP43 is one of the recombinant plasmids and is depicted in Figure 4. Isolation and characterization of cell fractions Cell envelopes were isolated by differential centrifugation after ultrasonic disintegration of the cells (Lugtenberg et al., 1975). A fraction containing outer membrane proteins was isolated after Triton X- 100 extraction of the cell envelopes in the presence of Mg2+ ions (Schnaitman, 1971). Peptidoglycanassociated proteins were isolated after extraction of the cell envelopes at 60°C in the presence of 2% SDS (Rosenbusch, 1974). Periplasmic proteins were isolated by a modification (Tommassen and Lugtenberg, 1980) of the EDTAlysozyme method described by Willsky and Malamy (1976). The protein patterns of the cell fractions were analyzed by SDS-polyacrylamide gel electrophoresis (Lugtenberg et al., 1975). For the identification of hybrid proteins on gels, the gel radioimmunoassay technique was used (Poolman and Zanen, 1980; Overbeeke and Lugtenberg, 1980b). Acknowledgements We thank Dolf Evenberg for 1251-labeled Protein A, C.P.Hollenberg for anti,3-lactamase serum and Nico Overbeeke for anti-PhoE protein serum and for the information on the exact location of the PstI sites in phoE and ompF before publication. This work was supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). References Bachmann,B.J. and Low,K.B. (1980) Microbiol. Rev., 44, 1-56. Bedouelle,H., Bassford,P.J.,Jr., Fowler,A.V., Zabin,I., Beckwith,J. and Hofnung,M. (1980) Nature, 285, 78-81. Birnboim,H.C. and Doly,J. (1979) Nucleic Acids Res., 7, 1513-1524. Bolivar,F., Rodriguez,R.L., Green,P.J., Betlack,M.C., Heynecker,H.L. and Boyer,H.W. (1977) Gene, 2, 95-113. 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(1979) in Inouye,M. (ed.), Bacterial Outer Membranes: Biogenesis and Functions, Wiley-Interscience, NY, pp. 361-407. Overbeeke,N., Bergmans,H., Van Mansfeld,F. and Lugtenberg,B. (1983) J. Mol. Biol., 163, 513-532. Overbeeke,N. and Lugtenberg,B. (1980a) FEBS Lett., 112, 229-232. Overbeeke,N. and Lugtenberg,B. (1980b) J. Gen. Microbiol., 121, 373-380. Poolman,J.T. and Zanen,H.C. (1980) FEMS Microbiol. Lett., 7, 293-2%. Pugsley,A.P. and Schnaitman,C.A. (1978) J. Bacteriol., 135, 1118-1129. Rosenbusch,J.P. (1974) J. Biol. Chem., 249, 8019-8029. Schnaitman,C. (1971) J. Bacteriol., 108, 545-552. Silhavy,T.J., Bassford,P.J.,Jr. and Beckwith,J.R. (1979) in Inouye,M. (ed.), Bacterial Outer Memnbranes: Biogenesis and Functions, Wiley-Interscience, NY, pp. 203-254. Sutcliffe,J.G. (1978) Proc. Nat!. Acad. Sci. USA, 75, 3737-3741. Tanaku,T. and Weisblum,B. (1975) J. Bacteriol., 121, 354-362. Tommassen,J. and Lugtenberg,B. (1980) J. Bacteriol., 143, 151-157. Tommassen,J. and Lugtenberg,B. (1981) J. Bacteriol., 147, 118-123. Tommassen,J., Overduin,P., Lugtenberg,B. and Bergmans,H. (1982a) J. Bacteriol., 149, 668-672. Tommassen,J., Van der Ley,P., Van der Ende,A., Bergmans,H. and Lugtenberg,B. (1982b) Mol. Gen. Genet., 185, 105-1 10. van Alphen,W., Van Boxtel,R., Van SeIm,N. and Lugtenberg,B. (1978) FEMS Microbiol. Lett., 3, 103-106. Van den Hondel,C.A.M.J.J., Keegstra,W., Borrias,W.E. and Van Arkel, G.A. (1979) Plasmid, 2, 323-333. Verhoef,C., de Graaff,P.J. and Lugtenberg,E.J.J. (1977) Mol. Gen. Genet., 150, 103-105. Willsky,G.R. and Malamy,M.H. (1976) J. Bacteriol., 127, 595-609. 1279