osmoregulation of OmpF-OmpC synthesis is discussed. OmpF and OmpC are major outer membrane proteins of. Escherichia coli K-12 and are coded for by the ...
JOURNAL OF BACTERIOLOGY, Aug. 1984, p. 688-692
Vol. 159, No. 2
0021-9193/84/080688-05$02.00/0 Copyright C) 1984, American Society for Microbiology
Mutation Causing Reverse Osmoregulation of Synthesis of OmpF, Major Outer Membrane Protein of Escherichia coli
FUTOSHI NARA, KAORU INOKUCHI, SHIN-ICHI MATSUYAMA, AND SHOJI MIZUSHIMA*
Laboratory of Microbiology, Faculty of Agriculture, Nagoya University, Nagoya 464, Japan Received 30 March 1984/Accepted 23 May 1984
Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between maLA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.
MATERIALS AND METHODS Bacteria, bacteriophages, and plasmids. Bacteria, bacteriophages, and plasmids used in this study are listed in Table 1. All bacterial strains were derivatives of E. coli K-12. In strain FN101 recA, the recA mutation was introduced with pheA by conjugation from strain KL16-99 Nalr. The recA conjugant was scored for UV sensitivity. Media, chemicals, and enzymes. All experiments concerning the switching of OmpF and OmpC protein syntheses were carried out in medium A supplemented with different concentrations of sucrose as described previously (13). Medium A contained per liter 7 g of nutrient broth, 1 g of yeast extract, 2 g of glycerol, 3.7 g of K2HPO4, and 1.3 g of KH2PO4. Phage sensitivity was tested on medium A plates with and without 20% (wt/vol) sucrose. Transduction and transformation experiments were carried out in L-broth. M9 was used as the minimal medium. Maltose-MacConkey plates were used to examine maltose-utilizing ability. When required, ampicillin, chloramphenicol, and nalidixic acid were added at concentrations of 50, 25, and 10 p.g/ml, respectively. For solid cultivation, media were supplemented with 1.5% agar. N-Methyl-N'-nitro-N-nitrosoguanidine (NTG) was purchased from Sigma Chemical Co. Staphylococcus aureus V8 protease was purchased from Miles Laboratories, Inc. Restriction endonucleases were obtained from Takara Shuzo Co. Phage sensitivity test. The OmpF and OmpC proteins are receptor components for phages Tula and TuIb, respectively (7). The presence of the OmpF or OmpC protein in the outer membrane was examined preliminarily based on the sensitivity of cells to these phages. The sensitivity was tested by cross-streaking on medium A plates with or without 20%
OmpF and OmpC are major outer membrane proteins of Escherichia coli K-12 and are coded for by the ompF and ompC genes, respectively. These proteins have many properties in common. The homologies in the coding sequences and the deduced amino acid sequences between the ompF and ompC genes are 69 and 61%, respectively (12, 20). Both proteins exist as trimers that are resistant to sodium dodecyl sulfate (SDS) (24, 25, 39). Furthermore, they play important roles not only as proteins responsible for passive diffusion of small hydrophilic molecules across the outer membrane (26, 27) but also as basal constituents for stabilizing the cell surface structure (28, 36, 37). Despite the similarities between the two proteins, the promoter structures of the two genes are quite different (12, 20), and biosynthesis of the two proteins is affected in opposite directions by high concentrations of substances like sucrose which increase the osmolarity of media (13, 33). An increase in sucrose results in switching the protein synthesis from OmpF to OmpC. Studies with hybrid genes including ompF-lacZ and ompC-lacZ fusions (9, 10) and ompF-ompC chimeric genes (17) revealed that the promoter region is primarily responsible for the osmoregulatory switching. Two genes, the ompR and envZ genes, in the ompB operon are involved in the synthesis of these two proteins (11, 22). Mutations in this region exhibit complicated phenotypes, including OmpF+ OmpC-, OmpF- OmpC+, and OmpFOmpC- (8-10, 32). In addition, a genetic locus that codes for the micF RNA that is involved in osmoregulation of the ompF gene was reported recently (21). Expression of the micF RNA is also controlled by the ompB locus. Regulation of ompC expression by the ompF gene has also been observed (29). Despite the accumulation of such a large amount of evidence, the total view of the mechanism of the osmoregulation, including the role of the ompB operon, is still unclear. To study the regulation, we tried to isolate mutants in which the OmpF and OmpC synthesis are osmoregulated in the direction opposite from normal. Here we report the isolation of a mutant in which OmpF synthesis is in the direction opposite that in normal regulation. The mutation was mapped in the ompR region. *
Transduction, transformation, and conjugation. Generalized transduction was carried out with phage Plkc as described by Miller (18). Transformation was carried out by the method of Dagert and Ehrlich (6). Procedures for bacterial conjugation were those described by Verhoef et al. (34). Isolation of mutants causing reverse switching of OmpF synthesis in terms of medium osmolarity. E. (oli W4626 Phewas mutagenized with NTG (200 pg/ml) at 37°C for 30 min as described by Adelberg et al. (1). Mutagenized cells were incubated in medium A with 20% sucrose at 37°C overnight. The culture was then transferred to 50 volumes of fresh
Corresponding author. 688
REVERSE OSMOREGULATION OF OmpF
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TABLE 1. Bacteria, bacteriophages, and plasmids Strain, phage, or
plasmid E. coli K-12 strains W4626 PheFN101 FN101 maIA+ FN101 recA KL98 nalA YA21 AB2847 KL16-99 Nalr MH19 MH70 MH760 MH1461
Plasmids pACYC184 pBR322 pMAN014 pMAN015 pAT2002
F- purE pheA trp lac-85 gal-2 malA mtl xyl-2 ara rpsL (A) W4626 Phe- ompR20 (res) maIA+ transductant of FN101; donor, YA21 recA pheA+ conjugant of FN101; donor, KL16-99 Nair Hfr gyrA F- leu met X- rel F- aroB tsx malA supE X- Ar Hfr thi recA Nalr F- AlacUl69 rpsL relA thiA pyrD malQ7fibB MC4100 (M. J. Casadaban ) malQ7 MC4100 ompR472 (ompR2) MC4100 envZI I
19 This study This study This study B. Lugtenberg 23 T. Nakae Yamagata et al. (38) Hall and Silhavy (10) Hall and Silhavy (9)
Receptor; OmpF and lipopolysaccharide Receptor; OmpC and lipopolysaccharide Used for generalized transduction
7 7 Our laboratory stock
CmrTcr Apr Tcr Cmr; vector, pACYC184; cloned gene, ompB Apr; vector, pBR322; cloned gene, ompR Apr; vector, pIN-III; cloned gene, envZ
4 2 This study
Hall and Silhavy (10) Hall and Silhavy (10)
This study T. Mizuno (unpublished data)
a Abbreviations: Cm, chloramphenicol; Tc, tetracycline; Ap, ampicillin.
medium, and the cultivation continued for 4 h at 37°C. Then it was infected with phage TuIb at a multiplicity of infection of 10. After another 60 min of incubation, samples (0.1 ml) were spread on a medium A-20% sucrose on which phage TuIb (109 to 1010 PFU) had been spread. After cultivation at 37°C for 24 h, colonies that appeared were isolated and purified. All of the strains thus purified were tested for sensitivity to phage Tula, and strains sensitive to phage Tula on medium A-20% sucrose plates were selected. Preparation of cell envelopes, solubilization with Triton X100, and urea-SDS-polyacrylamide gel electrophoresis. Bacteria were grown at 37°C in 5 ml of medium A containing different concentrations of sucrose. Preparation of envelope fractions and subsequent solubilization with Triton X-100 were carried out as described (17). The Triton X-100insoluble fraction was washed once with 20% ethanol and used as the outer membrane proteins. The amounts of proteins in envelope preparations were determined by the method of Lowry et al. (14). Urea-SDS-polyacrylamide gel electrophoresis of outer membrane proteins. The outer membrane proteins were analyzed on 8 M urea-SDS polyacrylamide gel. The method was basically that described previously (23) with slight modifications. Briefly, about 120 ,ug of outer membrane proteins was dissolved in 100 ,ud of 1% SDS-1% ,B-mercaptoethanol solutiof and heated at 100°C for 5 min. Then the solution was mixed with 100 ,ul of a urea solution containing 10 M urea, 1% SDS, 1% ,B-mercaptoethanol, and 0.005% bromophenol blue. About 30 pu1 of the mixture (15 to 25 p.g of protein) was applied onto a gel (0.5 by 9.0 cm) containing 8% acrylamide, 0.13% N,N'-methylenebisacrylamide, 0.5% SDS, 8 M urea, 0.1 M sodium phosphate buffer (pH 7.2), and 0.6 ,ul of N,N,N',N'-tetramethylethylenediamine per ml. The polymerization of the gel was initiated with ammonium persulfate (1.2 mglml). Electrophoresis was first performed at 4 mA per tube for 30 min at room temperature in 0.1 M sodium phosphate (pH 7.2)-0.1% SDS. Then, the current was in-
creased to 6 mA per tube. Gels were fixed with 20% sulfosalicylic acid and stained with Coomassie brilliant blue by the method of Maizel (15). Analysis of proteolytic fragments of the OmpF protein. Cell envelopes obtained by sonication were treated with SDS at 60°C as described by Rosenbush (30), and the OmpF protein was isolated from the insoluble residue on a urea-SDSpolyacrylamide slab gel. Analysis of proteolytic fragments of the OmpF protein was carried out on SDS-polyacrylamide gels by the method of Cleveland et al. after digestion with V8 protease from S. aureus (5). Construction of a plasmid carrying the ompB operon and of one carrying the ompR gene. The ompB operon, consisting of the ompR and envZ genes, has unique BamHI and Sall sites at its upstream and downstream regions, respectively (22). The 5.3-kilobase fraction, supposed to contain the BamHISall fragment, was isolated from the chromosome of strain MH70 by agarose gel electrophoresis and was cloned into plasmid pACYC184, which in turn was used to transform strain MH760 recA (ompR2). Transformants were first selected for chloramphenicol resistance and then for TuIb sensitivity. Gel electrophoretic analyses revealed that transformants thus selected possessed the OmpC and OmpF proteins. A restriction map of the BamHI-SalI fragment from one of the plasmids (pMAN014) was constructed with HindIII, PvuII, and EcoRI. The result was consistent with the map of the ompB locus determined by Mizuno et al. (22). From these results, we concluded that plasmid pMAN014 carries the ompB operon. The ompR gene was obtained as a BamHI-EcoRI fragment from pMAN014 and was subcloned into plasmid pBR322. The plasmid thus constructed (pMAN015) was assumed to be the same as pEW007 described previously (35). RESULTS Isolation of switching mutants. We isolated mutants that showed abnormal osmoregulation of ompF and ompC
NARA ET AL.
expression. W4626 Phe- was mutagenized with NTG, and 2,000 colonies that were resistant to phage TuIb on medium A-20% sucrose plates were isolated. Among them, 143 were sensitive to phage Tula on the same plates. Six colonies out of the 143 were resistant to phage Tula on medium A plates, suggesting that osmoregulation of OmpF synthesis is in the reverse direction in these mutants. On the other hand, they were resistant to phage TuIb regardless of the sucrose concentration. Reverse osmoregulation of OmpF synthesis. The six mutants thus isolated were analyzed on polyacrylamide gels, and all of them were found to exhibit osmoregulation of OmpF synthesis in the reverse direction. A typical example of the outer membrane protein profiles is shown in Fig. 1. As expected from the phage sensitivity test, none of them produced a significant amount of the OmpC protein, regardless of the amount of sucrose in the media. Possible slight synthesis of the OmpC protein in the presence of sucrose will be discussed later. Since the phenotypic expressions of all the six mutants were indistinguishable, we used one of them (FN101) for further analyses, and the mutation was tentatively designated as the res (reverse switching) mutation. Evidence that the reverse regulated protein is OmpF. The protein in question was proved to be OmpF based on the following. The migration position was exactly the same as that of the authentic OmpF protein. Strain FN101 was sensitive to phage Tula, which required the OmpF protein as
receptor component, only when this protein was expressed. Finally, proteolytic fragments after V8 protease digestion of this protein and those of authentic OmpF gave the same profile on SDS-polyacrylamide gel (data not shown). Genetic mapping of the res mutation. Four genes are known to be involved in the OmpF synthesis: amzpF, oampR. envZ, and micF. Although the oampF gene is the structural gene of the OmpF protein (12), the other three genes are involved in the regulation of the oanpF expression (11. 21). The rnicF gene is located right upstream of the oamipC gene (22). We tried to determine whether the res mutation is linked to one of these genes. The oampF region of FN1I1 (pyrD+) was transduced by phage Plkc into strain MH19 (pyrD omnpFj) by selecting for pvrLD. All pyirDt transductants remained unchanged with respect to the osmoregulation of the OmpF protein. Similarly, the mnicF-ompC region of strain KL98 nalA (mnicF+ oanpCx gvrA) was transduced into FNIOI (gyrA t) by Plkc by selecting for nalidixic acid resistance (gp'A). All gvrA transductants had the -es phenotype. These results indicate that the res mutation was not linked to the omnpF, tnicF, or oanpC gene. The ompB region of strain YA21 (mna/A' oampB ) was transduced by Plkc into strain FN101 (mai/A). About 48% (56 out of 116) of the mai/At transductants lost the Eres phenotype and exhibited normal osmoregulation for both ompF and ompC expression, indicating the close linkage between the m/ilA and m-es loci. It should be noted that restoration of normal osmoregulation of ompF expression and the restoration of ompC expression, which was also regulated normally, always took place together upon transduction. This indicates that the res mutation is responsible for the abnormal expression of both genes in FN101. Fine-mapping analyses of the linkages among aroB, res, a
and ma(/A were carried out as shown in Table 2 with strain FN 101 mailA (araoB' res mclA/A) as the donor and strain AB2847 (aroB res' mna/A) as the recipient. The results are consistent with the gene order aroB-res-mnalA. The omnpB
I* lo 1
FIG. 1. Influence of sucrose in growth media on polyacrylamide gel profiles of outer membrane proteins of E. (coli W4626 Phe and FNIOI. The parent strain. W4626 Phe (A), and a switching mutant, FN101 (B), were grown in medium A supplemented with 0% (lane 1), 10% (lane 2), and 20% (lane 3) (wt/vol) sucrose. Outer membriane proteins were analyzed on urea-SDS-polyacrylamide gels. The positions of the OmpC, OmpF and OmpA proteins are indicated.
operon has been mapped between the atroB and malA genes (31). In addition, the cotransduction frequencies of o)npB with aroB and mal/A are similar to those obtained for re.x listed in Table 2 (31). These results suggest that the res mutation is in the omipB operon. Complementation tests. To determine whether the r es mutation is located within the ompB operon, whcih consists of the ompR and envZ genes, we examined suppression of the res mutation by plasmids that carried either one or both of the genes. The res mutation was suppressed by both pMAN014 and pMAN015. which carry the entire oinpB operon and the omt2pR gene, respectively. i.e., transformation by these plasmids of FN101 recA resulted in restoration of normal osmoregulation of OmpF synthesis with concomitant restoration of OmpC synthesis that was also normally regulated (Fig. 2). Figure 2 also shows the results with the cloned envZ gene. Plasmid pAT2002 carries the envZ gene, which is under the control of both the Ipp promoter and the lac promoteroperator (16; T. Mizuno and M. Inouye, unpublished observations). Although this plasmid also carries the lal gene, it TABLE 2. Three-point transductional mapping of the l es
mutation" Selected marker
les molA+ re.s+ ma/JlA res ma/lA
i-es+ a-oB' rexs araoB+ res+ ar-oB re.s aroB
ductants 3 71 83
2 39 106 44
38 44 16 1 20( 56 23
Plkc grown on FN101 mo/0lA (aroI3B- rex tonlA -) was incubated with AB2847 (aroB r-es' mo/A), and the mixture was plated on both M9 mediuIm and maltose-MacConkey medium supplemented with 50 p.g each of tryptophan. phenylalanine. and tyrosine per ml.
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REVERSE OSMOREGULATION OF OmpF
A .SA MfIA
pmnmvl-o Plasmid -
MH1461 pAT2002 I
.. .. .. = . .I II
4_1 ___ _ 4__
4 9 10 1 2 3 3 4 5 6 7 8 FIG. 2. Complementation tests of the res mutation with the ompR and envZ genes. Strains FN101 recA (A) and MH1461 (B) were transformed with the indicated plasmids and grown in medium A supplemented with 0 or 20% (wt/vol) sucrose. In lanes 9 and 10, 0.05 mM IPTG was added to the culture media. Outer membrane proteins (20 ,ug) were analyzed on urea-SDS-polyacrylamide gels. The positions of the OmpC, OmpF, and OmpA proteins are indicated. Lane
did not suppress envZ expression completely. pAT2002 suppressed the envZ phenotype (OmpF- OmpC+) of strain MH1461 even in the absence of isopropyl-3-D-thiogalactopyranoside (IPTG) (Fig. 2B). On the other hand, pAT2002 did not suppress the res mutation in FNlOlrecA at all, either in the absence or presence of IPTG. (Fig. 2A, lanes 7 to 10). The addition of IPTG was even rather inhibitory toward the OmpF synthesis. From these results, we conclude that the res mutation is located in the ompR gene. Osmoregulation of ompC expression in the res mutant. Although OmpC synthesis was almost completely suppressed in the res mutant, the remaining OmpC synthesis seemed to be osmoregulated rather normally (Fig. 1). To study this further, strain FN101 was transformed with plasmid pMAN002, a high-copy-number plasmid carrying the ompC gene. The plasmid-harboring strain produced an appreciable amount of OmpC protein whose synthesis was osmoregulated normally (Fig. 3). Growth of FN101, To study the physiological importance of the osmolarity-dependent switching of the syntheses of these proteins, growth of strain FN101 was compared with that of its parent strain (W4626 Phe-). No significant difference in cell growth was observed when they were cultivated in medium A supplemented with 0 or 20% sucrose (data not shown). DISCUSSION We have isolated a mutant exhibiting reverse osmoregulation of OmpF synthesis with a concurrent lack of OmpC synthesis. Both phenomena were due to a mutation (res) which was mapped at about 75 min on the E. coli chromosome map, where the ompB operon (ompR and envZ genes) occurs. The mutation was suppressed by a plasmid carrying the ompB operon and one carrying the ompR gene alone, but not by one carrying the envZ gene alone. Therefore, we conclude that the mutation occurred in the ompR gene. The ompR-carrying plasmids we used possessed an additional 2kilobase polynucleotide upstream of the ompR gene. However, the possibility that this region is responsible for the suppression was excluded since the res mutation was suppressed by plasmid pAT2001, which has the ompR gene free from the upstream region (F. Nara and T. Mizuno, unpublished observation). The ompR gene in pAT2001 is under the control of the lac promoter-operator, as is the envZ gene in pAT2002, and is expressed weakly in the absence of IPTG. The res mutation was suppressed by the weak expression as well, suggesting that the suppression is not an artificial event caused by OmpR overproduction. Mutations in the ompR region exhibit two different phenotypes, depending on the locus at which they occur (11). Mutations in the ompRI locus result in a lack of both the OmpF and OmpC proteins, whereas those in the ompR2
locus suppress OmpC synthesis alone. The OmpF synthesis of the latter mutant was strongly constitutive. The res mutation we studied here did not coincide with any of these. However, as far as the protein species synthesized is concerned, the res mutation is OmpC- OmpF+, i.e., the ompR2 type. Furthermore, OmpC synthesis, although strongly suppressed, was osmoregulated normally (Fig. 1). OmpC synthesis directed by the ompC-carrying high-copy-number plasmid in the res mutant was also osmoregulated normally (Fig. 3). It was previously reported that OmpC synthesis directed by the plasmid-cloned ompC gene in the ompR2 mutant was normally osmoregulated (17). These results, taken together, suggest that the res mutation probably occurred in the ompR2 locus. Since we used NTG (200 ,ug/ml), it is possible that the reverse osmoregulation was the result of multiple closely linked mutations within this locus. A reversioh analysis to investigate the possibility, however, was not carried out owing to technical difficulty. The mechanism of the reverse osmoregulation of OmpF synthesis in the res mutant is still a matter of speculation. The OmpR protein is assumed to play a central role in the osmoreguiation. A locus in the ompF promnoter responsible for the regulation by ompR gene was defined (K. Inokuchi, H. Furukawa, K. Nakamura, and S. Mizushima, J. Mol. Biol., in press). A model has been proposed in which the OmpB protein can take two alternative structures depending on the medium osmolarity, and each structure positively regulates either OmpF or OmpC synthesis (11). Thus, a change in medium osmolarity results in a switching of the synthesis from one protein to the other. In the res mutant, however, both the OmpF and OmpC syntheses were osmoregulated in the same direction. It would be rather difficult, although not impossible, to explain the res mutation by this model. Nucleotide sequencing of the ompR region of the res mutant is being. performed in this laboratory to gain more insight into the mechanism of osmoregulation. Although OmpF synthesis is reversely osmoregulated, the res mutant grew as fast as the wild-type strain, regardless of 0mpC-r OmpF
FIG. 3. Influence of sucrose in growth media on osmoregulation of OmpC synthesis in the res mutant. Strains FN101 (lanes 1 and 2) and FN101 transformed with plasmid pMAN002 carrying the omnpC gene (lanes 3 and 4) were grown in medium A supplemented with 0 or 20% (wt/vol) sucrose. Outer membrane proteins were analyzed on urea-SDS-polyacrylamide gels. The positions of the OmpC, OmpF, and OmpA proteins are indicated.
NARA ET AL.
the amount of sucrose in the media. Consistent with previous observations with the promoter-exchanged ompF-ompC chimeric genes (17), the results suggest that osmoregulation is not important for cell growth in conventional media. ACKNOWLEDGMENTS We thank T. Mizuno for plasmids and helpful discussion, M. N. Hall and T. J. Silhavy for bacterial strains, and S. Teranishi for her excellent secretarial assistance. This work was supported by grants from the Ministry of Education, Science and Culture of Japan, the Science Technology Agency of Japan, and the Foundation for Promotion of Research on Medicinal Resources. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 18:788-795. 2. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 3. Casadaban, M. J. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104:541-555. 4. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P1SA cryptic miniplasmid. J. Bacteriol. 134:1141-1156. 5. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. 6. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23-28. 7. Datta, D. B., B. Arden, and U. Henning. 1977. Major proteins of the Escherichia coli outer cell envelope membrane as bacteriophage receptors. J. Bacteriol. 131:821-829. 8. Garrett, S., R. K. Taylor, and T. J. Silhavy. 1983. Isolation and characterization of chain-terminating nonsense mutations in a porin regulator gene, envZ. J.. Bacteriol. 156:62-69. 9. Hall, M. N., and T. J. Silhavy. 1979. Transcriptional regulation of Escherichia coli K-12 major outer membrane protein lb. J. Bacteriol. 140:342-350. 10. Hall, M. N., and T. J. Silhavy. 1981. The omnpB locus and the regulation of the major outer membrane porin proteins of Escherichia coli K-12. J. Mol. Biol. 146:23-43. 11. Hall, M. N., and T. J. Silhavy. 1981. Genetic analysis of the ompB locus in Escherichia coli K-12. J. Mol. Biol. 151:1-15. 12. Inokuchi, K., N. Mutoh, S. Matsuyama, and S. Mizushima. 1982. Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12. Nucleic Acids Res. 10:6957-6968. 13. Kawaji, H., T. Mizuno, and S. Mizushima. 1979. Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins 0-8 and 0-9 of Escherichia coli K-12. J. Bacteriol. 140:843-847. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Maizel, J. V., Jr. 1966. Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science 151:988-990. 16. Masui, Y., T. Mizuno, and M. Inouye. 1984. Novel high-level expression cloning vehicles: 104-fold amplification of Escherichia coli minor protein. Biotechnology 2:81-85. 17. Matsuyama, S., K. Inokuchi, and S. Mizushima. 1984. Promoter exchange between ompF and ompC, genes for osmoregulated mnajor outer membrane proteins of Escherichia coli K-12. J. Bacteriol. 158:1041-1047. 18. Miller, J. H. 1972. Experiments in molecular genetics, 201-205.
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