JOURNAL OF BACTERIOLOGY, Jan. 1991, p. 156-160

Vol. 173, No. 1

0021-9193/91/010156-05$02.00/0 Copyright C 1991, American Society for Microbiology

Molecular Characterization of an Enterobacter cloacae Outer Membrane Protein (OmpX) JOKE STOORVOGEL,' MARIO J. A. W. M. VAN BUSSEL,' JAN TOMMASSEN, 2 AND JOS A. M. VAN DE KLUNDERTl* Department of Medical Microbiology, University Hospital, P.O. Box 9600, 2300 RC Leiden,' and Department of Molecular Cell Biology, State University of Utrecht, 3584 CH Utrecht,2 The Netherlands Received 20 March 1990/Accepted 15 October 1990

A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The ompX gene was located by insertions of the y8 sequence into the recombinant plasmid. The polarity of the gene was determined by in vitro transcription and translation of the ,y8-containing plasmids. The nucleotide sequence of the ompX gene was elucidated by using both inverted terminal repeats of the yB sequence as starting points for M13 dideoxy sequencing. The gene was found to encode a precursor of the OmpX protein consisting of 172 amino acid residues with a molecular mass of 18.6 kDa. The protein contains an N-terminal signal sequence of 23 amino acid residues. The exact cleavage point was established by sequencing the N-terminal part of the mature protein. The OmpX protein has several characteristics in common with outer membrane proteins of gram-negative bacteria. The protein is rather hydrophilic and is devoid of long hydrophobic stretches. On the basis of these results, we present a model for the OmpX protein folding in an outer membrane.

The outer membranes of gram-negative bacteria obtain their nonspecific permeability for hydrophilic solutes from the presence of porins, a class of proteins that form waterfilled diffusion channels (22). Lack of these proteins leads to resistance to most beta-lactam antibiotics and quinolones (1, 21). In a previous report (28), we described the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that was associated with resistance to beta-lactam antibiotics in transformants both of Escherichia coli and of the parental E. cloacae strain. In these transformants, the amount of the porin proteins OmpF and OmpC present decreased, and an outer membrane protein of approximately 18 kDa was overproduced. In vitro transcription and translation of the recombinant plasmid showed that this protein was encoded by a gene on the cloned fragment. The function of the protein is still unknown, but its overproduction clearly interferes with the presence of OmpF and OmpC in the outer membrane. We propose the name OmpX for this protein, as long as its proper function is obscure. In the accompanying paper (29), the biological characterization of OmpX is presented. In this report, we present the nucleotide sequence of the ompX gene and the deduced amino acid sequence. From these data, some physical properties of OmpX are derived.

ers. Transformation of competent bacterial cells with plasmid DNA was carried out as described by Maniatis et al.

(18). Preparation and characterization of outer membrane proteins. Outer membrane proteins were isolated as described by Matsuyama et al. (20). The proteins were analyzed on 14% polyacrylamide gels containing 0.2% sodium dodecyl sulfate (SDS) as described by Lugtenberg et al. (17) except that the gels were supplemented with 4 M urea. Gels were stained with fast green FCF. Isolation of plasmids carrying an inserted y8 sequence. Insertions of the transposable element -yb (11) in plasmid pJS04 were obtained as follows. DNA of plasmid pJS04 was introduced into the F' lac+-containing E. coli CE1304 by transformation. Transformants were selected on Iso-Sensitest agar (Oxoid CM471) containing chloramphenicol (34 ,ug/ml). Several transformants were subsequently mated with E. coli W3110 by filter mating on agar plates for 18 h at 37°C. Cells were collected from the filter, and the mixture was plated on Iso-Sensitest agar plates containing chloramphenicol and nalidixic acid (80 ,ug/ml). Transconjugants were screened subsequently for growth on Iso-Sensitest agar plates containing cephalothin (10 ,g/ml). The positions of the -yb sequence insertions were localized by restriction enzyme digestions. Analysis of plasmid-encoded proteins. Plasmid-encoded proteins were synthesized in vitro in a procaryotic DNAdirected transcription-translation system (Amersham, U.K.). L-[35S]methionine [NEN Research Products, Du Pont]-labeled proteins were detected by autoradiography after SDS-polyacrylamide gel electrophoresis as described by Laemmli (16). DNA sequence analysis. DNA restriction fragments were subcloned into the multiple cloning site of bacteriophage vectors M13mpl8 and M13mpl9 and sequenced by the dideoxy-chain termination method (26) with an M13 sequencing kit (Boehringer Mannheim Biochemicals) as prescribed by the manufacturer. [a-32P]dCTP was from NEN

MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains, plasmids, and bacteriophages used are listed in Table 1. Growth conditions. Bacteria were grown at 37°C in brain heart infusion broth (Oxoid Ltd.) supplemented when required with chloramphenicol (34 jig/ml). DNA techniques. Plasmid DNA was prepared as described by Birnboim and Doly (3). Digestion of DNA with restriction endonucleases (Boehringer Mannheim Biochemicals) and ligation (T4 DNA ligase from Promega Biotec) of DNA fragments were performed as specified by the manufactur*

Corresponding author. 156

MOLECULAR CHARACTERIZATION OF OmpX

VOL. 173, 1991 TABLE 1. Bacterial strains, plasmids, and phages Strain, plasmid, or phage

pJS04

pJS04::,y pJS05 Phages M13mpl8 M13mpl9

b

c

d

e

f

Reference or origin

Relevant characteristics"

Bacterial strains E. coli K-12 HB101 W3110 CE1304(F' lac+ -y8) E. cloacae 2249-1 Plasmids

a

157

OmpF :C*

4 5 7 28

F- Ndr

Clinical isolate

-42 -

31

- 21

OmpX . -14

Cmr ompX pJS04 carrying a yb insertion Cmr; ompX deletion derivative of pJS04

28 This study

M13 sequencing vector M13 sequencing vector

32 32

a Abbreviations: Ndr, resistance to nalidixic acid; chloramphenicol.

Cmr,

FIG. 2. SDS-polyacrylamide gel electrophoresis pattern of outer membrane proteins of E. coli W3110 transformed with pJS05 (lane a), pJS04::-y8-3 (lane b), pJS04::-y8-5 (lane c), pJS04::-y8-6 (lane d), pJS04: :yb-7 (lane e), and pJS04 (lane f). Positions of molecular mass standards are indicated at the right (rabbit muscle phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; hen egg white ovalbumin, 42.7 kDa; bovine carbonic anhydrase, 31.0 kDa; soybean trypsin inhibitor, 21.5 kDa; hen egg white lysozyme, 14.4 kDa). Positions of OmpF, OmpC, and OmpX are indicated at the left.

28

resistance to

RESULTS Location of oompX on plasmid pJS04. The gene encoding OmpX was locaited by -y5 sequence insertion mapping. pJS04 plasmids carryiing a y& sequence were isolated. Since the physical map off the -y8 sequence is known (10, 11), the sites of the insertion s in or near ompX could be determined by using BamHI-, EcoRI-, and HindlIl-generated restriction profiles (Fig. 1) Susceptibility &ita for transconjugants showed that about 92% of them weere able to grow on agar plates containing 10 ,ug of cephalothhn per ml, indicating that the insertion was located outside the gene. On the other hand, transconjugants containing a pla smid with a yyb sequence in a region of 500 bp between the tw o KpnI sites of pJS04 had lost their cephalothin resistance. Analysis of the outer membrane proteins of the transconjug ,ants by SDS-polyacrylamide gel electrophoresis demonstraated that the 18-kDa protein band could be detected only irn resistant strains (Fig. 2). Polarity of thee gene. To determine the polarity of the ompX gene, the synth iesis of proteins encoded by pJS04::yb-3, -5, -6, and -7 was s tudied in an in vitro transcription-translation system, followezd by SDS-polyacrylamide gel electrophoresis (Fig. 3). Pla Lsmid pJS04 (lane f) and plasmid pJS04::-yb-7 (lane e) produc(ed a protein of approximately 19 kDa. Since in such an in viitro system no processing occurs, this result implies that OnmpX is synthesized as a precursor protein, proOmpX. Plassmid pJS04::-yb-3 yielded no OmpX protein

Research Products, Du Pont. Fragments obtained from restriction digestion of plasmid pJS04 were sequenced by using the universal M13 primer. The fragments obtained from restriction digestion of plasmid pJS04 carrying -yb sequences were sequenced by using a 20-mer primer (TTC CATTGGCCCTCAAACCC) homologous to the inverted terminal repeats of the -y5 sequence (10) according to Van de Klundert (30a). The labeled fragments were analyzed on 0.4-mm-thick sequencing gels containing 6% polyacrylamide and 8 M urea (6). The gels were exposed to X-ray films (Fuji RX) for autoradiography at -80°C. Analysis of the sequence data. Nucleotide sequences were analyzed using the computer programs of the University of Wisconsin Genetics Computer Group (8). Amino acid sequence analysis of the N-terminal part of the mature OmpX protein. A membrane fraction containing the mature OmpX protein was loaded on a 12% polyacrylamide gel and electrophoresed as described by Laemmli (16). The gel was electroblotted on a polyvinylidene difluoride membrane (19). This membrane was stained with 0.1% Coomassie brilliant blue R-250. The protein band was cut out and processed in a Protein Sequenator (Applied Biosystems model 470A) equipped with on-line PTH (phenylthiohydantoin) analysis, using a model 120A PTH analyzer. Nucleotide sequence accession number. The nucleotide sequence of the ompX gene has been deposited in the EMBL data library under accession number M33878. RI

S04 pJpJSO

Ca

_

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KpnI

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Hin dill

0.5 kb -

ciai

Cll

1

2

3

Kpni

Kpnl

75-6 456 VV Kpv

Kp~Inl

0.1 kb

FIG. 1. Schematic representation of the restriction map of plasmid pJS04 and the lyb sequence insertion sites. pJS04 is linearized by EcoRI. Vector pACYC184 DNA is indicated by the solid bar. The yyb insertion sites are numbered 1 to 7 and marked to indicate beta-lactam-sensitive (V) or -resistant (V) phenotypes. Light arrows indicate the direction and extent of each DNA sequencing run. 0, DNA sequences that begin at a -yb sequence insertion; 0, DNA sequences that begin at the KpnI restriction site. The location of ompX and the direction of transcription are indicated by the heavy arrow.

158

J. BACTERIOL.

STOORVOGEL ET AL. a

b

d

c

e

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21

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*

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~~~~~~~~~~~~~~~.... ...

FIG. 3. Autoradiogram of L-[35S]methionine-labeled proteins from an in vitro transcription-translation system. Plasmids added to the incubation mixtures: lane a, pJS05; lane b, pJS04::-y5-3; lane c, pJS04::yb-5; lane d, pJS04::-yb-6; lane e, pJS04::yb-7; lane f, pJS04. Positions of molecular mass standards are indicated at the left (see legend to Fig. 2). Positions of the precursor of OmpX (proOmpX) and chloramphenicolacetyltranferase (CAT) are indicated at the right.

(lane b), whereas plasmid pJS04::-yb-5 produced a truncated form of this protein (lane c). pJS04::-y5-6 directed the synthesis of an almost full-length protein in vitro (lane d). Remarkably, the transconjugant containing this plasmid was sensitive to beta-lactam antibiotics in vivo, and no OmpX protein band could be detected among the outer membrane proteins of this strain (Fig. 2, lane d). From these data we conclude that insertion -6 mapped at the end of the gene and that the direction of transcription of the gene is as indicated in Fig. 1. Nucleotide sequence of the ompX gene. The data obtained from the yb sequence insertion mutations were used to design the sequencing strategy shown in Fig. 1. The KpnI restriction site inside the gene and restriction endonuclease fragments from several -y8 sequence insertion plasmids were used to construct M13 sequencing templates. The nucleotide sequence of the ompX gene is shown in Fig. 4. The open reading frame appeared to code for a protein of 172 amino acid residues with a calculated molecular mass of 18.6 kDa. The ompX RNA is composed of codons corresponding to abundant tRNAs (13). Our sequencing strategy, using both ryi termini as priming regions, made it possible to establish that the -yb sequence of pJS04::-yb-6 was inserted about 9 bp before the stop codon. The nucleotide sequence preceding the ATG start codon of the ompX gene contains a putative promoter sequence (12) and a ribosome binding site (27) (Fig. 4). The proposed stop codon TAA (nucleotides 517 to 519) is followed by a stable hairpin structure (nucleotides 538 to 567; AG037 = -14.6 kcal [ca. -61.1 kJ]/mol [9]) ending with a T track. Such a hairpin structure is characteristic of rho-independent terminators of transcription in procaryotes (25). Comparison of the ompX sequence with those stored in GenBank and the EMBL data library did not reveal homology with other known genes. Signal sequence of OmpX. The derived amino acid sequence of the OmpX protein suggested that the protein is synthesized as a precursor with an N-terminal signal peptide, characteristic of exported proteins (24, 31). It starts with two basic lysine residues (positions -22 and -21), followed by a stretch of hydrophobic amino acid residues. The exact cleavage point was determined by sequencing the N-terminal part of the purified mature protein (underlined in Fig. 4). We conclude that the precursor protein is cleaved between the alanine -1 and alanine +1 residues. The resulting mature OmpX protein consists of 149 amino acid residues with a calculated molecular mass of 16.5 kDa,

~~ATTTTTTTGATATATS~ -23 Met Lys Lys .SfD ATG AAA AAA CGGACTTACTTGAAGCACATT7 ~GGTT

ATCGTGAATGACAA.AGA~

Cys Leu TGT CTT RpnI Gly Thr GGT ACC

Ser Ala Leu Ala TCA GCA CTG GCA -1 +1 Ala Val AlaAAla GCT GTA GCG GCA

~TA -32

Ile Ala ATT GCA

15

Ala Val Leu Ala Val Ser Ala GCT GTT CTG GCT GTT TCC GCA

54

Thr Ser Thr Val Thr Gly Gly ACT TCT ACT GTA ACT GGT GGT

93

Tyr Ala Gln Ser Asp Met Gln Gly Val Met Asn Lys Thr

TAC GCT CAG AGC GAT ATG CAG GGC GTG ATG AAC AAA ACC

132

Asn Gly Phe Asn Leu Lys Tyr Arg Tyr Glu Gln Asp Asn AAC GGT TTC AAC CTG AAG TAC CGT TAC GAG CAA GAC AAC

171

Asn Pro Leu Gly Val Ile Gly Ser Phe Thr Tyr Thr Glu AAC CCG CTG GGT GTG ATC GGT TCT TTC ACT TAC ACC GAG

210

Lys Asp Arg Thr Glu Asn Gly Ser Tyr Asn Lys Gly Gln AAA GAT CGT ACT GAA AAT GGC TCT TAC AAT AAA GGT CAG

249

Tyr Tyr Gly Ile Thr Ala Gly Pro Ala Tyr Arg Leu Asn TAC TAC GGC ATC ACC GCG GGT CCT GCT TAC CGC CTG AAT

288

Asp Trp Ala Ser Ile Tyr Gly Val Val Gly Val Gly Tyr GAC TGG GCA AGC ATC TAC GGT GTT GTA GGT GTT GGC TAC

327

Gly Lys Phe Gln Gln Thr Glu Asn Gln Gly Leu Asn Arg GGT AAA TTC CAG CAG ACC GAA AAC CAG GGT CTG AAC CGT

366

Thr Ala Ser Asn Ser Asp Tyr Gly Phe Ser Tyr Gly Ala ACT GCA AGC AAC AGC GAC TAC GGT TTC TCC TAT GGC GCA

405

Gly Met Gln Phe Asn Pro Ile Glu Asn Val Ala Leu Asp GGT ATG CAG TTC AAC CCA ATC GAA AAC GTT GCT CTG GAC

444

Phe Ser Tyr Glu Gln Ser Arg Ile Arg Asn Val Asp Val TTC TCC TAT GAG CAG AGC CGT ATC CGC AAC GTT GAC GTT +149 Gly Thr Trp Ile Ala Gly Val Gly Tyr Arg Phe *** GGC ACC TGG ATC GCG GGC GTA GGT TAC CGC TTC TAA TCA

CTTCGGTGAGCCAGTAAAAAATCCGC§CAATGT§GCGGATTTTTTTTGTTT

483

522 573

GTACCCTCT

FIG. 4. Nucleotide sequence of ompX. Putative -35, -10, and Shine-Dalgarno (SD) sequences are boxed. The transcription terminator is indicated by horizontal arrows. Nucleotide numbering is relative to the ATG start codon. The sequence matching the amino acid sequence analysis of the N-terminal region of mature OmpX is underlined. The cleavage site between the signal peptide and mature OmpX is indicated by the vertical arrow. Numbering of the amino acid residues of the mature OmpX starts with +1, whereas the residues of the signal sequence have negative numbers. The asterisks mark the stop codon.

slightly less than the value estimated from SDS-polyacrylamide gel electrophoresis (18 kDa). Physical properties of OmpX. The OmpX protein contains 16.8% charged amino acid residues, which are scattered along with protein. Hydrophobic regions of the OmpX protein were traced by using a computerized version of the method of Kyte and Doolittle (15). A hydropathy plot of the OmpX protein is shown in Fig. 5. Apart from the signal sequence, no stretches of more than 10 hydrophobic amino acids were found. The derived amino acid sequence and hydropathy plot of the OmpX protein showed some striking features, also occurring in known outer membrane proteins

(30).

Characteristic are the amino acid residues of the C-terminal part of the protein, Thr-Trp-Ile-Ala-Gly-Val-Gly-TyrArg-Phe, following the consensus sequence found in many outer membrane proteins of X-Z-X-Z-X-Z-X-Tyr-X-Phe (X is any amino acid residue; Z is a hydrophobic amino acid

VOL. 173, 1991

MOLECULAR CHARACTERIZATION OF OmpX

159

4 3K

2-

0 C c

k

0I

0

XI0A1

t\

A4I

10

4.

a

0 L..

-17

la

Ib -3

1

50

25

75

100

125

150

Amino Acid Residue Number FIG. 5. Kyte-Doolittle (15) hydropathy plot of the deduced amino acid sequence of OmpX. Each value was obtained by averaging over a window including seven amino acids. Hydrophilic regions are indicated by positive values, and hydrophobic regions are indicated by negative values. An arrow indicates the cleavage point between the signal peptide and mature OmpX.

residue) (29a). Outer membrane proteins with a totally different primary structure appear to be arranged in a membrane in a rather similar way (30). The rules governing this folding are (i) the hydrophilic maxima of the protein are exposed at the cell surface, (ii) these maxima are separated by approximately 40 amino acid residues, and (iii) the membrane-spanning segments are folded in amphipathic P sheets 9 to 10 residues long. By applying these criteria, it is possible to predict the topology of OmpX in an outer membrane as shown in Fig. 6. This model will be used as working model in further studies.

cellsurface G

Q L

Q

M

E T

S Y

M

N

R

N

N E T Q Q F

D

K

D

K

K

K

G

G

D

Y G

Y G

N G

G V

S

T

0

N

A

G

Y

G G

T T T

A

F

Y

L

F

Y

G

Y

V

Q D

L P

N

T

K

V S

E T

R E NN

S I

G

0

Y

Y

V

I

V

A

G

G

T

G

G

A

P

I

Y R L

A W D N

V

N R T A S

S

N

N V R D I V

F

Q

Y

Y

A

F

S

G

Y

G

S

Q

M

L

F N P

V N E

R S

G T

E

I

W

S

G

D

G

A

R

A V Y

F

I

periplas m

FIG. 6. Model for the topology of the OmpX protein in an outer membrane. The OmpX sequence is shown in the one-letter amino acid code.

DISCUSSION In a previous paper (28), we have shown that overproduction of the OmpX protein in the parental E. cloacae 2249-1 and E. coli HB101 strains causes a decrease in the concentration of the porin proteins OmpF and OmpC, which results in beta-lactam resistance. In this study, we have elucidated the nucleotide sequence of the gene coding for the precursor of the OmpX protein. The codons used in this gene correspond to the class of abundant tRNAs. It has been postulated by Ikemura (13) that E. coli genes encoding abundant proteins selectively use codons corresponding to tRNAs that are present at relatively high levels. This hypothesis has subsequently been confirmed by other authors (2, 14). Nevertheless, in nontransformed cells under the culturing conditions described in Materials and Methods, the OmpX protein is not present in massive amounts. This could mean that under more physiological conditions, the ompX gene is subject to up-regulation, as is the case for the phoE gene, which is expressed in large amounts only under phosphate limitation (23). Such an up-regulation of the ompX gene would lead to lower susceptibility to beta-lactam antibiotics. In pJS04::-y-6, a yyb sequence is inserted about 9 nucleotides before the stop codon, leading to an almost full-length protein in vitro (Fig. 3, lane d). It is interesting that this insertion resulted in a beta-lactam-sensitive transconjugant and that no overproduction of the OmpX protein could be detected in vivo (Fig. 2, lane d). Similarly, it has been shown that PhoE lacking the Phe residue at the C terminus of the protein is no longer tncorporated in the membrane (29a). These results indicate that the OmpX protein modified in this way is no longer able to be properly processed. Further study will be needed to reveal the fate of this truncated

protein. The OmpX protein has an N-terminal signal sequence characteristic of periplasmic and outer membrane proteins (24, 31). Furthermore, the hydropathy analysis reveals that OmpX has four hydrophilic regions, which could be at the cell surface, separated by amphipathic regions representing membrane-spanning domains of the protein. These data strongly suggest that OmpX is an integral outer membrane

160

STOORVOGEL ET AL.

protein. In the accompanying paper (29), we present more data on the localization of OmpX and on its role in the loss of the porin proteins. ACKNOWLEDGMENTS We thank R. Amons and J. de Graaf for sequencing the N-terminal part of the mature OmpX protein. We thank M. A. E. Groothuis for assistance with the computer programs of the University of Wisconsin Genetics Computer Group. The continuing support of R. P. Mouton during this study was greatly appreciated. Amino acid sequences were determined at the Gas Phase Sequenator Facility (Department of Medical Biochemistry, State University of Leiden, The Netherlands), which is supported by the Netherlands Organization for the Advancement of Pure Research (NWO) through the Netherlands Foundation for Chemical Research (SON). This work was supported by the Stichting ter Bevordering van Medisch Microbiologisch Onderzoek, Leiden, The Netherlands.

ADDENDUM IN PROOF After submission of the manuscript, the sequences of two outer membrane proteins were published: the Ail protein of Yersinia enterocolitica (V. L. Miller, J. B. Bliska, and S. Falkow, J. Bacteriol. 172:1062-1069, 1990) and the Lom protein of Escherichia coli (X) (J. J. Barondess and J. Beckwith, Nature (London) 346:871-874, 1990). Both proteins show striking homologies to OmpX. Variations are found mainly in regions exposed at the cell surface according to the model of Fig. 6. These findings firmly support our model for the folding of OmpX in an outer membrane. REFERENCES 1. Aggeler, R., R. L. Then, and R. Ghosh. 1987. Reduced expression of outer-membrane proteins in P-lactam-resistant mutants of Enterobacter cloacae. J. Gen. Microbiol. 133:3383-3392. 2. Bennetzen, J. L., and B. D. Hall. 1982. Codon selection in yeast. J. Biol. Chem. 257:3026-3031. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Boyer, H. W., and D. Roulland-Dussoix. 1969. A complementation analysis of the restriction and modification of DNA in

Escherichia coli. J. Mol. Biol. 41:459-472. 5. Campbell, J. L., C. C. Richardson, and F. W. Studier. 1978. Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Proc. Natl. Acad. Sci. USA 75:2276-2280. 6. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic methods in molecular biology, p. 264-267. Elsevier Science Publishing Co., New York. 7. De Geus, P., I. van Die, H. Bergmans, J. Tommassen, and G. de Haas. 1983. Molecular cloning of pldA, the structural gene for outer membrane phospholipase of E. coli K-12. Mol. Gen. Genet. 190:150-155. 8. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 9. Freier, S. M., R. Kierzek, J. A. Jaeger, N. Sugimoto, M. H. Caruthers, T. Neilson, and D. H. Turner. 1986. Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA 83:9373-9377. 10. Goto, N., A. Mochizuki, Y. Inagaki, S. Horiuchi, T. Tanaka, and R. Nakaya. 1987. Identification of the DNA sequence required for transposition immunity of the y8 sequence. J. Bacteriol. 169:4388-4390. 11. Guyer, M. S. 1978. The -yb sequence of F is an insertion sequence. J. Mol. Biol. 126:347-365. 12. Hawley, D. K., and W. R. McClure. 1983. Compilation and

J. BACTERIOL.

analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237-2255. 13. Ikemura, T. 1981. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J. Mol. Biol. 151:389-409. 14. Ikemura, T. 1982. Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. J. Mol. Biol. 158:573-597. 15. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 17. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L. van Alphen. 1975. Electrophoretic resolution of the major outer membrane protein of Escherichia coli K12 into four bands. FEBS Lett. 58:254-258. 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035-10038. 20. Matsuyama, S.-I., K. Inokuchi, and S. Mizushima. 1984. Promoter exchange between ompF and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coli K-12. J. Bacteriol. 158:1041-1047. 21. Nikaido, H. 1988. Bacterial resistance to antibiotics as a function of outer membrane permeability. J. Antimicrob. Chemother. 22:17-22. 22. Nikaido, H., and M. Vaara. 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49:1-32. 23. Overbeeke, N., H. Bergmans, F. van Mansveld, and B. Lugtenberg. 1983. Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. J. Mol. Biol. 163:513-532. 24. Perlman, D., and H. 0. Halvorson. 1983. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J. Mol. Biol. 167:391-409. 25. Platt, T. 1981. Termination of transcription and its regulation in the tryptophan operon of E. coli. Cell 24:10-32. 26. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 27. Shine, J., and L. Dalgarno. 1974. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71:1342-1346. 28. Stoorvogel, J., M. J. A. W. M. van Bussel, and J. A. M. van de Klundert. 1987. Cloning of a beta-lactam resistance determinant of Enterobacter cloacae affecting outer membrane proteins of Enterobacteriaceae. FEMS Microbiol. Lett. 48:277-281. 29. Stoorvogel, J., M. J. A. W. M. van Bussel, and J. A. M. van de Klundert. 1991. Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX). J. Bacteriol. 173:161167. 29a.Struyve, M., M. Moons, and J. Tommassen. Submitted for publication. 30. Tommassen, J. 1988. Biogenesis and membrane topology of outer membrane proteins in Escherichia coli, p. 352-373. In J. A. F. Op den Kamp (ed.), Membrane biogenesis. NATO ASI Series, vol. H16. Springer-Verlag KG, Berlin. 30a.van de Klundert, J. A. M. Unpublished data. 31. von Heine, G. 1983. Patterns of amino acids near signalsequence cleavage sites. Eur. J. Biochem. 133:17-21. 32. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33:103-119.