Motecuiar Microbiotogy (1993) 10(5), 1029-1035

A novel ompC mutation of Escherichia coii K-12 thatreduces OmpC and OmpF levels in the outer membrane

Rajeev MisraDepartment of Microbiology, Arizona State University,Tempe, Arizona 85287-2701, USA.

Summary

A novel ompC mutation was isolated that not onlylowered the amount of its own product, OmpC27, butalso reduced the level of OmpF present in the outermembrane. ompC27 codes for a mutant OmpC pro-tein that contains two non-native cysteine residues.The ompC27 allele confers phage resistance by low-ering the level of OmpC present in the outer mem-brane. This effect on OmpC27 was manifested at thelevel of assembly as a result of disulphide bond for-mation between the two cysteine residues. This disul-phide bonding in OmpC27 also produced a novelphenotype by specifically influencing OmpF levels.The effect of OmpC27 on OmpF was partly a result of alowering of ompF transcription, and partly a result ofan effect at the post-transcription level. The transcrip-tional effect is likely to be brought about by a defectivemembrane as a result of the insertion of the disulphidebond containing OmpC27. The post-transcriptionaleffect of OmpC27 on OmpF could be due to interfer-ence at the assembly level. In a dsbA::kan1 back-ground where the in vivo disulphide bonding abilitywas dramatically reduced, the OmpC27-mediatedeffects were also curtailed.

Introduction

The outer membrane of Escherichia coli K-12 containstwo major proteins, OmpC and OmpF, which form water-fitled channets to allow diffusion of hydrophitic solutes,including certain antibiotics, into the cell (for a review onchannel-forming proteins, see Benz, 1988). In addition,these proteins serve as a receptor for various bacterio-phages. Other components of the outer membrane, suchas lipids and lipopolysaccharide (LPS), interact with outermembrane proteins. This interaction provides functionaland structural integrity to the outer membrane (for areview, see Nikaido and Vaara, 1985).

Received 18 May. 1993; revised 13 August, 1993; accepted 16 August,1993. *For correspondence. Tel. (602) 965 3320; Fax (602) 965 0098.

OmpF and OmpC are subject to osmoregulation, whichis controlled at the transcriptional level by OmpR andEnvZ (Hall and Silhavy, 1981). tn addition, several othermutations and growth conditions affect ompF and ompCexpression. One such mutation, tolC::Jn10, alters ompFexpression primarily by exerting its effect at the post-tran-scriptional level (Morona and Reeves, 1982; Misra andReeves, 1987). In addition, tolC.TniO dramaticallychanges the permeability properties of the outer mem-brane, which affects the osmoregulation of OmpF andOmpC by OmpR and EnvZ (Misra and Reeves, 1987).Mutations in the rfa locus, which is responsible for thesynthesis of LPS molecules, influence the assembly ofouter membrane proteins (Reid et ai, 1990; Laird andMisra, unpublished results).

In this study, the characterization of a novel ompCmutation, which interferes with the biogenesis of bothOmpC and OmpF, is described. This mutation, ompC27,was isolated from OmpC-specific bacteriophage-resistantmutants. The mutant protein contains two cysteineresidues in the mature portion of the protein. The wild-type OmpC protein contains no cysteine residues. Thepresence of ompC27 dramatically reduces OmpC levels.This effect of ompC27was shown to be at the level of pro-tein assembly. In addition, ompC27 reduces the amountof OmpF present in the membrane. This effect of ompC27on OmpF levels was found to be partly the result ofreduced ompF transcription, and partly the result of anaffect at the post-transcriptional level. The mechanismsby which ompC27confers these effects are described.

Results and Discussion

Isolation, genetic mapping and DNA sequence analysisofompC27

OmpC is an outer membrane protein of E. coli, which, inaddition to facilitating the diffusion of small solutes intothe cell, serves as a receptor for certain bacteriophages,namely Hy2 and SS4. The ompC27mutation was isolatedamong mutants that conferred resistance to OmpC-spe-cific phages. This isolation of phage-resistant mutants isbriefly described here. To avoid the isolation of ompC nullmutations, phage-resistant mutants were isolated using avariant ompC allele. We have previously reported the iso-lation of mutant ompC alleles that permitted growth on

1030 R. Misra

OinpC'OnipF 'OmpA'

Fig. 1. SDS-PAGE analysis ol envelopes prepared from strains growneither on Ltjria broth (lanes 1-3) or minimal medium containing glucose(lanes 4-6}. The vanous ompC alleles present were: lanes 1 and 4,ompC*; lanes 2 and 5, ompC234\ lanes 3 and 6, ompC27. Only the rele-vant portion of the Coomassie brilliant blue-stained gel is shown. The run-ning gel contained 4M jrea.

maltodextrin medium in the absence of a functionalLamB, which normally allows transport of maltodextrins(Misra and Benson, 1988b). These ompC{Dex) allelesresulted in increased channel size without affecting otherproperties of the protein. One such allele, ompC234,which contained a single alteration in ompC that put acysteine in place of arginine at position 74 of the matureOmpC protein (R-74-C), was used here. Starting with astrain carrying ompC234, bacteriophage-resistant mutantswere isolated by spreading bacteria on maltodextrinplates containing either Hy2 or SS4. This double selectionstrategy favoured the isolation of missense ompC muta-tions. However, the spontaneous rate of mutagenesiswas too low to obtain any resistant mutants. Therefore,the bacterial cultures were mutagenized with NTG andthen plated on the selection plates as above.

Several phage-resistant mutants isolated by utilizingthe above selection scheme were further characterized todetermine whether the mutation mapped in ompC. P1lysates prepared on these strains were used to transducean ompC-linked (50% cotransducible) Tn 10 marker into arecipient strain carrying 0{ompC'-iacZ^) and lackinglamB, by selecting for growth on a medium containingtetracycline and Xgal. The presence of Xgal permitted thedetection of Lac^ or Lac" colonies. Tetracycline-resistant(Tc'̂ ) transductants that have lost the function (whitecolonies) owing to its replacement by an ompC allele fromthe donor, were purified and tested for their Dex andphage phenotypes. In all cases, Tc" white colonies dis-played phage resistance. This showed that the mutationrendering phage resistance mapped in ompC.

To confirm that the phage-resistant mutations indeedmap within cmpC and to determine the nature of geneticalterations, mutant ompC alleles were cloned and thenucleotide sequence was determined. The mutant ompCalleles were cloned onto a low-copy number plasmid vec-tor utilizing an in wVo cloning technique as described pre-viously (Misra and Benson, 1988b; see the Experimentalprocedures). The entire cmpC gene was sequencedusing six complementary primers. In all but one case, onlyone nucleotide change was detected. Of the eleven inde-pendently isolated mutants analysed, five contained a

transition mutation in ompC that put a cysteine in place ofglycine at position 154 of the mature protein (G-154-C).These mutants that now possessed two cysteine residuesin OmpC displayed a pleiotropic phenotype. Characteri-zation of one such allele, ompC27, is described here indetail.

The presence of ompC27 reduces OmpC and OmpFleveis

When envelopes from various strains were examined bySDS-PAGE, the level of OmpC was found to be signifi-cantly reduced in strains containing ompC27 comparedwith those bearing either wild-type ompC or the parentalompC234 allele (Fig.1). This was true for all indepen-dently isolated strains carrying the ompC mutation thatresulted in a G-154-C substitution. This effect of ompC27on OmpG levels was independent of growth medium (Fig.1). Thus, growth on Luria broth or minimal medium sup-plemented with glucose produced the same effect.

Surprisingly, the presence of ompC27 also loweredOmpF levels present in the membrane (Fig. 1). This wasnot true for the parental ompC alleles. This effect ofompC27 on OmpF was growth medium-dependent.When envelopes prepared from cultures grown in Luriabroth were examined, the effect of ompC27 on OmpFlevel was noticeable. However, growth in minimalmedium supplemented with glucose produced noompC27 effect on OmpF, although it maintained the neg-ative effect on OmpC27 level (Fig.1, compare lanes 3and 6). These results suggest that the effect of ompC27on OmpF is dependent on the level of ompC expression.Growth on Luria broth facilitates wild-type ompC expres-sion, whereas its expression is significantly reduced whenexamined from cultures grown on minimal medium sup-plemented with glucose. This was also the case withompC27although the protein levels were reduced overall.The media-dependent fluctuation in ompC expression isregulated at the transcription level (data not shown).

The ompC27 effect on OmpF discussed above wasvery specific. As can be seen in Figs 1 and 2, the level ofOmpA was not affected. The level of neither inducible

OmpF -OmpA"

Fig. 2. SDS-PAGE analysis of envelopes prepared from strains carryingeither a dsbA' (lanes 1 and 3) or dsbA.:i<an1 allele (lanes 2 and 4). TheompC234 allele was present in strains analysed in lanes 1 and 2, whileompC27was present in strains analysed in lanes 3 and 4. Only the rele-vant portion of a Coomassie briliant blue-stained SDS-polyacrylamide(4M urea) gel is shown.

Assembly-defective ompC mutants 1031

OmpC27 [OmpF -

Fig. 3. The effect of |i-mercaptoethanol on the mobility of OmpC27 on aSDS-polyacrylamide gel. OmpC, solubilized from [^^S]-methionine-labelled cells, was immunoprecipitaled with OmpC antibodies, tmmuno-precipitates were split into two tubes, one of which contained [l-mercap-toeihanol. Lane 2 holds samples containing |i-mercaptoGthanol. Afterboiling tor 3 min, samples were analysed by SDS-PAGE, The autoradio-graph is shown. OmpF was also brought down by OmpC antibodies.

LamB nor other outer tTiembrane proteins was altered bythe presence of ompC27 (data not shown). In a separatestudy, Catron and Schnaitman (1987) reported the isola-tion of a novel ompC mutation that exerted an effect on allsecretory proteins. Thus, these two ompC mutations con-ferred very different phenotypes, suggesting that themechanisms by which they exert their negative effectsare different.

The role of two cysteine codons o/ompC27 on OmpClevels

The wild-type porin proteins OmpC and OmpF do notcontain a cysteine residue. In the case of ompC234, thepresence of an R-74-C substitution in OmpC functionallyenlarges the pore size without affecting other cellularfunctions and properties of the protein (Misra and Ben-son, 1988b). However, in the case of ompC27, the pres-ence of two cysteine residues, resulting from R-74-C andG-154-C (obtained from the phage-resistant selection)substitutions, not only affected its protein level but alsoaltered the cell's surface structure as evident by anincrease in sensitivity to certain hydrophobic antibiotics(see below). It is conceivable that the two cysteineresidues engage in disuiphide bonding. This interactioncould result in the production of structurally altered OmpCmonomers which could lead to a defect in the assembly oftrimeric OmpC, thus resulting in reduced levels of OmpCin the outer membrane. If disuiphide bond formation inOmpC is responsible for the defect it would be possible toalleviate this defect by preventing the formation of thesebonds. This was achieved by inactivating a gene, dsbA,whose periplasmic product has been shown to promote invivo disuiphide bond formation in extracytoplasmio pro-teins containing native cysteine residues (Bardwell et al,1991). The appropriate strains were constructed by trans-ducing a null dsbA allele, dsbA::kan1, into cells containingeither the parental ompC234 allele (R-74-C) or the two-cysteine-containing ompC27 allele (R-74-C, G-154-C).Envelopes prepared from these strains were analysed by

SDS-PAGE followed by Coomassie brilliant blue staining(Fig. 2). The results showed that the removal of a func-tional dsbA gene increased the amount of the two-cys-teine-containing OmpC almost back to the parental level(Fig. 2, compare lanes 3 and 4). These results demon-strated that the two cysteine residues present in themutant OmpC indeed engage in disuiphide bond forma-tion in vivo. This provides a likely explanation for thereduction in mutant OmpC levels in strains containing twocysteines in OmpC.

Apart from the results presented above, which showedin vivo disuiphide bonding in the mutant OmpC protein, invitro evidence was also obtained. It has been shown thatthe gel mobility of a protein can be influenced by its abilityto form disuiphide bonds (Scheele and Jacoby, 1982). Ingeneral, proteins under oxidized conditions can formdisuiphide bonds, which results in a more compact struc-ture that migrates faster than the reduced form, on SDS-polyacrylamide gel. This was the case with mutant OmpCcontaining two cysteines (Fig. 3).

Effect of on\pC27 on OmpF levels

Since the ompC27 effect on OmpC levels is dependenton the ability of the mutant protein to form disuiphidebonds, it was of interest to examine whether the disui-phide bonding also resulted in the decrease in OmpFlevel. If the effect of mutant OmpC is indeed broughtabout by disuiphide bonding of OmpC27 residues, theremoval of disuiphide bonds and thus normalization ofOmpC27 levels in a dsbA null background should restoreOmpF levels. This was not readily apparent from the gelshown in Fig. 2. The complexity was generated by thefinding that the presence of dsbA:.kan1 itself reduced thelevel of OmpF (compare lanes 1 and 2 in Fig. 2). OmpFdoes not contain any cysteine residues, therefore theeffect of dsbAv.kani on OmpF must be imposed indirectlyvia some other cellular component that is affected by theabsence of the dsbA gene product.

Recently, Pugsley (1993) has shown that thedsbA..kan1 effect on OmpF is due to a reduced ompFtranscription. To ensure that the dsbAv.kani still reducesompF transcription in mutant otDpC backgrounds, p-galactosidase activities of an ompF-lacZ transcriptionalfusion were measured (Table 1). The data showed thatthe presence of dsbAv.kani in either the ompC234 orompC27 background reduced p-galactosidase activitiesby 50% of that seen in the parent strain (ompC234dsbA*). Surprisingly, the presence of ompC27 itself in adsbA* background resulted in a 25% reduction in (i-galac-tosidase activity. Thus, both dsbAv.kani and ompC27reduce ompF transcription, although when these muta-tions were put into the same genetic background theircombined effects on ompF transcription were no greater

1032 R. Misra

than that produced by dsbA::kan1 alone. Thus, the twomutations probably exert their effect on ompF transcrip-tion through the same regulatory pathway.

The reductions in p-galactosidase activity and OmpFprotein level observed in a dsbA:.kan1 ompC234 strainwere similar to that found in a dsbA::kan1 ompC27 strain(Table 1). However, in a strain bearing a dsbA^ ompC27composition, a slightly larger reduction in OmpF levelcompared to p-galactosidase activity was observed; aroughly 50% reduction in OmpF level correlated with onlya 25% reduction in )3-galactosidase activity (Table 1). Thisindicated that the ompC27effect on OmpF cannot be sim-ply explained by the reduction in ompFtranscription. Thefollowing possibilities can account for the OmpC27 effecton OmpF.

Since the OmpC27 effect on OmpF was specific, it isreasoned that the biogenesis of these proteins may sharea common step. Gehring and Nikaido (1989) providedbiochemical evidence of mixed trimer formation betweenthe porin proteins, OmpF and OmpC. For this to happen,these proteins must interact at a level prior to trimerizationas the trimerized molecule is extremely stable. Theycould interact at either the monomer-monomer ormonomer-dimer level. The transdominant nature ofOmpC containing two cysteines can be explained byproposing that either mutant OmpC monomers or dimerssequester OmpF monomers or dimers into a futile assem-bly pathway that renders them susceptible to degrada-tion. This effect can be reversed by either the removal ofOmpC or lowering of its expression by growth on minimalmedium. These results are consistent with the biochemi-cal evidence noted above.

OmpC234OmpF

OmpC27OmpF

mmmwdMk ^riiS ^^^ 1^^

*

^HB i^^m i0i^ # 1 ^

UO

80

Rn

40

•

O OtnpCa34

• OmpC 27

_ _ - — 1

1

0 5 10 15 20 25 :30 35 40 45

CHASE PERIOO (min)

Fig.4. Pulse-chase experiments with (reshly grown cells (glucose mini-mal medium) carrying either ompC^34 or ompC27allele. Celis werepulse labelled with [^^S]-methionine for 20 s and chased wilh cold methio-nine in the presence of lOOngml"' chloramphenicol. Cells, pelleted fromsamples withdrawn al various chase times, were resuspended in SDSlysis buffer (2% SDS, 20 mM Tris-HCi, 5% |l-mercaptoethanol) and boiledfor 5 min. A portion of this extract was used to immunoprecipitate OmpC.Immunoprecipitates were resuspended in sample buffer and analysed bySDS-PAGE followed by autoradiography. Lanes 1 to 7 contain sampleswithdrawn after 1, 2, 5.10. 20. 30 and 45 mm ot chase, respectively.OmpF, brought down by OmpC antibodies, was used as an internal con-trol. The amount of OmpC was determined from the OmpC/OmpF ratio.The highest ratio was arbitrarily chosen to be 100, and remaining ratioswere extrapolated from this. These numbers were plotted in the graphshown.

An explanation for the effect o^ompC27 on OmpC levels

It is conceivable that the mutant OmpC protein couldexert an effect on itself at the level of transcription, trans-lation or signal sequence processing through a feed-backmechanism. To examine this, cells carrying the one- ortwo-cysteine-containing OmpC protein were pulse-

Table 1. Effects of dsbA:kan 1 and ompC27on OmpC and OmpF levels,and ompFtranscription,

ompC dsbA OmpC"

ompC234ompC234ompC27ompC27

dsbA'dsbA.kanidsbA-dsbA.kani

1.40(100)1.33(95)0.56 (40)1.17(83)

|l-gal units"

0.60(100) 615(100)0.28(46) 335(54)0.32 (53) 460 (75)0.26(43) 322(52)

a. Protein bands from the gel shown in Fig. 2 were quantified. The levels otOmpC and OmpF, relative to OmpA, are shown. Numbers in parenthesesare percentages ot the protein level present in the parent strain.b. Beta-galaclcsidase activities of an ompF-iacZ fusion are shown inMiller units. Numbers in parentheses are percentages of the |i-galactosi-dase activity present in the parent stratn.

labelled with p^S]-methionine and chased with an excessof non-radioactive methionine in the absence of furtherprotein synthesis. OmpC, extracted from samples with-drawn at various chase points, was immunoprecipitatedusing OmpC antibodies, Immunoprecipitates were anal-ysed by SDS-PAGE followed by autoradiography. Theresults showed that both the mutant and parent OmpCproteins were synthesized in an identical manner (Fig, 4,lane 1), Thus, transcription, translation or signal sequenceprocessing of OmpC27 was not affected. This suggeststhat either OmpC27 assembly is defective or its assem-bled trimers are unstable. It is hard to envisage how thelatter scenario can be reconciled with the specific effect ofOmpC on OmpF since there is no evidence that OmpFand OmpC specifically interact with each other afterassembling in the membrane. Therefore, it appears likelythat the assembly process of the mutant OmpC is defec-tive and this defect interferes with the normal assembly of

Assembly-defective ompC mutants 1033

OmpF. The following experiments were performed to testthis.

A defect in assembly may lead to the degradation ofunassembled intermediates but not the final assemblyproduct. In the pulse-chase experiment shown in Fig. 4,no degradation of the one-cysteine-containing OmpC234was noted. The two-cysteine-containing OmpC27 proteinwas synthesized normally. However, at later chase pointsdegradation of earlier synthesized OmpC27 moleculeswas apparent and by 15 min one-third of the protein hadbeen degraded (Fig. 4). The kinetics of OmpC degrada-tion was biphasic, i.e. after 15 min no further degradationof the protein was noted. This result showed that it is notthe final product (stable trimers) but the process ofassembly that is defective in mutant OmpC27. We havepreviously reported results similar to this while analysingan assembly-defective LamB protein (Misra etai, 1991).

The OmpC antibodies used in these experiments alsorecognize OmpF as a result of substantial structural simi-larities between the two proteins. Therefore, both OmpCand OmpF were brought down using OmpC antibodies(Fig, 4). In these labelling experiments, similar OmpF lev-els were present in strains carrying either parent ormutant OmpC. This is because, for labelling experiments,cells were grown on minimal medium and, as noted ear-lier (Fig.1), OmpC27 imposes no negative effect onOmpF under these growth conditions. The degradation ofOmpC was also observed when strains devoid of OmpFwere used (data not shown), suggesting that theOmpC27-mediated assembly defect is independent ofOmpF.

OmpC27 causes membrane defects

Bacterial cultures of mutants carrying the ompC27 alleleshowed some degree of lysis. It is conceivable that thecell lysis is caused by the insertion of a defective OmpCin the outer membrane resulting in structural damageto the membrane. This was supported by the findingthat mutants carrying OmpC27 exhibited a significantincrease in sensitivity to certain hydrophobic antibiotics,e.g. erythromycin, novobiocin and rifampicin (Table 2). Anincrease in sensitivity to hydrophobic antibiotics is an indi-cation of a defective outer membrane, as it has been pro-posed that these antibiotics penetrate the membranethrough the lipidic environment that is normally shieldedfrom the outside (Nikaido and Vaara, 1985). Results pre-sented above suggested that the formation of a disul-phide bond between the two cysteine residues ofOmpC27 led to various phenotypic changes in the cell. Itwas of interest to see whether the increase in sensitivityto hydrophobic antibiotics was also caused by this mech-anism. Antibiotic sensitivity tests were performed in dsbA*and dsMnull strains containing the ompC234 ot ompC27

Table 2, Disk sensitivity and EOP assays of strains carrying ompC234 orompC27m dsbA* and dsbA null backgrounds.

Genotype

ompF* ompC dsbA"ompF' ompC dsbA.kaniompF* ompC234 dsbA'ompF' ompC234 dsbAwkaniompF* ompC27dsbA'ompF* ompC27dsbA.kani

Zone of Inhibition

Ap

171722222222

Nb

79'9

111712

Em

7.,20

7121812

Ra

7979

109

E

SS4

1111

10-'1

EOP

HY2

1111

10-^0.5

a. Ap, ampicillin (10^.ig); Nb, novobiocin (30ng); Em, erythromycin (15fig); Ra, rifampicin (5 tig).b. Zones ol inhibition in mm. The diameter of the paper disc was 7 mm.This experiment was performed in triplicatec. Only very faint inhibition zones were observed.

aliele. The presence of dsbAwkani resulted in a decreasein the OmpC27-mediated sensitivity to hydrophobicantibiotics (Table 2). These results suggest that the disul-phide bonding forms a structurally altered OmpC27whose misinsertion damages the membrane, thus caus-ing increased antibiotic sensitivity. It should be noted thatOmpC27 channel size was not enlarged compared to thatof OmpC234 since both caused a similar sensitivity toampicillin, a hydrophilic antbiotic that penetrates the cellthrough water-filled OmpC channels (Nikaido and Vaara,1985).

The ompC27effect on ompFtranscription is likely to becaused by the membrane defect. This membrane defectmay change the normal osmoregulation of OmpF, whichis controlled by OmpR and EnvZ, two transcriptional regu-lators of ompF (Hall and Silhavy, 1981). We have previ-ously reported a to/C-mediated effect on ompFtranscrip-tion which occurs by a similar mechanism (Morona andReeves, 1982; Misra and Reeves, 1987). TolC is a minorenvelope protein that plays no direct role in the regulationof ompF transcription (Misra and Reeves, 1987), Thepresence of to/C:;Tn^O results in a major membranedefect, which makes cells hypersensitive to a variety ofchemicals (Morona and Reeves, 1982). In addition, thismembrane defect resulted in a two- to threefold reductionin ompFtranscription. Since the ompC27effect on mem-brane permeability was not as dramatic as that mediatedby to/C::Tn 10, only a 25% reduction in ompFtranscriptionwas observed.

ompC27 confers phage resistance owing to the reducedOmpC level

As mentioned earlier, ompC27 was isolated amongmutants that conferred resistance to the OmpC-specificphages, HY2 and SS4, Since the presence of two cys-teines in OmpC27 affected its level in the outer mem-brane, it is conceivable that the reduced efficiency of

1034 R. Misra

plaque formation (EOP) displayed by these mutants is theresult of lower OmpC27 levels and not due to the substitu-tion of a speoific amino acid residue. To examine this,EOPs of strains with a dsbAv.kani background weredetermined. In this genetic background the level ofOmpC27 present was similar to that of the parentalOmpC234 protein (Fig, 2), The results showed that, in thepresence of dsbAv.kani, mutants containing two cysteineresidues have EOPs against HY2 and SS4 similar to thatdisplayed by the parent strain (Table 2). Thus, G-154does not play any role in phage binding or infection.

Experimental procedures

Bacterial strains and growth conditions

All strains used in this study were derived from MC4100 (F~araD139 A{argF-lac)U139 rpsL150 relAI flbB5301 ptsF25deoC1 thi-1 rbsR) (Casadaban, 1976). The parent strain,RAM271, used for the isolation of phage-resistant mutants isMC4100 AlamB106 AompF80 ompC234 (Misra and Benson,1988b). Minimal medium (M63) and Luria broth (LB) were pre-pared as described previously (Silhayy etal., 1984). Maltodex-trin was purchased from Pfanstiehl Laboratories, Inc. and wasfurther purified as described previously (Misra and Benson,1988a). [^^S]-methionine was obtained from Du Pont-NewEngland Nuclear, Other chemicals were of analytical grade.

Isolation of phage-resistant mutants and binding assays

Bacterial cultures were mutagenized with nitrosoguanadine(NTG) as described previously (Silhavy et al., 1984), and thenplated on selection medium that consisted of minimal platescontaining maltodextrins, and spread with either HY2 or SS4.Phage-resistant mutants were obtained after 3d incubation at37"C. Many resistant colonies were mucoid in appearance.These colonies were avoided since they may contain a defec-tive rfa locus that is responsible for the synthesis oflipopolysaccharide. Non-mucoid mutants were purified andtested for sensitivity to the phages.

Genetic mapping, cloning and nucleotide sequencedetermination

Genetic mapping of phage-resistant mutants was carried outby PI transductions as described in the Results and Discus-sion section. Mutations that map in ompCwere further charac-terized. The cloning of ompC alleles onto a plasmid was carriedout by an in vivo allele switching method as described previ-ously (Misra and Benson, 1988b). Briefly, this involved trans-formation of mutants cells with a plasmid, pRAM1006, contain-ing wild-type ompC. The recombination event that crossed themutant (chromosomal) ompC allele onto the plasmid resultedin the appearance of large Dex* colonies as a result of anincrease in mutant ompC(Dex"') copy number. Control experi-ments established three requirements for this in vivo cloning ofthe chromosomal ompC altele onto a low-copy-number plas-mid: (i) the host strain must be recA*; (ii) the host strain must

contain a mutant (Dex*) ompC allele in the chromosome; and(iii) the plasmid must contain the wild-type ompC gene. Theentire ompC gene was sequenced from plasmid clones by thedideoxy method (Sanger et al., 1977) using six internal comple-mentary ompC primers.

Isolation of envelopes and SDS-FAGE analysis

Envelopes were prepared from bacterial cultures, grown eitherin LB or minimal medium containing glucose, by the Frenchpress lysis procedure as described previously (Misra ef al.,1991), Envelope samples were analysed by SDS-PAGE(Lugienberg etal., 1975). When a better separation of OmpCand OmpF was required, solid urea (4M) was added in theseparation gel solution. Visualization of proteins bands wasachieved by staining gels with Coomassie brilliant blue-R250,

Pulse-chase experiments and immunoprecipitations

Labelling of cells with [^^S]-methionine and immuoprecipitationwere carried as described previously (Misra e/a/., 1991). Driedgels were autoradiographed at -70°C and bands were quanti-fied by scanning the gel using a Hoeffer gel scanner.

Acknowledgements

I thank Leanne Misra for critically reading the manuscript andJames Bardwell for supplying the dsbAv.kani mutant strain.This work was supported from the NIH Grant GM48167.

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Assembly-defective ompC mutants 1035

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