Vol. 132, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Oct. 1977, p. 23-27 Copyright C 1977 American Society for Microbiology

Genetic Locus (ompB) Affecting a Major Outer-Membrane Protein in Escherichia coli K-12 VIMALA SARMA* AND PETER REEVES Department of Microbiology and Immunology, The University of Adelaide, Adelaide, SA. 5000, Australia

Received for publication 6 May 1977

Three multiply colicin-tolerant mutants in Escherichia coli K-12 from the TolIV, TolXIV, and TolXV phenotypic groups, all lacking or having only trace amounts of protein 1, a major outer-membrane protein, were mapped by Hfr crosses, and the position on the chromosome was confirmed by cotransduction with nearby markers. The mutations were located near malQP in the 74-min region of the E. coli chromosome. This locus is designated ompB, and analysis of data from two three-point crosses determined the linear sequence of genes to be aroB-ompB-malQP-glpD.

The outer membrane of Escherichia coli K12 contains at least three major or abundant proteins, designated 1, 3a, and 3b (12, 20). Protein 1 exists as two forms of differing electrophoretic mobilities, la and lb (3, 12), which give similar cyanogen bromide peptides, suggesting that the two forms may arise by modification of a single precursor protein. In addition, a fourth protein, the tsx protein, is a major protein in our strain of E. coli K-12 (13). Some genetic data are now available for these proteins. The ompA gene (for outer-membrane protein), previously known as con (21), tolG (7), or tut (11), is located at 21.5 min and is apparently the structural gene for protein 3a (11). The tsx mutation (8) leads to the loss of the tsx protein (13). However, genetic information relating to protein 1 suggests that at least three loci may be involved in the control of protein 1 (see Discussion). In this paper we present data on a class of mutants that lack both la and lb. The strains used, P530, P692, and P686, are three of a range of mutant strains isolated by Davies and Reeves (4) showing multiple colicin tolerance. These strains were also shown by J. K. Davies (Ph.D. thesis, University of Adelaide, Adelaide, Australia, 1974) to lack protein 1. With the better techniques now available, we were able to show that both proteins la and lb are absent or deficient in these strains. These data together with a map position for these mutations are presented in this paper.

ble strength plus 5 mg of sodium chloride per ml; nutrient agar was blood agar base (Difco), prepared as directed, without the addition of blood. Minimal agar was prepared by the addition of 20 g of agar per liter (Difco) to minimal liquid medium (21). Glucose was added as a carbon source at a final concentration of 5 mg/ml, and glycerol was added to a final concentration of 1%. Growth supplements were added at a concentration of 20 ug/ml. Maltose tetrazolium agar was as described previously (1). Streptomycin was added where indicated at a concentration of 100 ,ug/ml. Genetic methods. Mating methods were essentially those of Verhoef et al. (22). In a cross between HfrC RC740 and auxotrophic malA recipients carrying colicin tolerance, selection was for a proximal marker, argE, using nalidixic acid (20 ytg/ml) counterselection against the donor. Transduction was mediated by bacteriophage PlcmlclrlOO (16). Cultures of recipients were grown to late log phase, harvested, and suspended in 1 ml of MC buffer (0.1 M MgSO4-0.005 M CaCl2), and 0.1 ml of recipient cells was incubated with various dilutions of phage lysates for 20 min at 37'C, after which sodium citrate was added to 0.5 M in order to prevent subsequent phage adsorption. The contents of the tube were plated on the appropriate selection medium. In the case of the malA (see footnote, Table 1) transduction, 3 ml of nutrient broth and 0.1 ml of 20% maltose were added to 0.4 ml of the transduction mixture of recipients and phage. Subsequent incubation with shaking at 37°C for 30 min allowed expression of the malA allele. The cells were then centrifuged, suspended in 0.1 ml of 10 mM MgSO4, and 0.2 ml of 1010 plaque-forming units of bacteriophage Xvir per ml was added. The cells were further diluted and incubated for 3 h, and 0.1 ml of this mixture was plated on maltose tetrazolium agar plates (1). The Mal colonies were isolated as transductants. The recombinants and transductants were isolated and purified, after which the colicin tolerance

MATERIALS AND METHODS Bacterial strains. The strains used are listed in Table 1. nalA mutants were isolated by the method of Miller (16). The colicinogenic strains have been described previously (4, 6). Media. Nutrient broth (Difco) was prepared dou23

24

J. BACTERIOL.

SARMA AND REEVES TABLE 1. Bacterial strainsa

Strain

Relevant properties

Sex

Source or reference

A. L. Taylor thr leu proA his argE thi rpsL lacY galK ara Fintl xyl supE This laboratory FnalA derivative of AB1133 P1761 (4) FTolXIV derivative of AB1133 P530 This laboratory FTolXIV nalA of AB1133 P1770 This laboratory FTolXIV malA of AB1133 P1696 (4) FTolIV derivative of AB1133 P692 This laboratory FTolIV nalA of AB1133 P1771 This laboratory FTolIV malA of AB1133 P1698 (4) FTolXV derivative of AB1133 P686 R. Clowes HfrC metB RC740 M. Schwartz HfrC asd U483 K. B. Low FmalA KL833 E. C. C. Lin F95 glpD A. J. Pittard F+ aroB malA AB2847 a All strains are E. coli K-12 derivatives. Genetic symbols are according to Bachmann et al. (2) except for malA, which refers to any mutation in the malQPI operon.

AB1133

phenotype was scored, using an overlay technique (4) with cross-streaking against colicinogenic strains CA31, CA42, K12-CA38, K235, JF246, P15, and K-12-(ColX-K235) (4), producing colicins A, E2, E3, K, L (14), S4, and X, respectively. The growth requirements and the sugar fermentation patterns were determined by replica plating or by spotting onto the appropriate plates. A few transductants were tested for resistance or sensitivity to the ktw group of phages (9), i.e., K2, K20, K21, and K29, by cross-streaking against high-titer phage stocks. The glpD strain, strain 95, was resistant to colicins A and L and partially resistant to colicin K. Care was taken to distinguish this pattern from that of TolIV, TolXIV, and TolXV when cross-streaking the Glp+ transductants. Outer-membrane preparation and polyacrylamide gel electrophoresis. Outer membranes were prepared from cells grown in nutrient broth with vigorous aeration at 37°C and harvested in log phase at two-thirds of the maximum growth yield. The outer-membrane fraction was the Triton X-100insoluble component of the cell envelope as described by Schnaitman (19, 20). Slab gel electrophoresis and sample preparation were essentially as described by Lugtenberg et al. (12). However, to obtain good resolution of all relevant bands we had to use a mixture of sodium dodecyl sulfate of grades 30175 BDH and 30176 BDH (15). The batches differ in purity. Slabs were stained with Coomassie brilliant blue according to Fairbanks et al. (5).

RESULTS

Polyacrylamide gel electrophoresis of both the outer-membrane fraction and whole-cell envelope preparations showed that the outer membrane of the mutant strain P530 lacked both the la and lb forms of protein 1. This result was obtained with several independent preparations. However, the outer membrane of strain P692 contained trace amounts of the la

form but no lb form of protein 1, and strain P686 preparations contained barely detectable amounts of the la form and also no lb form (Fig. 1). The whole-envelope samples showed the same effect with la and lb but were otherwise not distinguished from those of the parent strain. Hfr crosses analyzed by gradient of gene transfer (22) placed the colicin tolerance mutations of P530 and P692 approximately between the xyl and rpsL loci. Further analysis by phage P1-mediated transduction showed that the mutations in strains P530 and P692 were cotransducible with malA, glpD, and asd (Table 2). The map distances between Tol and the various markers were derived from their respective cotransduction frequencies by substitution into the equation (23): cotransduction frequency = 3 ( 1 -distance between 2 markers J length of transducing particle that the mutations are 0.2 suggest results The min from malA, on the opposite side to glpD at approximately 73.8 min, for both the TolXIV strain (P530) and the TolIV strain (P692) mutations, with reference to the recalibrated map of Bachmann et al. (2). The data from the two three-point crosses (Table 3) unambiguously determine the gene order. In the first cross, cross A (MalA GlpD+, colicin tolerant with MalA+ GlpD, colicin sensitive), the least frequently occurring class of recombinants with the MalA+ GlpD+, colicintolerant phenotype can be assigned to a doublecrossover event if the gene order is tol-malAglpD as shown in Fig. 2. Again, in the second cross, cross B (AroB+ MalA+, colicin tolerant

GENETIC LOCUS FOR OUTER-MEMBRANE PROTEIN

VOL. 132, 1977 c;

-A

'w

#.:'. .

-&

CA)

w,

AB1133

P530

P686

P692

25

with AroB MalA, colicin sensitive), the frequencies of the various classes of recombinants were only consistent with the hypothesis for gene order shown in Fig. 2. Strain P686 was included in the second cross. The three-point cross data together with the cotransduction frequency of colicin tolerance in strain P686 with glpD (i.e., 41.5%) gave the approximate location of the mutation in strain P687 as 73.7 min. Several of the colicin-tolerant transductants of strains 95 and AB2847 were examined by polyacrylamide gel electrophoresis of their outer membranes, as well as for resistance to phages K2, K20, K21, K29, and the relevant colicins. In every case the transductants showed the same pattern of resistance to colicins and phages as the appropriate donors, and there was absolute correlation between the absence of protein 1 and the resistance to phages TABLE 2. Cotransduction frequencies between ompB and nearby markers Cotransductiona of colicin tolerance :om Recipient Selected genetic strain: strain

marker

P530

AB1133

P692

4 1.4 asd+ 61 48 glpD+ 77 malA+ 79 a Plcmlclr carrying tol and asd+, glpD+, or malA + were incubated with recipient strains and subsequently plated on selective media. One hundred transductants were scored in each case. The cotransduction frequencies are averages of two experiments.

U483 95 KL833 FIG. amide

1. Sodium

dodecyl sulfate-slab gel polyacrylof the Triton-insoluble outer

electropho'resis

membranes

of the parent

strain (AB1133) and the

mutant strains (P530, P686, P692). Proteins la and

correspond to protein bands b and c, r-espectively, of Lugtenberg et al. (12). Only the major outer-membrane protein region of the gel is shown.

lb

TABLE 3. Three-point crosses a Parental phenotype

Cross A Donor: GlpD+ MalA, colicin tolerant Recipient: GlpD MalA+, colicin sensitive

Marker s lected

Recombinant phenotypic classes

% Transductants TolXV TolIV doTolXIV oX TlVd-TII nor

donor

48.1 31.2 0.8 19.8

56.6 28.7 1.5 11.7

donor

Glp+

MalA, colicin tolerant MalA, colicin sensitive MalA+, colicin tolerant MalA+, colicin sensitive

Cross B AroB+ 20.5 14.1 17.0 MalA, colicin tolerant Donor: AroB+ MalA+, 52.0 41.5 42.4 MalA, colicin sensitive colicin tolerant 37.5 27.0 40.4 MalA+, colicin tolerant Recipient: AroB MalA, 0.5 4.0 3.0 colicin sensitive MalA+, colicin sensitive a Three-point crosses were performed by incubating PlcmlclrlOO phage lysates of donors with recipient cells and plating out on selective media. In cross A, P1698 (TolIV) and P1696 (TolXIV) were used as donors and strain 95 was used as recipient, whereas in cross B, P692 (TolIV), P530 (TolXIV), and P686 (TolXV) were donors and AB2847 was the recipient. In each cross, about 200 transductants were isolated, purified, and scored for colicin tolerance and maltose fermentation characteristics.

26

J. BACTERIOL.

SARMA AND REEVES

is P686, P530, P692. All the mutants are very resistant to colicin X, with only occasionally a very narrow hazy inhibition zone being observed. Sensitivity to colicin El is absolute and consistent, as is sensitivity to group B colicins (4). Resistance to bacteriophages has also been reexamined, and again the effects are not always absolute. Thus, whereas all three mutant strains are resistant to bacteriophages K2, K20, K21, and K29, strain P692 is less resistant. Both the phage resistance of strain P692 and the resistance of P692 to colicins K, S4, and X were not noted in the previous work (4, 9).

73-45IFaro B *37 tol IV -48 tOI XIV *33 tol XV

A. omp

B 24 tol IV *23 tol XIV *27 tol XV

I

b^d.L

we 73-995 mal A

*43 tOl IV *31 tol XIV *51 toI XV

132 tot IV 1-52 tol XIV 74-1

[gip D

74-3Fasd FIG. 2. Map of the 74-min region of tAke E. coli K12 chromosome. All distances in minu tes (23) are calculated from data in Tables 2 and 3. Where there is more than one set of data, the distanc,es are averaged. The arrowhead represents the selec ted marker.

and colicins. It would appear that a single mutation gives rise to the pleiotropic effects in these mutants. During the course of this study, the colicin resistance pattern of these mutants was tested on many occasions and can now be de.fined more precisely than before (4; Davies, Ph..D. thesis). Results of plate overlay tests are 4consistent, with the exception that some of thLe colicinogenic strains do not always appear to produce the same amount of colicin. Resistanices to colicins E2 and E3 are always similar , and both colicins produce narrow hazy zones (Dn the mutants, with strain P692 consistently Inore sensitive. Resistance to colicin K zones iEs again incomplete, with the zone typically naLrrowed for all three mutants. Resistance to ci olicin L is often absolute, but strain P692 may have an inhibition zone. Strains P530 and P6186 are usually resistant to colicin A, and P692 iis partially resistant (hazy narrow zone). S4 coli4 cin production is erratic, and all strains show resistance: the order of increasing sensitivity of the strains

DISCUSSION Transduction results essentially show that the three phenotypic groups of colicin-tolerant mutants, TolIV, TolXIV, and TolXV, are all due to mutations located in the 73.7-min region of the chromosome. The cotransduction of the mutations with the asd marker is much lower than expected. We have no explanation for the low results obtained in two independent experiments. The three phenotypic groups were originally distinguished on the basis of their colicin tolerance patterns (4, 9), but a reappraisal of the phenotypes shows the differences to be quantitative rather than qualitative. The three mutations thus have similar, though not identical, colicin tolerance patterns and phage resistances, and a likely hypothesis is that they are all located in the same gene, which we propose to call the ompB locus.

Although the protein 1 defect of these mutant strains has been known for some time (Davies, Ph.D. thesis) and the strains have been used by others (3, 10, 11), it is only now that they have been mapped that it is appropriate to designate a genetic symbol. We have chosen omp (outermembrane protein) because at present the outer-membrane protein loss seems to be the fundamental aspect of the mutant phenotype. The alleles have been designated ompBlOl

(P530), ompB102 (P692), and ompB103 (P686). However, if later work shows that there is more than one locus involved, then ompBlOl should be considered as the type mutation for ompB. The role that the ompB gene plays in the expression of protein 1 is uncertain and will require further study. Two additional genes have been recently reported to be involved in protein 1 expression: the tolF mutants, which lack protein la (J. Foulds, personal communication), and the par mutants, which lack protein lb. These two loci may be involved in regulation of the two forms of protein 1, as discussed by Bassford et al. (3), since tolF par

GENETIC LOCUS FOR OUTER-MEMBRANE PROTEIN

VOL. 132, 1977

double mutants produce protein la. Of the three loci, ompB is the only one with properties compatible with its being the structural gene for protein 1, but further studies are required to resolve the role of ompB in the expression of proteins la and lb on the outer membrane. LITERATURE CITED 1. Achtman, M., N. Willetts, and A. J. Clark. 1972. Conju-

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10. 11.

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27

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