Cellular control of conjugation in Escherichia coli K12

J. Mol. Biol.

(1982) 161, 13-31

Cellular

Control

Effect of Chromosomal LII)IA

SAMBVCETTI,

of Conjugation cpx Mutations LILLIAS

EOYAN(:

in Escherichiu

cofi K12

on F-Plasmid

Gene Expression

ANI)

&LVERMANt

PHILIP

M.

Department of Molecular Biology, Division of Biological Sciences Albert Einstein College of Medicine, Bronx, N.Y. 10461, U.S.A. (Received

5 April

1982)

DNA donor activity and surface exclusion of Escherichia coli Ff and Hfr strains require expression of both F-plasmid and chromosomal genes. The plasmid genes are contained in the 35,000 base tru region. where most of them are organized as a co-regulated gene block designated the traY -+ 2 operon. The chromosomal genes have been identified among chromosomal mutants that fail to express donor activity and surface exclusion, even when they carry normal F-plasmid DNA. We show here that mutations in two chromosomal genes, cpzA and cpxB, together reduce the abundance of tm operon mRNA to 15% or less of the value in otherwise isogenic cpxil + cpxB+ cells. The cpxBI mutation alone had no effect on the tra operon messenger RNA level, in agreement with previous evidence that this allele by itself is cryptic. We attribute the effect of both cpx mutations on tru operon mRNA to a transcriptional defect resulting from the inability of mutant cells to accumulate the hd gene product. a 24,000 M, outer membrane protein that is also required for efficient tru operon expression in viva. Ultraviolet light-irradiated cpxA2 cpxBl mutant cells infected with a hp(traJ) transducing bacteriophage that contains an intact traJ gene and its normal control sequences failed to accumulate the TraJ protein as a 24,000 M, polypeptide, whereas cpxA+ cpxBl cells, otherwise isogenic, did. In the same experimental system. both cpxil2 cpxB1 and cpxA+ cpxB1 cells infected with the /\p(truJ) bacteriophage accumulated comparable levels of RNA complementary to trd. Moreover, cpzA2 cpxB1 and cpxA+ cpxB1 cells synthesized comparable levels of fl-galactosidase from a traJlac2 protein fusion. These results show that the cpx mutations do not reduce transcription or translation initiation at trd sequences. They define a new cellular contribution to conjugation, which we propose is related to the translocation of the TraJ protein to the outer membrane.

1. Introduction Bacterial cells harboring any of a number of large plasmids, collectively designated conjugative plasmids, transfer DNA to other cells by the process of conjugation, and they are poor conjugal DNA recipients, a phenomenon designated surface exclusion (Lederberg et al., 1952; for reviews, see Achtman, 1973; Willetts & Skurray, 1980). For the conjugative plasmid F and F-like plasmids, all the plasmid genes required for the expression of these properties are organized in a continuous t Author to whom all correspondence should be addressed 00X2-2836/82/290013-19

$03.00/O

13

I%)1982 Academic Press Inc. (London) Ltd.

14

L. SAMBUCETTI.

L. EOYANG

AND

P. M. SILVERMAN

block of DNA, about 35 kbt long and containing at least 23 tra and related cistrons as well as the origin of conjugal DNA transfer, oriT (Fig. 1; Sharp et al., 1973; Willetts & Skurray, 1980; Johnson & Willetts, 1980). Most of the tra cistrons are organized as a co-regulated gene block designated the tray -+ 2 operon (Helmuth & Achtman, 1975; Fig. 1). Both DNA donor activity and surface exclusion require the expression of genes within the operon (Fig. 1). Neither activity is expressed by cells lacking an active TraJ protein, which is required for maximal operon transcription (Willetts, 1977). At least four chromosomal genes of Escherichia coli are also required for DNA donor activity and surface exclusion, insofar as mutations in these genes prevent the expression of both properties in cells containing a normal F-plasmid (Lerner & Zinder, 1979 ; Beutin & Achtman, 1979 ; McEwen & Silverman, 1980a,h ; Silverman et al., 1980). Two of these genes, sfrA (or fez) and sfrB, have been suggested to encode transcriptional control factors, the former active on transcript’ion initiation at trd and the latter on the tray + 2 operon itself, apparently as an antiterminator (Beutin et al., 1981). Mutations in the other two genes, cpxA and cpxB, are required together for maximal reduction of DNA donor activity and surface exclusion (McEwen & Silverman, 1980a,b). A cpxA mutation alone significantly reduces both activities, but a cpxB mutation has no significant effect and is therefore cryptic by itself. In addition, the effect of the cpx mutations on DNA donor activity is more severe at 41°C than at 34°C (McEwen & Silverman, 19806). This temperature sensitivity appears to be a property of the cpzA mutant alleles (McEwen & Silverman, 1980a). In this paper, we show that cpxA cpxB double mutant cells fail to express DNA donor activity and surface exclusion because these cells are deficient in tra operon mRNA. Furthermore, we attribute this deficiency to the inability of mutant cells to accumulate the TraJ protein, which is required in viva for maximal tra operon expression. Finally, we show that the failure of mutant cells to accumulate the TraJ protein cannot be attributed to a defect either in transcription initiation from from traJ mRNA sequences. By the traJ promoter or in translation initiation analogy to the effects of the cpx mutations on other envelope proteins (McEwen & Silverman, 1982), we propose that the cpx mutations prevent or retard the translocation of the TraJ protein to the outer membrane.

2. Materials and Methods (a) Bacterial

strains,

These are described in Table 1 and Fig. nutrient broth were as described (McEwen by Miller (1972) and B2 (low phosphate) The bacteriophages were isolated from lysogen XK5462 (obtained from Dr Karin medium to an optical density (660 nm) of 60°C waterbath. Incubation was resumed aeration for 90 min. Chloroform (05%, t Abbreviation

bacteriophage

and plamids

1. Vogel-Bonner minimal medium, LB broth and & Silverman, 1980a,b). M9 medium is described medium by Studier (1973). lysates prepared by heat induction of the hp (traJ) Ippen-Ihler). Cells were grown aerobically in LB 96 at 30°C and warmed to 43°C by immersion in a at 43°C for 15 min and then at 37°C with vigorous v/v) was added and debris removed by centri-

used: kb, lo3 base-pairs; p.f.u., plaque-forming

units

trn GENE

EXPRESSION

IN cpz MVTANTS TAHLE

OF E. coli

15

1

Escherichia coli Kl2 strains Strain AE2 o ) AE2038 AK2089 AE2091 AE2129 AE2132 AE2118 AE3195 AE3115 AE3117 Hfr H KL647 AE1031 AElOl9 .4ElOlO

Relevant FFFFF-

genotype

cpxA+ epsB1 leu-6 rpsL104 cpxA2 cprB1 h-6 rpsL104 cpx.4 ’ cpxB1 malA + (A”) cpz42 cpxB1 m&A+ (X5) leu+ d(lac-pro),,,, cpxA+ cpzBl malA+ (h’) F- IPU+ d&c-pro),,,, epx.42 cpxB1 molA + (h”) F- cpd’ cpdl malA+ h+ F- cpxA+ cpxB2 matA+ (h’)/Rl F- cpxA+ cpxB1 leu-6 rpsLlO4/pRSSl F- epxd42 cpxBI leu-6 rpsL104/pRS31 leu + rpsL+ Hfr C leu’ d (Zac-pro),, , , Hfr cpxil+ cpzB+ Hfr cpzAZ cpzBl Hfr epzA+ cpxB1

Reference or source McEwen & Silverman (19806) J. McEwen; from AE2000 This work; spontaneous Mal+ of AE2000 This work ; spontaneous Mal+ of AE2038 This work; from AE2089 by conjugation with the Cavelli-type Hfr stram KL647 This work : from AE2091 by conjugation with KLM7 This work ; A+ lysogen of AE2089 This work; from AE2089 by conjugation This work ; from AK2000 by transformation This work ; from AE2038 by transformation Laboratory collection Brooks Low McEwen & Silverman (1980a) McEwen & Silverman (1980b) McEwen & Silverman (1980b)

All of the AE strains in the Table, including the Hfr strains AE1031, AE1019 and AElOlO, are essentially isogenic. except as indicated in the Table. They were all derived from AE2000, whose complete genotype was described by McEwen & Silverman (1980b), by Pl transduction, conjugation, or as indicated in the Table (see McEwen & Silverman. 1980a,b). A complete geneologg of these strains and the specific methods used in their construction will be made available upon request.

fugation in the cold (lO,OOOg, 15 min). The bacteriophage particles were concentrated by precipitation with NaCl/polyethylene glycol (Yamamoto et al., 1970) and extracted in h buffer (001 M-Tris .HCI (pH CO), 20 mM-MgSO,, 5 mM-CaCl,). Solid CsCl was added to give a refractive index (25°C) of 1.3800. The bacteriophage were concentrated by isopycnic banding in the Beckman 50Ti rotor for 24 h at 40,000 revs/min. The lower band, containing the hp (truJ) bacteriophage, and the upper band, containing bacteriophage lacking traJ (Fig. 2) and otherwise simular to the h parental bacteriophage, were separately removed, dialyzed against h buffer, and stored at 4°C. Titers on strain C600 were generally 10” to 10” p.f.u./ml for both the Ap(tmJ) and parental bacteriophage preparations. As described by Ippen-Ihler (1978), the /\p(traJ) phage plated on a GLl strain with an efficiency of -02, and the parent bacteriophage with an efficiency of 001. Bacteriophage RNA was prepared by extraction with phenol, as described by Kaiser & Hogness (1960). pAE4030 was derived by digestion of pRS27 with P&I, religation, and transformation of strain AE2074; selection was for tetracycline resistance. pAE4029 was similarly derived from pAE4030. except the plasmid was digested with BgZII (see Fig. 1). The 1.0 kb RgZIIPstI fragment was prepared from pAE4029 by sequential digestion with BgZII and PstI followed by electrophoretic separation of the fragments on a 3.5% (w/v) polyacrylamide gel run in a Tris-borate buffer system (Peacock & Dingman. 1967). The smaller fragment was electroeluted from the gel (McDonell et al., 1977) and purified by 1 extraction with phenol and 2 precipitations with ethanol. Restriction fragment sizes were determined by agarose gel electrophoresis. using a HpaI digest of phage T7 DNA (Studier et al., 1979) as molecular weight markers. The trd-la& fusion plasmid pLS200 was constructed from pMC1403 (Casadaban et al.. 1980) and the 1.1 kb BgZII fragment containing oriT; traM; $nP,; and the trd promoter. translation initiation sequences, and the first 45 nucleotides of the tmJ coding sequence (.Johnson et al.. 1981; R. Thompson, personal communication). Plasmid pMC1403 DNA was

16:

I,. SAMBUCETTI,

L. EOYANG

AND

P. M. SILVERMAS

first digested with BarnHI, which opens the molecule at the eighth codon of the la& gene (Casadaban et al., 1980). The BglII fragment, electrophoretically purified from pAE4030 (Fig. 1) after BgZII digestion, was then ligated into the open BamHI site. The DNA was used to transform the cpzA+ cpxBl d (lac-pro) strain AE2129. The cells were plated on LB agar containing ampicillin (25 pg/ml) and the chromogenic /3-galactosidase substrate. a-bromo-4chloro-3-indolyl-p-n-galactoside (X-Gal ; Miller. 1972). About 0.2% of the ampicillinresistant colonies were blue after 18 h at 32°C. Several of these were repurified twice and shown to contain plasmid DNA. Analysis of this DNA by restriction endonuclease digestion confirmed the presence of the BgZII fragment derived from F (Sambucetti & Silverman, unpublished results). Plasmid DNA was used to transform the cpzA2 cpxBl A (lac-pro) strain AE2132, selecting for ampicillin resistance. AE2129 and AE2132 are isogenic except at the cpxA locus. (b) Analysis

of protein

synthesized

in irrndiated

cells infected

with h

Cultures (10 ml) were grown at 41°C in M9 medium containing 62% (w/v) maltose and amino acid supplements (40 pg/ml) to optical densities (660 nm) of 0.5. The cells were then concentrated by centrifugation to 2.5 ml of MS medium without amino acid supplements and irradiated with 10,000 ergs mm-’ at a fluence rak of 20 ergs mmm2 s-i. After 30 min incubation in the dark at 4l”C, the cultures were brought to 10 mM-MgSG, and infected with phage at a multiplicity of 5 to 10 p.f.u./cell. After 15 min at 41°C to allow phage infection, the cells were labeled for 15 min with a i4C-labeled amino acid mixture. The cultures were then chilled to 4°C and washed once by centrifugation with cold 1 &I-Tris. HCl (pH 7.5). collected again by centrifugation, and suspended in 0.1 ml of Laemmli sample buffer. The samples were heated at 100°C for 5 min and acid-precipitable radioactivit? was measured after mild base hydrolysis (Capecchi, 1966). Equal amounts of acid-precipitable radioactivity from each sample were applied to an SDS/polyacrylamide gel. The pattern of radioactive proteins separated by electrophoresis was visualized by fluorography, using commercial fluor preparations according to the manufacturer’s suggestions. (c) Analysis

of RNA

synthesizrd

in irradiatrd

cells injected

with h

Cultures (10 ml) were grown at 41°C in B2 medium, irradiated and infected as described above for protein labeling. After infection. cells were labeled for 10 min at 41°C with 1 mCi [32Y]phosphoric acid/ml (carrier-free) and then chilled at 4°C. The cells were collected by centrifugation and suspended in 0.75 ml of buffer containing 20 mM-sodium acetate (pH 5.2). @I M-iodoacetate, 1 mM-EDTA. Each sample was extracted twice with an equal volume of hot (55”C), buffer-saturated phenol. The combined aqueous phases were brought to 63 Msodium acetate and RNA was precipitated at - 80°C after addition of 3 vol. absolute ethanol, collected by centrifugation and precipitated again from buffer containing 0.3 Msodium acetate, The second RNA pellet was suspended in 0.2 ml of 2 x SSC containing 0.10,; (w/v) SDS (SSC is 15 mw-sodium citrate, 0.15 iv-NaCl). At this stage, more than 99% of the acid-precipitable radioactivity was rendered acid-soluble after incubation in 0.1 x-NaOH for 18 h at 37°C. Appropriate amounts of RNA were diluted to 2 ml with hybridization buffer (5 x SSC, 0.02 M-Tris. HCl (pH 7.5), 0.5% (w/v) SDS, 50% (v/v) formamide). The RNA was then incubated in sealed plastic bags with nitrocellulose filter replicas of agarose gels containing electrophoretically separated restriction fragments (Southern, 1975); the filters were soaked in hybridization buffer for 30 min at 37°C before use. The bags were incubated for 72 h at 37°C with gentle agitation. The filters were then removed. washed for 30 min at 37°C with gentle agitation in 0.2 x SSC containing 62% (w/v) SDS, and then for 30 min at 37°C in 92 x SSC containing 20 pg ribonuclease/ml. After treatment with ribonuclease, the filters were rinsed for 60 min at 37°C in 2 x SSC containing 1% (w/v) SDS, and then dried. Hybridization was determined qualitatively by autoradiography. For quantitative analysis, sections of the filter containing hybridized RNA were removed and radioactivity measured by liquid scintillation spectrometry. For each filter, 4 sections of comparable width but not containing hybridized RNA were also analyzed. The average of these was taken to be background radioactivity and subtracted to give the data shown in Table 3.

trn GENE

EXPRESSION

IN cpr

(d) Liquid

MUTANTS

OF E. coli

17

hybridization

RNA was prepared from 200 ml of cultures grown at 41°C to an optical density (660 nm) of 08 to @9 by extraction with phenol (Miller, 1972) followed by precipitation in the cold in the presence of 2 M-LiCl. RNA insoluble in 2 M-LicI was collected by centrifugation, dissolved in buffer, and precipitated with ethanol, as described above. The RNA precipitate was of collected b.y centrifugation and dissolved in -01 ml of water at a concentration 50 mg/m 1. pRS31 DNA was prepared from strain Ml889 (McEwen & Silverman, 1980b; obtained from Dr P. Manning). [32P]DNA ( N lo8 cts/min per pg) was prepared by nick-translation. Hybridization mixtures (7 ~1) contained 0.2 M-sodium phosphate (pH 6?3), 05% (w/v) SDS. 6000 cts/min of radioactive DNA, and @l to 1OOpg of RNA in flame-sealed capillary micropipettes. Duplicate samples were heated to 100°C for 2 min, incubated at 65°C for 19 h. and then expelled into 1 ml of buffer containing @l iv-sodium acetate (pH 4.3), 10 pg of heatdenatured calf-thymus DNA, @l M-NaCl, I mM-ZnS04. Total acid-precipitable radioactivity was determined in @2 ml of each sample. The remaining 0.8 ml was incubated at 45°C for 60 min with 2000 units of S, nuclease, and acid-precipitable radioactivity was measured. The percentage of nuclease-resistant radioactivity in each sample was calculated from these data. These were corrected for nuclease-resistant radioactivity in samples lacking RNA, but, treated otherwise identically, and duplicate samples were averaged to yield the percentage hybridization (e) fl-Galactosidaae

and /3-lactamasr

assays

Strains AE2129 and AE2132 containing the truJlacZ fusion plasmid pLS200 were grown at 41°C for at least 5 generations in LB medium containing ampicillin (25 pg/ml). Portions (1 ml) of the culture (optical densities between @2 and 15) were then removed for enzyme assay. fl-Galactosidase activity was determined and units expressed as described by Miller (1972). fi-Lactamase assays were carried out by a modification of the iodometric method described by Sykes & Nordstrom (1972). Cells (1 ml) were collected by centrifugation in an Eppendorf centrifuge and the medium was removed by aspiration. The cell pellets were suspended in 25 ~1 of water, followed by addition of @2 ml of a solution containing 075 Msucrose, 10 mM-Tris.HCl (pH 8), and then 50 ~1 of a solution containing lysozyme (2 mg/ml) and 1.5 mm-EDTA (pH 7). After 2 min at 4°C. the samples were brought to 1 ml with 1.5 mmEDTA. left at 4°C for 20 min, and subjected to 3 cycles of freezing in an ethanol/solid CO, bath and thawing at 30°C. These extracts were used to measure /3-lactamase activity. Reaction mixtures (@25 ml) contained 0.1 M-potassium phosphate (pH 6), 1 InM-ampidin. and sufficient extract to provide about lo-* units of fl-lactamase activity (usually 2 to 5 ~1). After 10 min at 3O”C, the reactions were stopped by addition of 2 ~1 of 50% (w/v) trichloroacetic acid. The ampicilloic acid formed was measured by adding 25 ~1 of reaction mixture to 1 ml of a starch/iodine solution (Sykes & Nordstrom, 1972). These samples were incubated for 30 min at 30°C to allow complete oxidation of the ampicilloic acid (Sykes & Nordstron, 1972), after which the absorbance at 620nm was measured. Ampicilloic adid was determined by loss of absorbance compared to a sample lacking enzyme, assuming a ampicilloic acid. One unit of fl-lactamase activity is the amount AA 620nm of -004/nmol required to form 1 pmol of ampicilloic acid/min. For comparison with the /3-galactosidase measurements. specific activities were expressed as units/optical density unit of the cell. culture. (f) Other

methods

Slab gel electrophoresis of proteins was performed with the buffer system described by Laemmli (1970) using 12.5% acrylamide resolving gels and 3.5% acrylamide stacking gels. Agarose slab gel electrophoresis and visualization of DNA was performed as described by Aaij & Borst (1972). Nick-translations were as described by Rigby et al. (1977), except that the DNAase concentration was reduced 50-fold for nick-translation of the 1.0 kb BgZII-I-‘&I fragment (see Fig. 2). Plasmid DNA was prepared by dye-buoyant density sedimentation as

18

I,. SAMBUCETTI,

L. EOYANG

AND

I’. M. SILVERMAN

described by Cohen & Miller (1969) for loo-ml cultures or Humphregs rt al. (1975) for 1 1 cultures. Cleavage of DNA catalyzed by restriction enzymes were carried out according to the manufacturer’s suggestions for each enzyme. (g) Materials Restriction enzymes HpaI, BgZII, Sal1 and EcoRI, DNA polymerase I and phage T4 DNA ligase were obtained from Bethesda Research Labs ; restriction enzyme PstI was provided by Dr Kei Amemiya. Bovine pancreatic DNAase I (code DPFF, electrophoretically purified) and ribonuclease A were obtained from Worthington Biochemicals and nuclease S, from Boehringer-Mannheim. [32Y]phosphoric acid (carrier-free) and Enhance (for fluorography) were purchased from New England Nuclear; reconstituted i4C-labeled amino acid mixture (“Schwarz mixture”), from SchwarzMann and [n-321’]dCTI’ (400 to 600 Ci/mmol), from Amersham. X-ray film was obtained from Kodak (X-Omat AR) or from DuPont (Cronex 4). Nitrocellulose paper sheets (BA85) were purchased from Schleicher and Schuell, and formamide from MCB Manufacturing Chemists. Inc. Ampicillin was obtained from the Sigma Chemical Co. We found this material suitable for fl-lactamase assays, using the method described above, but it contains significant quantities of material that bleaches starch/iodine, presumably ampicilloic acid. All other materials were obtained from standard commercial sources.

3. Results (a) Effect of the cpx mutations

on expression

of the tray

+ Z operon

We used pRS31 as a hybridization probe to examine directly the effect of the cpxA2 and cpxB1 mutations on the amount of cellular tra operon mRNA. pRS3l is a recombinant plasmid containing three contiguous EcoRI fragments of F cloned in pSClO1 (Skurray et al., 1976). The cloned F DNA contains part of the traG gene, intact traS, traT, traD, traI and traZ genes and a smaller region of additional DNA (Willetts & Skurray, 1980). Altogether, about 75% of the F DNA in pRS31 consists of known coding sequences and these account for 45% of the DNA assigned to the tray-+ 2 operon (Fig. 1). pRS31 DNA labeled in vitro by nick-translation was incubated with different amounts of total RNA isolated from Hfr cpxA+ cpxB+, Hfr cpxA+ cpxB1, Hfr cpxA2 cpxB1 and F- cells. Hybridization to tra mRNA sequences from the cpxA+ pRS31 DNA to an Si nuclease-resistant form cpxB + strain AE1031 converted (Fig. 2). The extent of hybridization approached 30% of the DNA; this is a theoretical maximum, since 60% of the pRS31 DNA is derived from F, most of this F DNA consists of coding sequences (see above), and only one tra DNA strand is 1974). Little or no hybridization occurred with transcribed (Vapnek & Spingler, RNA derived from the F- strain AE2000, especially at Rot-f values of less than 1000, and no DNA-RNA hybridization at all occurred when pSClO1 DNA was used in place of pRS31 DNA (data not shown). Given a maximum of 30% hybridization, we estimate the Rot+ value for hybridization of AE1031 RNA to the F sequences of pRS31 to be 300. Similarly, we determined an Rot+ value of 260 for the cpxAf cpxB1 strain AElOlO (not shown), consistent with our findings that the cpxB1 mutation in cpxA+ strains, such as AElOlO, is cryptic (McEwen & Silverman, 198Oa,1982). For this reason, subsequent experiments compare cpxA+ cpxB1 and cpxA2 cpxB1 cells. t &t, molar concentration of RNA nucleotide x seconds of hybridization

trn GENE

EXPRESSION

IN c,ppx MUTANTS

OF E. coli

19

t~~~+fi~plosmid

4

I

-

H

I

pAE4030

1-h

pAE4029 Xp (+oJ)

/t--t----H’/ SO/I

-

AfI

Frc:. 1. The transfer region of the conjugative plasmid F. The top of the Figure (not drawn to scale) shows the order of the tra cistrons and oriT, between F co-ordinates 6.5 and 100. and the position of lr:coRI restriction sites in this region (from Willetts & Hkurray, 1980). This part of the Figure also denotes the position of the trd and traT genes, required for the expression of surface exclusion (Achtman et nl.. 1980). within the traY +Z operon (Helmuth & Achtman, 19’75). The presumptive promoter for this operon is designated traY,. Secondary promoters within the operon are not indicated (see the text). The combined effect of %nl’, and $4 gene products (the latter supplied by an p’ plasmid) to repress expression of trctJ at the fraJ promoter (tdp), and the effect of the trd gene product to promote expression of the tray + 2 operon are indicated at the top of the Figure. The F DNA contained in relevant plasmids and bacteriophage are also indicated : these are described in detail as appropriate in the text

FIG. 2. tra mRNA abundance in Hfr cpxA+ cpxB+ (strain AE1031), Hfr cpzAZcprB1 (strain AE1019), and F- (strain AE2000) cells. Liquid hybridization of total cell RNA with 13’l’]DNA of pRS31 were carried out as described in Materials and Methods. (A) RNA from strain AK1031 ; (0) RNA from strain AE1019: (0) RNA from strain AE2090.

Using a value of 115 kb for the sequence complexity of tru mRNA complementary to the F DNA of pRS31 and a value of 10m3 for the Rota of hemoglobin mRNA (sequence complexity of 1.2 kb under these hybridization conditions; A. Skoultchi, personal communication), we calculate an Rot+ value of

20

L.

SAMBUCETTI.

L.

EOYANU

AND

I’.

M.

SILVERMAN

9.6 x low3 for pure tra mRNA complementary to the F DNA of pRS31. Given the observed Rot+ values, the steady-state level of this tra mRNA is 3.2 x 10d3% of total cell RNA in AE1031 or about 0.1% of total cell mRNA, assuming mRNA is 3% of total cell RNA (Kennell, 1968). This estimate is about tenfold lower than estimates based on pulse-labeling experiments (Beutin et al., 1981), perhaps owing to an unusually short half-life of tm mRNA. The cpxA2 and cpxB1 mutations together caused a significant increase in the Rot+ value for hybridization of RNA to the F DNA of pRS31, and hence a decrease in the proportion of this RNA in the cell (Fig. 2). Assuming an equivalent reduction in all sequences, we estimate that the cpxA2 cpxB1 mutant strain AE1019 contained only about 15% of the amount of tra mRNA in the cpxA+ cpxB+ strain AE1031 or the cpxA+ cpxB1 strain AElOlO. However, this is a minimum estimate for the effect of both cpx mutations on tra operon expression, because traI and traZ may also be expressed from an internal promoter (Willetts & Skurray, 1980), though it is not clear whether this promoter is under traJ control. There is, however, evidence for traT expression that is independent of traJ (Ferrazza & Levy, 1980; Achtman et al., 1980; Moore et al., 1981a,b). pRS31 contains the traS and traT genes of F, and cells containing this plasmid express surface exclusion (Achtman et al., 1977). However, traS and traT gene expression from pRS31 must occur from a promoter other than tray,, which the plasmid lacks (Fig. 1). Hence, if the cpx mutations alter the expression of the tra operon, and have no direct effect on the proteins encoded by operon genes, then cpxA2 cpxB1 and cpxA+ cpxB1 cells containing pRS31 should express comparable levels of surface exclusion (Beutin et al.. 1981), whereas in Hfr strains the cpxA2 and cpxB1 mutations together reduce surface exclusion by more than two orders of magnitude (McEwen & Silverman, 1980a,h). Both cpxA2 cpxB1 cells and cpxA+ cpxB1 cells containing pRS31 express comparable levels of surface exclusion (Table 2) and of TraT protein in their outer membrane (data not shown). These experiments show that a cpxA mutation in a cpxB1 mutant strain described substantially reduces expression of the tray -+ Z operon. The experiments below indicate that this effect of the mutation is indirect, in that mutant cells fail which is required in vivo for efficient operon to accumulate the TraJ protein, transcription (Willetts. 1977).

Effect of the cpx mutations Recipient strain AE2000 AE3115 AE2038 AE3117 t Measured $ Recipient

as Leu+ activity

on surface exclusion

Genotype

cpxA + cpxB1 cpxA+ cpxBl/pRS31 cpxA2 cpxB1 cpxA2 cpxBlJpRS31 St? recombinants/ml with Hfr of strain lacking pRS3l/recipient

Recipient activityt 1.4 252 6.0 2.6

x x x x

lo6 lo3 lo6 lo3

by pSR31

&ruins

Surface exclusion indext 636 SO8

H as donor (Silverman & McEwen. 1980n). activity of strain containing pRS31.

trrr (:ENE

(b) TraJ

EXPRESSION

protein

synthesis

IN cpz MUTANTS in ultraviolet

PI

OF E. coli

light-irradiated

cells

The TraJ protein has not been consistently detected in normal Hfr or F’ cells (Achtman et al., 1977,198O; Beutin et al., 1981; Moore et al., 1981a,b). We therefore used ultraviolet light-irradiated cells infected with a hp(traJ) bacteriophage (IppenIhler. 1978) to examine the effect of the cpx mutations on TraJ protein synthesis. As we show in this section, this bacteriophage carries F DNA that includes the entire traJ gene, its normal promoter and regulatory sequences, and a functional jS’, locus (Fig. 1). The relevant region of F DNA is defined by a 15 kb SalI-PstI restriction fragment (Fig. 1). The Sal1 site lies within traM and the PstI site, within tray (Thompson & Achtman, 1978,1979). As shown (Fig. 3), the hp(trd) bacteriophage contains a SalI-PstI fragment of the expected size that is not present in the parental /\ bacteriophage. A fragment of the same size is contained within the F DNA of plasmid pAE4030, a derivative of pRS27 (Skurray et al., 1978), which also contains F DNA from the traM-tray region (Fig. 1). The 1.5 kb fragments are identical, insofar as each one hybridized to a 1.0 kb BgZII-PstI restriction fragment prepared from nick-translated plasmid pAE4029 (Fig. 3). This smaller fragment contains all but 45 nucleotides of the traJ coding sequence and a portion of tray (Thompson & Achtman, 1978,1979; R. Thompson, personal communication). When irradiated and infected with the hp(traJ) bacteriophage in the presence of radioactive amino acids, cpxA+ cpxB1 cells elaborated a protein that was not synthesized in uninfected cells or in cells infected with the /\ parental phage (Fig. 4, lanes 1 to 3) and whose apparent molecular weight ( ~24,000) corresponds to that of the Tra,J protein (Kennedy et al., 1977; Ippen-Ihler, 1978; Manning & Achtman, 1979). Furthermore, synthesis of this 24,000 iM, protein was specifically repressed in irradiated cells containing the J;’ R plasmid, Rl (Fig. 4, lanes 4 and 5). The Ji’ plasmids contain a locus, JinO, that F itself lacks and that supplies a trans-acting component that, in conjunction with the JinP product of F dfinPF), represses transcription of traJ (Finnegan & Willetts, 1971,1972,1973; Willetts, 1977). In addition. the 24.000 M, protein was the major product synthesized in a I\+ lysogen infected with the hp(traJ) bacteriophage (Fig. 4, lane 6). These data confirm that the 24,000 M, protein is the product of the traJ gene and show that it is synthesized in this system primarily from its own, rather than from a bacteriophage, promoter, This is not unexpected, since the method used by Ippen-Ihler (1978) to isolate the hp(traJ) bacteriophage placed the gene to the left of the PL promoter, but oriented such that, the direction of traJ transcription is opposite to that of transcription from P, i&elf. (c) Effect

of the cpx mutations on TraJ ultraviolet light-irradiated

protein

synthesis

in

cells

In contrast to cpxA+ cpxB1 cells, cpxA2 cpxB1 cells grown, irradiated and infected with the Ap(traJ) bacteriophage at 41°C did not accumulate the TraJ protein (Fig. 5). The amount of TraJ protein synthesized in cpxA+ cpxB1 cells varied with different preparations of the hp(traJ) bacteriophage (compare Fig. 5, lane 2 and Fig. 4, lane 3), but in more than ten experiments with several different

1,. SAMBUCETTJ.

I

2

L. EOYANG

3

AND

P. M. SILVERMAN

4

5

6

FK:. 3. Analysis of DNA from A parental and h(~nr./) bacteriophage and plasmid pAE4030. DNA (2 pg) was digested with MI and P&I, and the restriction fragments were separated by electrophoresis through a 1% agarose gel (lanes 1 to 3). The fragments were then transferred to nitrowllulose paper (lanes 4 to 6) and hybridized to [32P]DNA prepared in vitro by nick-translation of the 1.0 kb BgZII-P&I restriction fragment of pAE4029 (see Fig. 1). Lanes 1 and 4. parental phage; lanes 2 and A, hp(traJ) phage; lanes 3 and 6, pAE4030. DNA-DNA hybridization was performed as described by Raboy et cd. (1980).

trcl GENE

EXPRESSION

-

67-

-

43-

-

-

2

OF E. co/i

13

30-

T~OJ -

-

I

IN cpx MUTANTS

20-

3

4

5

6

VII:. 4. tm.J expression in ultraviolet light-irradiated cells. Cells were infected and labeled with 14Clabeled amino acids, and labeled proteins were analyzed by gel electrophoresis. as described in Materials and Methods. Lanes I through 5 are from the same experiment, and lane 6 from a different experiment. The mokcular weights ( x 10m3) of proteins with the indicated mobilities are shown between lanes 3 and 4 for t,he tirst experiment, and to the right of lane 6 for the second experiment. The position of the trcc.J gene product (TraJ) is also indicated, as described in the text. The strains used (Table 1) were: AE2089 (lanes I to 3); AE3195 (lanes 4 and 5); AE2118 (lane 6). Lane 1, no phage; lane 2, parent h phage; lane 3. A(tmJ) phage: lama 4. parent h phage/Rl + cells: lane 5. h(tm.J) phage/Rl + cells: lane 6, h(tm.J) phage ‘A+ Iysogen.

/\p(traJ) preparations we never detected TraJ protein in cpxA2 cpxB1 cells at 4172, nor could we detect TraJ protein fragments when we infected a cpxA2 cpxB1 lysogen (data not shown). Only when double mutant cells were grown and maintained at 34°C could we detect a small amount of TraJ protein synthesis (data not shown). At this temperature, mutant cells partially recover DNA donor activity (McEwen & Silverman, 19806). (d) Effect of the cpx mutations

on traJ

transcription

We used two approaches to evaluate the effect of the cpx mutations on traJ transcription. In the first, we used DNA-RNA hybridization to examine [32P]RNA synthesized in irradiated cells after infection with the hp(truJ) bacteriophage, for direct comparison with the protein synthesis data shown above. In the second, we used a traJ-1acZ fusion plasmid to examine transcription and translation initiation from traJ sequences in unirradiated cells. Both approaches lead to the same conclusion ; the cpx mutations reduce neither transcription initiation at traJ, nor translation initiation from the traJ mRNA sequences derived from the traJ-1acZ fusion plasmid. Irradiated cpzA+ cpxB1 and cpxA2 cpxB1 mutant cells were infected with Ap (trod) and the RNA synthesized after infection was labeled with “P as described

14

L.

SAMRUCETTI.

1,. EOTANG

-

I

2

TraJ

AND

P.

111. SILVERMAS

-

3

4

FIN:. 5. traJ expression in epxA+ cpxB1 and cpnl:! cp.rBl mutant cells. See Materials and Methods and Fig. 3 for details. Lanes 1 and 2. cpx-d+ cpsBl strain AE2089: lanes 3 and 4. cpxcd2 cprR1 strain AE2001. Lanr 1. parent h phage: lane 2. h(~ro.l) phage: lane 3. parent h phage; lane 4. h(lrrrJ) phage.

in Materials and Methods. Purified [32P]RNA was hybridized to BgZII-PstI restriction fragments of pAE4029 that had been separated by gel electrophoresis and transferred to nitrocellulose paper. When cleaved with these two enzymes, pAE4029 yields only two fragments : the 1.0 kb fragment, which contains nearly all of the traJ coding sequence (see Fig. 3), and a larger fragment containing the entire pSClO1 cloning vector and remaining F sequences. We have taken RNA hybridized to the former as a measure of transcription initiation at traJ (see Discussion). For comparison (see below), a filter containing a PstI digest of h DNA was included in each hybridization. phage As shown in Figure 6, cpxA+ cpxB1 cells infected with the h parental synthesized RNA complementary to h DNA, but not to any of the F sequences of

trn

GENE

EXPRESSION

IN

cpz

MITTANTS

OF

E.

25

coli

d

b-

I

2

3

4

5

6

7

6

l0c:. 6. /rcr.l mRNA synthesis in cpxA+ epzH1 and rpxA2 epxB1 mutant cells. The preparation of “I’ RNA from infected cells and the hybridization protocol are provided in Materials and Methods. The odd-numbered lanes contain the 3 BgIII-Pstl restriction fragments of pAE4029 (see Fig. 1) separated b? agarose gel rlrctrophoresis and transferred to nitrocellulose paper. (The position of the 2 fragments can prepared. Iw swn in lane .5.) The even-numbered lanes contain I?ctI fragments of h’ DNA. identically Lanes 1 and 2 wtw incubated with [3ZP]RNA from uninfected AIUOXR: lanes 3 and 4. with [“P]RNA from Al~2089 infected with the h parent phage: lanes 5 and 6. with [ 32P]RNA from AE2089 infected with the hp(trcr.1) phage: and lancx 7 and 8 with [321’]RNA from the cpz mutant strain AE2091 infected with the Xp(tro.1) phage. For quantitative analysis (see Table 3). the bands designated a (lanes 5 and 7) and ewlosrd I)!- brackets (lanes 6 and 8) were excised (see Materials and Methods).

pAE4029 (lanes 3 and 4). In contrast, the same cells infected with the hp(trti) phage synthesized RNA complementary to h DNA, to the 1.0 kb BgZII-PstI fragment of pAE4029 (labeled a in the Figure), and to the F sequences remaining with the pSClO1 vector in the larger RgZII-PstI restriction fragment (labeled b). This last’ RNA must be a complementary to F DNA sequences between the leftmost EcoRI site and the BgZII site of pAE4029 (Fig. l), because hp(trd) does not carry F DNA to the right of traA (Ippen-Ihler, 1978). This RNA may include the message for protein 6d (Thompson & Achtman, 1979). We also observed hybridization to the 1.0 kb fragment of pAE4029 using [ 32P]RNA from cpxA2 cpxB1 mutant cells infected with hp(lruJ) (Fig. 6, lanes 7 and 8). Quantitative comparisons of the amount of radioactive RNA hybridizable

2ti

I,.

SAMBUCETTI.

L.

EOYANG

ANI)

P. M.

SILVERMAN

to this fragment and synthesized in cpxA+ cpxB1 cells with the amount synthesized in cpxA2 cpxB1 cells are shown in Table 3. These comparisons indicate that cpxA2 cpxB1 cells synthesized no less, and in relation to h transcription somewhat more, RNA complementary to traJ than did cpxA+ cpxB1 cells (Table 3), even though accumulation of the TraJ protein occurred only in the cpzA+ cells. In an independent analysis, we examined the effect of the cpx mutations on transcription and translation initiated at traJ sequences by using the traJ-1acZ fusion plasmid pLS200. We constructed pLS200 from the Zac expression vector pMC1403 (Casadaban et aZ., 1980) and the 1.1 kb BgZII fragment containing oriT: traM; $nPF; and the traJ promoter, translation initiation sequences. and the first 45 nucleotides of the TraJ protein coding sequence (Johnson et al.. 1981 : Fig. 1). Sequence data provided by Dr Russell Thompson (personal communication) and by Casadaban et al. (1980) indicated that the BglII fragment cloned into the BamHI site of pMC1403 in the correct orientation would fuse the TraJ protein coding sequence and the /3-galactosidase coding sequence in the correct reading frame to produce a hybrid enzyme whose synthesis would be dependent on the traJ promoter and translation initiation sequences. This fusion plasmid, designated pLS200, was constructed as described in Materials and Methods. Comparison of /% galactosidase levels in cells containing pLS200, pLS200 and the J;’ plasmid RlOO, and pLS200 and RlOO-1, a derepressed mutant’ of RlOO. show that /3-galactosidase control (Xambucetti & synthesis from pLS200 is under ,finO JinPr repression Silverman, unpublished results), as expected for the traJ promoter. pLS200 was used to transform the cpxA+ cpxB1 strain AE2129 and the cpxA2 cpxB1 strain AE2132, both of which are deleted for chromosomal Zac DNA. Both strains maintain substantial levels of fl-galactosidase at 41°C. the non-permissive temperature for TraJ protein accumulation (Table 4). The level of /I-galactosidase in the cpxA2 cpxB1 strain was about twice that in the cpxA+ cpxB1 strain. This difference could not be attribut’ed to different plasmid copy numbers in the two strains. As estimated from plasmid-coded fl-lactamase levels (Meacock &, Cohen.

Quantitative analysis of traJ expression CprC lrnd Cpx mutant cells Strain

Genotype cts/min

AE2089 AE2091

epxrl + cprR1

8120

cpxA2 cpxB1

8280

in

tm.lmRN.4 0” of h mRNA

7.6 15%

traJ mRNA was estimated from the radioactivity in the bands marked a in Fig. 6. and h mRNA from the bands enclosed by the brackets. Background radioactivity (in cts/min) for strain AE2089 was 166+29 (mean f standard deviation; see Materials and Methods). and for strain AE2081. 258+39. Total cts/min in &a,/ mRNA mere estimated from the measured radioartivit.v and the volume of each RNA preparation used in the hybridization (6 $/6O ~1 for ,4E2089 and 4 ,I/60 ~1 for AE2091). The amounts of trclJ mRNA relative t,o X mRNA were estimated from the ratios of the measured radioactivities.

trrr GENE

EXPRESSION

IN cpx Ml’TANTS

OF &. coli

27

1980), both strains maintained the same copy number of pLS200 (Table 4). Thus, as we concluded from measurements of truJ mRNA synthesis in irradiated cells, cpxA2 cpxB1 cells are at least as active in ivitiation of transcription at traJ as cells, and possibly somewhat more active. In addition, this cpxA + cpxB1 experiment shows that the traJ translation initiation sequences are also active in cpxA2 cpxB1 cells.

4. Discussion Chromosomal cpxA and cpxB mutations together reduce both DNA donor activity and surface exclusion in cells containing normal F DNA (McEwen & Silverman, 1980a,6). Since the mutants were isolated only on the basis of their inability to elaborate functional F pili, which are not required for surface exclusion, we suggested that at least one effect of the cpx mutations is to reduce transcription of the tray+ 2 operon (McEwen & Silverman, 1980b), which contains genes essential both for the formation of F pili and for surface exclusion (Helmuth & Achtman, 1975). As we have shown here, this is in fact the case. The cpxA2 cpxB1 Hfr strain AE1031 contained only 15% of the amount of tra operon mRNA as otherwise isogenic cpxA+ cpxB+ or cpxA+ cpxB1 cells. Moreover, this effect of the cpx mutations appears to be indirect, and a consequence of the inability of mutant cells to accumulate the TraJ protein. This plasmid gene product has been shown to be required for maximal transcription of the tray -+ 2 operon in viva (Willetts, 1977; Achtman et al., 1980), though its mechanism of action remains unclear (Manning & Achtman, 1979). Since traJ is the only plasmid gene required for tray+ 2 operon expression (Willetts, 1977; Achtman et al., 1980), the inability of cpx double mutants to accumulate the TraJ protein explains the reduction in tray -+ Z mRNA sequences in cpxA2 cpxB1 Hfr cells, but, we cannot totally exclude the possibility that the mutations also act directly on tra operon expression or that they affect one or a few tra gene products in addition to the TraJ prot’ein. We used ultraviolet light-irradiated cells infected with Ap(traJ) transducing bacteriophage to determine whether or not the cpx mutations affect, accumulation of the TraJ protein. Judged by its ability to complement F’ traJ mutants for DNA t’ransfer. this bacteriophage contains an intact, functional traJ gene that is

Effect of the cpxA2 mutation on /3-galactosidase expression from the traJ-1acZ fusion, plasmid pLS200 Strain AE2129/pLS200 AE2132/pLS200 t Mean + standard dwiation 1 Mean k standard deviation

Genot,vpe

cpxrl + epxRl cpxAP cpsB1

8.Galactosidnse fi-Lacwlxw (units/o.l).,,,) 311+4(it 769+ 1931

of 8 samples from 3 different cultures. of 16 samples from 6 different cultures

cN+ 1.47 (ia+ 1.31

28

I,. SAMBVCETTI.

L. EOYANG

AND

I’. hl. SILVERMAN

expressed in a hp(troJ) lysogen (Ippen-Ihler, 1978). Moreover, we have shown that the bacteriophage DNA contains the 1.5 kb SalI-PstI restriction fragment that includes the entire traJ coding sequence, the traJ promoter and an active jinP, gene. Ippen-Ihler (1978) showed that irradiated cells infected with t’his bacteriophage elaborate a 24,000 M, protein, the synthesis of which we have shown to be specifically repressed in irradiated cells containing the $’ plasmid Rl. Furthermore. a 24,000 M, protein is the major product synthesized when the irradiated cells are derived from a /\ lysogen. Finally, experiments employing traJ(am) mutants showed that the electrophoretic mobility of the traJ gene product corresponds to a 24,000 M, protein (Kennedy et al., 1977 ; Manning & Achtman, 1979). Of the other tra genes encoding products expected to have a comparable electrophoretic mobility (traK, trap, traF and traT), only traJ can plausibly be part of the bacteriophage DNA (see Ippen-Ihler, 1978: Willetts & Skurray, ‘1980). Our assignment’ of the 24,000 M, protein as the product of traJ is based on these criteria. Whereas both cpxA+ cpxB1 and cpxA2 cpxB1 cells irradiated and infected with hp(traJ) synthesized h proteins, only the former accumulated the TraJ protein as a 24,000 M, species. Moreover, whereas the TraJ protein accumulated as the major product synthesized in an infected cpxA+ cpxB1 A lysogen, no proteins if the accumulated to a comparable level in a cpzA2 cpxB1 h lysogen. Therefore, complete TraJ protein is synthesized at all in irradiated cpxA2 cpxB1 cells, it is turned over rapidly. In contrast to their effect on accumulation of the TraJ protein. the cpx mutations did not reduce the quantity of traJ mRNA synthesized in irradiated cells. The DNA restriction fragment used to detect traJ mRNA by DNA-RNA hybridization contained all of the traJ coding sequence, except for the first 45 nucleotides (Thompson & Achtman, 1978,1979; R. Thompson. personal communication); this sequence should correspond to about 96 kb of the 1.0 kb fragment. The remaining 0.4 kb consists of whatever regulatory sequences precede traY, the first gene of the tray + Z operon, and part of tray itself. Hence, at least 60% and probably more of the transcribed DNA of the fragment consists of traJ. For this reason. we conclude that most of the RNA synthesized in the mutant’ cells and hybridizing to the fragment is complementary to traJ, especially in view of the fact’ that transcription from the tray promoter should depend on the TraJ prot,ein (Willetts, 1977). which fails to accumulate in the mutant cells. Furthermore, since the DNA restriction fragment used in the DNA-RNA hybridizations lacks the first 45 nucleotides of the traJ coding sequence (R. Thompson. personal communication), transcription in the cpxA2 cpxB1 cells must continue at, least part of the way into the gene. These conclusions were confirmed by experiments utilizing cells containing pLS200. in which the expression of /3-galactosidase originates from the traJ promoter. These experiments showed, in addition, that traJ translation initiation sequences are active in cpxL42 cpxB1 cells. It thus appears that cpxA cpxB double mutants initiate transcription at, the traJ translation at trod promoter. extend transcription into the gene, and initiate mRNA sequences from the traJ-la& fusion mRNA of pLS200. Yet, irradiated mutant cells fail to accumulate the TraJ protein. Because the TraJ protein is

tro GENE EXPRESSION

IN epz MCTANTS OF E. eo2i

29

required for tray + 2 operon transcription (Willetts, 1977), the reduced level of traY-+ Z operon mRNA sequences in mutant Hfr cells is consistent with the absence of TraJ protein. Much evidence indicates that the TraJ protein is localized in the outer membrane of E. coli (Kennedy et al., 1977; Manning & Achtman, 1979; Achtman et al., 1979; Moore et al., 1981a). In view of the other cellular effects of the cpx mutations, we propose that the cpx mutations affect the TraJ protein as a cell envelope component. As we have described elsewhere (McEwen & Silverman, 1982), the cpx mutations alter the protein composition of both the inner and outer membranes. Among the proteins that are absent or deficient in the mutant cell outer membrane are the murein lipoprotein and the OmpF matrix porin (McEwen & Silverman, 1982). Pulse-labeling experiments show that both of these proteins accumulate in mutant cells over short labeling intervals (McEwen & Silverman, unpublished results). On the basis of these observations, we propose that the cpx mutations prevent or retard the translocation of certain envelope proteins, including the Fplasmid TraJ protein, to the envelope site(s) where they function. The failure of mutant cells to accumulate the TraJ protein suggests either that the complete protein is unstable if translocation does not occur efficiently, or that translocation is coupled to translation, and possibly to transcription (Beher & Schnaitman, 1981), such that one or both processes is prematurely terminated and the complete TraJ protein is not synthesized. An important and interesting implication of this hypothesis is that the genetic regulatory role of the TraJ protein is indirect, and requires the protein to be properly localized in the outer membrane or at least properly processed as an envelope component. In any case, the functions of the TraJ protein in genetic regulation and as an envelope component have not been reconciled. Two ot,her chromosomal genes, in addition to cpxA and cpxB, have been examined for their effects on the cellular expression of F-dependent DNA donor activity and surface exclusion. These are sfrA (fez) and sfrB (Beutin & Achtman. 1979). Genetic evidence shows that all four of these genes are distinct (Beutin & Achtman. 1979; McEwen & Silverman, 1980a), and the experiments described above, along with data reported by Beutin et al. (1981), indicate that they act differently to block expression of F tm functions. in transcription of the tray -+ Z Mutat,ions in sfrB cause a graded reduction operon. Genes distal to tray are expressed in sfrB mutants less well than genes proximal to tra,Y. Since the effect of sfrB mutations on DNA transfer functions was suppressed by a rho mutation, Beutin et al. (1981) suggested that the sfrB gene product is an anti-terminator. In any case, they reported no effect of sfrB mutations on accumulation of trd mRNA or on TraJ protein synthesis. In cont’rast, sfrA mutations uniformly reduced transcription of genes within the tmY -+ % operon and reduced trad transcription about, threefold. Beutin et al. (1981) suggested that the .sfrA gene product is a positive control protein required for efficient transcription initiation at traJ and possibly at the tray + Z operon as well. It, thus appears that the cell makes specific contributions to different stages in the expression of DNA donor and related cellular activities. The relation between

30

L. SAMBIJCETTI,

L. EOYANG

ANI)

Y. M. SILVERMAN

these contributions and other cellular processes remains to be elucidated, but a critical factor appears to be the involvement of the cell envelope in conjugation. Nearly all of the tra proteins appear to be envelope components (Manning & Achtman, 1979). Moreover, sfrB mutations are allelic with rfaH mutations, which block lipopolysaccharide core synthesis (Sanderson & Stocker, 1981), and the relation between the cpx mutations and the cell envelope is described above. Both the cpx and sfr mutations thus emphasize conjugation as a cellular property, requiring the integrated activities of both chromosomal and plasmid gene products, rather than a property uniquely dependent on plasmid proteins. We are indebted to Drs Russell Thompson, Karen Ippen-Ihler, Malcolm Casadaban and by Paul Manning for generously providing essential materials. This work was supported grants CA-1330, GM-11301 and HDO-7154 from the National Institutes of Health, and by grant PCM 80-14274 from the National Science Foundation. One author (P.M.S.) is a Career Scientist Awardee of the Irma T. Hirsch1 Trust.

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138, 779-795. Beher. M. & Schnaitman, C. (1981). J. Bacterial. 147. 972-985. Beutin. I,. & Achtman. M. (1979). .I. Bocteriol. 139, 730-737. Beutin. I,., Manning. P.. Achtman, M. & Will&s, N. (1981). .J. Bacterial. 145, 840-844. Capecchi, M. (1966). J. Mol. Biol. 21, 173-193. Casadaban, M., Chou, J. &, Cohen, S. (1980). J. Bncteriol. 143, 971-980. Cohen, S. & Miller, (I. (1969). A’ature (London). 224, 127331277. 144, 149-158. Ferrazza. D. & Levy, S. (1980). .J. Bacterial. Finnegan, D. $ Willetts, N. (1971). Mol. Gn. (I’enet. 111, 256-264. Finnegan, D. & Willetts, N. (1972). Mol. Gen. Genet. 119, 57-66. Finnegan, D. & Willetts, N. (1973). Mol. C:ew. Oevcrt. 127, 307-316. Helmuth, R. & Achtman, M. (1975). ,Vaturr (London). 257, 652-656. Humphreys. G., Willshaw, G. 8: Anderson. E. (1975). B&him. Biophys. Acta. 383, 457-463. Ippen-Ihler, K. (1978). In Microbiology-197X (Schlessinger, D.. Ad.), pp. 1466149, American Society for Microbiology, Washington D.C. Johnson, D. & Willetts, N. (1980). J. Bacterial. 143, 1171-1178. Johnson, I>.. Everett, R. & Willetts. N. (1981). J. Mol. Biol. 153, 187-202. Kaiser, A. I). & Hogness. D. (1960). J. Mol. Biol. 2, 392-415. R., Rahmodorf. I‘. & Herrlich. Y. (1977). Kennedy, N.. Beutin. I,.. Achtman, M.. Skurray. LTaturr (London), 270, 580--585. Kennell, D. (1968). J. Mol. Biol. 34, 855103. Laemmli, UJ. (1970). Nature (London), 227, 680-685. Lederberg, J., Cavelli. I,. & Lederberg. E. (1952). Genetics, 37. 720-730. Lerner. T. & Zinder, N. (1979). J. Bacterial. 137. 106331065. Manning, P. & Achtman, M. (1979). In Bacterial Outw Membran,e (Inouye, M., ed.). pp. 4099 447, Wiley, New York. McDonell, M., Simon, M. & Studier, F. (1977). ,J. Mol. Biol. 110, 119-146. McEwen. ,J. & Silverman, I’. (1980a). J. Bacterial. 144, W-67.

trn GENE

EXPRESSION

IN cps MUTANTS

OF E. coli

31

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Edited

by S. Rrenner