Gene, 122(1992)1-7 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00 GENE 06826

Involvement of the Escherichia coli RNA polymerase a subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins (Phage development;

lysogenization;

Grzegorz Wqgrz yn *, Robert qf Biochemistry.University

Department Received

by A.J. Podhajska:

rpoA mutants; complementation;

plaque morphology;

E. Glass

and Mark

positive control)

S. Thomas

of Nottingham Medical School, Queen’s Medical Centre. Nottingham NG7 ZUH, UK

12 June 1992; Revised/Accepted:

10 August/l

September

1992; Received

at publishers:

3 September

1992

SUMMARY

Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage 1, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cl in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage ;1 transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cl gene from the CI-activated promoter, pM in the rpoA341 background.

INTRODUCTION

Upon infection of E. coli, bacteriophage A can enter into one of two alternative pathways: a lytic path leading to the

Correspondence to: Dr. M.S. Thomas, versity of Nottingham

Department

of Biochemistry,

Uni-

Medical School, Queen’s Medical Centre, Notting-

ham NG7 2UH, UK. Tel. (44-602)421421 *Permanent Gdansk,

ext. 4473 1; Fax (44-602)422225

address:

Kladki

Abbreviations:

Department

aa, amino acid(s);

chloramphenicol;

plaque-forming

comprising

resistant/resistance; lysogen).

Ap, ampicillin; receptor

University

bp, base pair(s);

protein;

of

Cm,

eol, efficiency

from closed to open complex;

nt, nucleotide(s);

units;

Biology,

Tel. (48-8)310072.

of

isopropyl-p-D-thiogalactopyranoside; kb, kiloequilibrium constant for closed complex forma-

tion; k, rate of isomerisation plicity of infection;

wt, wild type;

Poland.

CRP, cyclic AMP

lysogenization; IPTG, base(s) or 1000 bp; K,,

promoter

of Molecular

24, 80-822, Gdatisk,

plac, promoter elements

moi, multi-

ori, origin of DNA replication; for the lac operon;

from the lacUV5

ts, thermosensitive;

[ 1,denotes plasmid-carrier

and trp promoters;

tsp, transcription

pfu,

ptac, hybrid R,

start point(s);

state; ( ), denotes prophage

(in

production of progeny virus and lysis of the host cell, or a lysogenic path leading to integration of the viral DNA into the host genome (Herskowitz and Hagen, 1980; Echols, 1986). Lysogenic development requires the action of two phage-encoded transcriptional activator proteins: CI (which also functions as a major 1 repressor) and CII. CII is required for commitment to the lysogenic mode of development by stimulating transcription from the otherwise quiescent phage promoters pE, p, and P,~. Promoters pE and pI, respectively, direct the synthesis of CI (which represses the lytic promoters pL and pR) and integrase (an enzyme required for integration of the phage DNA into the host chromosome), while transcripts arising from paq probably serve to antagonize expression of the Q gene (Hoopes and McClure, 1985; Ho and Rosenberg, 1985). The activator function of CI, on the other hand, is necessary after commitment to lysogenic development, whereupon it stimulates its own transcription from another phage promoter, pM, and thereby maintains a constant level of repressor for stabilization of the lysogenic state (Gussin et al., 1983).

2 The mechanism

of transcriptional

and CII-dependent promoters tensive investigation (Maurer 1980; Shimatake

activation

at these CI-

has been the subject of exet al., 1980; Meyer et al.,

and Rosenberg,

1981; Ho et al., 1986).

Their binding sites and the specific steps they modulate have been determined for each of the promoters (Johnson et al., 1981; Hawley and McClure, 1982; Shih and Gussin, 1983a,b; 1984; Ho et al., 1983; Ho and Rosenberg, 1985;

TABLE

I

Lysogenization

of rpoA341 mutant

by phage 1.

Strain*

eel’ - IPTG

+ IPTG

0.67

NT

WAMl06[pJMHl/pN01567]

0.48

NT

McClure and Hoopes, 1987). Based on these studies, it has been suggested that both activators exert their effects

WAMl06[pJMHlipJMH16]

0.73

0.61

through direct contact with RNA polymerase (Johnson et al., 1981; Guarente et al., 1982; Bushman et al., 1989; Ho et al., 1983). Indeed, the specific aa residues thought to

WAMIOS[pJMHl/pNO1567]

be involved in such contacts have been identified on CI (Guarente et al., 1982; Hochschild et al., 1983; Bushman et al., 1989). One prediction from such a model is the possible isolation of mutant derivatives of RNA polymerase which exhibit an altered response to either (or both) activator(s). Several RNA polymerase mutants have been described which are specifically impaired in their ability to transcribe certain genes subject to positive control (Sunshine and Sauer, 1975; Fujiki et al., 1976; Giffard and Booth, 1988; Slauch et al., 1991; Lombard0 et al., 1991). Furthermore, most of them affect the a subunit, leading to the suggestion that a may provide the contact site for at least some activator proteins (Venezia and Krakow, 1990; Igarashi and Ishihama, 1991; Thomas and Glass, 1991; Igarashi et al., 1991). One well-characterized LXmutant, rpoA341, confers on the host RNA polymerase a reduced ability to transcribe the mel, ara, and cys operons (Giffard and Booth, 1988) and presumably arises from a defective interaction between the respective positive regulators for these genes and RNA polymerase. Here we show that the rpoA341 mutation prevents lysogenization of E. coli by bacteriophage 1 and propose that this is the result of impaired transcription from at least two CII-dependent phage promoters. Evidence is also presented for decreased activity of the CI-stimulated promoter pM in the rpoA341 background.

RESULTS

AND DISCUSSION

(a) Mutation vpoA341 prevents lysogenization by phage A We investigated the efficiency of lysogenization of the rpoA341 mutant WAM105 under conditions favouring lysogeny, i.e., infecting starved cells at high moi. However, we failed to detect any lysogens among several hundred survivors tested whereas we routinely obtained 60-70% lysogens after infection of the isogenic rpoA + strain, WAM106 (Table I). In order to confirm that the observed effect is specific for the rpoA341 allele, we introduced into this mutant the r expression plasmid pJMH16 (Thomas and Glass, 1991).

WAMlOh(rpoA

+)

WAMlOS(rpoA

341)