PLEIOTROPIC ts 15 MUTATION OF E . COLI IS AN IS 1 INSERTION IN T H E rho STRUCTURAL GENE

Rho protein regulates transcription termination in E. coli. Some of the temperature-sensitive mutants defective in Rho protein, e.g., ts15, show remarkable pleiotropic phenotypes. The ts mutations map between the ilv and cya loci on the E. coli chromosome. We have cloned the gene that restores the wild-type phenotypes of these mutants. Genetic and biochemical characterizations have shown that the cloned DNA segment carries the structural gene for the Rho polypeptide. Analysis of the rhotsl5 mutation has revealed the presence of an IS1 insertion in the carboxy terminal segment of the rho cistron, thereby truncating the 52-kilodalton (kd) Rho polypeptide to a 50-kd size and also making it thermolabile. This provides an example of how an IS1 insertion mutation can cause a TS phenotype. We have also shown that the multiple phenotypes of the mutant cell, including the temperature sensitivity, are caused by a single mutation (rhotsl5::ISI) in the rho structural gene. How a rho structural gene mutation may cause such pleiotropy is discussed. HO protein of Escherichia coli catalyzes transcription termination at specific R sites on bacterial and bacteriophage DNA templates in vitro (ROBERTS 1969; DE CROMBRUCCHE et al. 1973). E. coli mutants in which transcription continues through some of these sites in viuo have been isolated and shown to produce defective Rho protein (RICHARDSON, GRIMLEY and LOWERY 1975; DAS, COURT and ADHYA 1976; KORN and YANOFSKY 1976; GUARENTE, MITCHELL and BECKWITH 1977; INOKO, SHICESADA and IMAI 1977). The mutations have been shown to be cotransducible, by generalized transducing phage P1, with both i lv and cya loci on the E. coli chromosome at frequencies of 80 and 50%, respectively (MORSE and GUERTIN 1972; S. A. and S . GARGES, unpublished results). The relative gene order is ilv-rho-cya (DAs, COURT and ADHYA 1976; UZAN et al. 1981). Highly defective rho mutations impart a remarkably complex set of phenotypes upon the cell (DAs, COURT and ADHYA 1978). Some of these properties of the rho mutants are described in RESULTS. The seemingly ’ Current address: Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R. e Cattedra di Microbiologia, Instituto di Patologia generale, I1 Facolta di Medicina e Chirurgia dell’universita di Napoli, 80131 Napoli, Ita1 ‘Current address: Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032. To whom reprint requests should be addressed. Genetics 105: 265-280 October, 1983. 266 E. CULLETTA, A. DAS AND S. ADHYA unrelated phenotypes of the rho mutants indicate the significance of the Rho protein and its gene in cell physiology and growth control. To examine the relationship between the rho gene structure and function, we have cloned and characterized the E. coli rho gene and its pleiotropic ts15 mutation. We show that an IS1 insertion in the rho structure gene is responsible for its temperature sensitivity and other pleiotropic defects. Recently, BROWN et al. (1982) have also characterized the E. coEi rho gene and determined its direction of transcription, and PINKHAM and PLATT (1983) have determined its complete nucleotide sequence. MATERIALS AND METHODS Phage and bacterial strains: X vector Charon 25 was constructed by F. BLATTNER and D. DANIELS (personal communication) and is shown in Figure 1 A. Other bacteriophage strains are described in RESULTS. P l c l r 1 0 0 : : T n P was used for phage P1 transductions (ROSNER 1972). The library of E. coli genomic fragments in Charon 25 was a gift from the Prokaryotic Genetics Laboratory of the University of Wisconsin (courtesy of D. DANIELS and F. BLATTNER). The library contains restriction endonuclease Hind111 fragments of E. coli DNA and Charon 25 DNA and was prepared by in vitro packaging. The immX region was crossed into Charon 25 phages (which are imm2I) as follows: the Ximm21 Charon 25 phage was grown lytically in SA431, which contains a Xc1857 prophage with a deletion of phage genes Q through the righthand terminus (ADHYA, CLEARY and CAMPBELL 1968). Deletion of the entire b region in SA431 ensures retention of the cloned DNA in the recombinants. Phages with the cI857 marker were then selected by plating the phage lysate on strain DC7, which is isogenic with SA431, but carries imm21. E. coli K-12 strains are described in Table 1. E. coli strain 159 (hind-), a UV-sensitive strain carrying the noninducible phage Xindwas used for protein-labeling experiments (YAMOMOTO and NOMURA 1979). Clonitzg of rho gene: Many rho mutations impart a heat-sensitive growth defect on the cell. Two such strains, AD1919 ( rhots l12) and AD1600 ( rhots l5) (DAs, MERRIL and ADHYA 1978), if plated on LB agar at 42", revert to temperature resistance (TR) at frequencies of and 5 X lo-*, respectively. To isolate the rho+ gene, r h o t s l l 2 and rhotsl5 cells were infected at a multiplicity of infection (moi) of lo-' with the phage vector Charon 25 (Figure 1A) population carrying random E. coli HzndIII fragments and plated on LB agar plates at 42". TR transductants were obtained at A t B J UIN R s + A. Charon25 10 20 lac, an30 C+ 45.4 _-40 I + + I [ r i E R H R B + t B. &Rho& ---Wm L ~ C , 30 an E R H R B H E R I _ _ c. ARho lOC--W/j/////b t 1 , t t D. ARho15 ---~/j<//// + 20 Lac5 30 att 40 20 Lac, 30 att 40 B R H R B H B R _-~~~~ ~~~~ t i + + , FIGURE 1.-Restriction maps of phage Charon 25 (A), Xrho& (B), XrholOO (C) and Xrhol5 (D). Restriction sites for BamHl (B), EroRI (R) and Hind111 (H) are shown by vertical arrows. Filled boxes represent the HindIII-replaceable region in the vector and the cloned DNA in the others. Open box (itunz21) and broken box ( n i f z deletion) are shown only in the vector. Boxes with crosshatch shows the lor DNA. The lines are not drawn to exact scale. rhol5::ISZ IS PLEIOTROPIC