The similarity of rho mutants to rep and ssb mutants in sensitivity to UV light and in recombination deficiency suggested that the function of the Rho protein might be related to that of Rep and Ssb. In support of that idea, we found that rho rep and rho ssb double mutants are either nonviable, or at best only marginally viable. Viability could be restored by suppressor mutations, one of which mapped either in the rho gene or close to its 5'-end. Rho may thus share a role with Rep and Ssb in replication and the structural maintenance of DNA; a multifunctional Rho protein could account for the diversity of the defects seen in rho mutants, some of which appear to have no relation to the defect in transcription termination.

Lethality of the double mutations rho rep and rho ssb in Escherichia coli.

A 0.7-kbp DNA fragment from bacteriophage P4 that contained the polarity suppression (psu) gene was cloned in an expression plasmid. Induction of the plasmid-borne psu gene resulted in the overproduction of a protein having the biological properties of the P4-induced polarity suppressor. In vivo, Psu protein acted in trans to suppress rho-dependent polarity in the late genes of an infecting P2 phage, in plasmid operons, and in the host chromosome. Psu action did not require the presence of other P2 or P4 phage genes. Psu caused efficient readthrough (antitermination) by Escherichia coli RNA polymerase at the rho-dependent terminators tR1 and TIS2, individually and in tandem, but did not affect termination at rho-independent sites. Neither the conserved antitermination sequence boxA nor any unique promoter or utilization sequence was required for Psu activity.

The Psu protein of bacteriophage P4 is an antitermination factor for rho-dependent transcription termination.

The plasmid pJSF6, a derivative of pBR327, could be maintained at 30° C in strains of Escherichia coli containing the strong rho mutation, rho-15. Plasmids extracted from rho-15 cells were always less negatively supercoiled than plasmids from rho + cells. Transduction experiments designed to separate the rho gene from possible extragenic suppressors showed that the rho allele consistently determined the degree of plasmid superhelicity. Comparison of the superhelicity of plasmids extracted from the rho-15 and from a gyrB mutant showed that at 30° C the negative supercoiling was reduced by the amounts ΔWrho=4.0±0.3 and ΔWgyr=6.0±0.3 turns; the effect of the rho-15 mutation on supercoiling was thus comparable to that of the gyrB mutation. A similar effect of the rho-15 mutation on the superhelicity of pBR329 was observed. The observation that the Rho protein has a role in determining DNA superhelicity (though not necessarily a direct role) provides a new point of view for studying the pleiotropic properties of rho mutants.

Reduced superhelicity of plasmid DNA produced by the rho-15 mutation in Escherichia coli.

The highly defective rho-15 mutant of Escherichia coli produces plasmid DNA that is 22% less negatively supercoiled than DNA from an isogenic wild-type strain (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We extended our measurements of plasmid superhelicity to additional rho mutants and to strains containing mutations that suppress rho transcription termination defects; the suppressor mutations were in the rpoB and the rho genes. The superhelicity of plasmid DNA was reduced by 11 and 10%, respectively, in the rho-702 and rho-201 mutants, both of which are less defective in Rho-mediated transcription termination than rho-15. Plasmid superhelicity was restored in all the suppressed rho mutants; in one rpoB mutant, plasmid DNA was even more negatively supercoiled than in rpoB+ cells, whether in a rho+ or rho mutant background. Suppression of rho mutants enabled them to maintain plasmids that could not be maintained in the mutants in the absence of the suppressor mutations. The results indicate that in addition to DNA gyrase, topoisomerase I, and Rho, RNA polymerase is also a determinant of DNA superhelicity, and its effect is modified by the Rho protein. We propose that Rho may increase the degree of DNA unwinding by the transcription complex, possibly at transcription termination sites.

Regulation of DNA superhelicity by rpoB mutations that suppress defective Rho-mediated transcription termination in Escherichia coli.

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-111 mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-111-bearing strain is the consequence of a single mutation. The rho-111 mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-111 mutation did not alter in vivo expression of either the tryptophan or histidine operons.

Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity.

Reduced recovery of plasmid transconjugants in crosses with Escherichia coli rho mutant recipients

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Reduced recovery of plasmid transconjugants in crosses with Escherichia coli rho mutant recipients