A novel rho promoter::Tn10 mutation suppresses and ftsQ1(Ts) missense mutation in an essential Escherichia coli cell division gene by a mechanism not involving polarity suppression.

An extragenic suppressor of the Escherichia coli cell division gene ftsQ1(Ts) was isolated. The suppressor is a Tn10 insertion into the -35 promoter consensus sequence of the rho gene, designated rho promoter::Tn10. The ftsQ1(Ts) mutation was also suppressed by the rho-4 mutant allele. The rho promoter::Tn10 strain does not exhibit rho mutant polarity suppressor phenotypes. In addition, overexpression of the ftsQ1(Ts) mutation does not reverse temperature sensitivity. Furthermore, DNA sequence analysis of the ftsQ1(Ts) allele revealed that the salt-remediable, temperature-sensitive phenotype arose from a single missense mutation. The most striking phenotype of the rho promoter::Tn10 mutant strain is an increase in the level of negative supercoiling. On the basis of these observations, we conclude that the ftsQ1(Ts) mutation may be suppressed by a change in supercoiling.     

Suppression of Amber and Ochre Mutants in Salmonella typhimurium by a Mutant F′-1-gal Factor Carrying an Ochre Suppressor Gene

A Salmonella typhimurium strain was given the amber mutation hisC527 by transduction, made galactose-negative by mutation, then infected with the F'-1-gal factor. Of 107 spontaneous and mutagen-induced histidine-independent mutants tested, 3 proved to result from suppressor mutations within the F' factor. The mutant F' factors, when transferred to S. typhimurium and E. coli auxotrophs, suppressed amber and ochre but not UGA or missense mutants, and are inferred to carry ochre suppressor genes. Attempts to isolate an F' amber suppressor mutant were unsuccessful. A suppressor F' factor was transferred to 14 rough mutants which had been isolated from LT2 hisC527 (amber) by selection for resistance to phage P22.c2. One rough mutant was partly suppressed, as shown by its acquisition of O agglutinability and by alterations in its phage resistance pattern. Phage P22h grown on the suppressed mutant contransduced its rf. gene with cysE(+) and with pyrE(+), and the affected locus is inferred to be rfaL. Both the original and the mutant F' factors conferred resistance to the rough-specific phage Br60, which is therefore "female-specific."     

Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F.

Previously we have described a mutant Hfr strain in which incompatibility between the integrated F factor and an autonomous F-prime (F') factor was abolished. The mutation (inc) was located in the integrated F factor. F-prime factors isolated from the mutant Hfr strain have the same incompatibility behavior as those isolated from normal Hfr strains. Reintegration of these F' factors into the chromosome restores the Inc- phenotype characteristic of the mutant Hfr. The inc mutation thus affects incompatibility between integrated F and autonomous F(Fi-Fa incompatibility) but not incompatibility between two autonomous F factors (Fa-Fa incompatibility). The implications of this finding for the mechanism of plasmid incompatibility are discussed.     

A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.     

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.     

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.     

Second-site rho mutation: Genetic linkage and polyC-dependent ATPase

Summary Rho has been purified to homogeneity from Escherichia coli double mutant rho-115 sur-38 cells, and from rho ⁺⁺ and rho-115 cells. The sur-38 mutation suppresses the original rho-115 phenotype. We observe that the polyC-dependent ATPases of these three rho preparations have the same specific activities. However, the ATPase of rho from the double rho-115 sur-38 mutant is extremely heat labile, while that from rho-115 shows a heat lability intermediate between the wild type and the double mutant.Transduction analysis suggests that sur-38 is closely linked to rho-115 in the order ilv-sur-38-rho-115-metE. These data are consistent with the model that the sur-38 mutation affects the structural gene for rho.Contact for offprints     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

Expression of the cloned uvrB gene of Escherichia coli: dependency on nonsense suppressors.

Recombinant plasmid pNP5, consisting of plasmid pMB9 on which the uvrB gene is cloned, fully complements for the defects due to chromosomal uvrB mutations in the presence of the amber suppressor sup-6 or supF. Correndonuclease II activity was also completely restored in in UvrB strains containing both plasmid pNP5 and amber suppressor sup-6, as compared with the parental UvrB+ strain. It is shown that the amber mutation which interferes with the expression of the cloned uvrB gene is located outside this gene. Apparently, the amber mutation exerts a polar effect on uvrB expression that is relieved by sup-6 or supF. Introduction of a rho mutation into suppressor-free UvrB strains, harboring pNP5, did not relieve the polarity caused by the amber mutation.     

Mutagenesis in cloned yeast genes. The effect of mutation rad2 on the frequency of gene mutation in plasmid and chromosome

The influence of rad2 mutation blocking incision of pyrimidine dimers on frequency of UV-light and 6-hydroxylaminopurine (6-GAP)-induced adenine-independent revertants was studied in the strains of Saccharomyces cerevisiae containing the same mutant allele of gene ADE2 in episomic plasmid and in chromosome. It was shown that the strains carrying the ade2 mutation in chromosome and in plasmid did not differ in sensitivity to lethal action of UV-light and 6-GAP. However, in the plasmid rad2 strain reversions were induced by UV-light more frequently (approximately 100 times), as compared to the chromosome strain. We observed no significant differences between reversion frequencies in plasmid and chromosome RAD strains. The tendency to enhanced 6-GAP-induced mutagenesis, less sharply expressed, was observed in the chromosome rad2 strain, as compared to the plasmid one. However, the plasmid RAD strain was characteristic of higher reversion frequency induced by 6-GAP, as compared to the chromosome strain. The possible mechanisms of these phenomena are discussed.