K. Brooks Low

Detection of transcribable recombination products following conjugation in Rec+, RecB− and RecC− strains of Escherichia coli K12

Abstract By crossing Hfr and F− strains of Escherichia coli which carry non-identical (but non-complementing) lacZ− mutations, the detection of β-galactosidase produced from LacZ+ recombination products is possible, beginning 60 minutes after transfer of the Hfr lac− allele. This system was used to show that when the F− cells carry recB−, almost normal amounts of LacZ+ enzyme are formed even though the number of viable recombinants is less than 1% of the Rec+ level. A similar result is found when the F− cells carry recC−. In contrast, LacZ+ enzyme activity is not detected either when RecA− F− cells are used or in a stable RecA− merodiploid carrying the two lacZ− alleles.

Derepression of single-stranded DNA-binding protein genes on plasmids derepressed for conjugation, and complementation of an E. coli ssb- mutation by these genes.

SummaryPlasmid single-stranded DNA-binding protein genes complement the E. coli ssb-1 mutation, and partially restore capacity for DNA synthesis, DNA repair (direct role as well as role in SOS induction) and general recombination. Plasmid mutants derepressed for fertility derived from R1, R64 and R222 show a higher level of complementation compared to the parental repressed plasmids. Derepressed mutants of R222 synthesize more RNA which hybridizes with the ssb gene of the F factor than does the original R222 plasmid. This indicates that plasmid ssb genes are regulated coordinately with fertility genes.

Suppression of a defective alanyl-tRNA synthetase in Escherichia coli: a compensatory mutation to high alanine affinity.

SummaryAmong temperature resistant revertants of a temperature sensitive E. coli alanyl-tRNA synthetase mutant a strain was found which contains an alanyl-tRNA synthetase with an additional mutation in the structural gene of the enzyme. This mutant enzyme has a 9 or 38 fold decreased Km value for alanine compared to that of the thermolabile parental enzyme or to wild-type enzyme, respectively. The alaS gene maps just counterclockwise from recA on the E. coli map (94% cotransduction frequency). It appears that the enzymes increased affinity for alanine is the mechanism of suppressing the temperature sensitive character of the cell. In addition, some coldsensitive temperature resistant revertants were found, where the cold-sensitive character mapped near strA, Presumably they are due to changes in ribosomal proteins as characterized by Ruffler et al. (1974).

Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli: I. Isolation and characterization of a mutant with elevated levels of tRNAGln1

Spontaneous revertants of a temperature-sensitive Escherichia coli strain bearing a thermolabile glutaminyl-tRNA synthetase have been selected for growth at 42°C. Of the 50 revertants tested, all have retained the thermolabile glutaminyl-tRNA synthetase and have elevated levels of tRNA Gln 1 . The genetic locus gln T, responsible for tRNA Gln 1 overproduction has been characterized as a duplication and mapped. The level of other tRNA species or of aminoacyl-tRNA synthetases is unaffected in this mutant. However, an increased amount of glutamine synthetase is found as a consequence of the gln T mutation.

Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli: II. Isolation of regulatory mutants affecting leucyl-tRNA synthetase levels

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase were selected for growth at 42°C. Of these, a large number still contain the thermolabile leucyl-tRNA synthetase. In four such revertants an increased production of the thermolabile enzyme was found. Two distinct genetic loci have been shown to be responsible for the overproduction of leucyl-tRNA synthetase. One locus, leuX, identifies a cis-dominant operator-promoter region adjacent to the structural gene for leucyl-tRNA synthetase, leuS; the other locus, leuY, maps far from leuS and codes for a protein. leuY− is recessive to leuY+.

Zygotic induction of the rac locus can cause cell death in E. coli.

SummaryConjugational transfer of the rac locus of E. coli K-12 into a Rac− recipient strain (i.e. rac+xrac−) results in the killing of a majority of the recipient cells. The efficiency of killing depends somewhat on the plating medium, and can be as high as 98%. The killing is not observed in the rac+xrac+, rac−xrac− or rac−xrac+ configurations. The rac locus, which has the properties of a cryptic prophage, may carry a function analogous to the kil function of bacteriophage lambda, or may instead cause killing by some replication related process.

Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. III. Biochemical characterization of regulatory mutants affecting leucyl-tRNA synthetase levels.

We have investigated the consequences of two mutations, leu X and leu Y, which affect the level of leucyl-tRNA synthetase on the amount of tRNA and other aminoacyl-tRNA synthetases and on the expression of the ilv and leu operons in Escherichia coli . Neither mutation appears to alter the cellular concentrations of other aminoacyl-tRNA synthetases or tRNA isoacceptor families. It was shown that leucyl-tRNA rather than leucyl-tRNA synthetase is the defective control element responsible for derepression of the leu and ilv operon in leu S31 strains. Steady-state levels of leucine, isoleucine, valine, ppGpp, pppGpp and leucyl-tRNA, and the rate of protein synthesis have been measured in leu S + , leu S − , leu S − leu X − , and leu S − leu Y − strains. The levels of magic spot compounds appear to correlate with the extent that protein synthesis is arrested rather than the concentration of limiting leucyl-tRNA.

Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli

SummarySpontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40°C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.