Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli.

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.     

Molecular characterization of ilvC specialized transducing phages of Escherichia coli K-12

A series of lambda defective ilvC specialized transducing phage has been isolated which carry regions of isoleucine and valine structural and regulatory genes derived from the ilv cluster at minute 83 on the linkage map of the chromosome of Escherichia coli K-12. The ilv genes carried by these phages and their order have been determined by transduction of auxotrophs. The ilvC+ lysogen of an ilvC- strain gave rise, after heat induction of the lysogen, to transducing particles which carried the wild-type allele of the cya-marker. Further experiments have shown that the lambda defective ilvC phages were able to cotransduce a rho-15ts mutation as well as a rep-5 mutation. Hence, the order of the clockwise excision of the ilv cluster was found to be ilvC-rho-rep-cya. Enzyme levels in strains carrying the lambda defective ilvC phages indicated the the ilvC gene was not altered by the insertion of lambda into the ilv cluster. The isolation and digestion of lambda defective ilvC DNA by EcoRI and HindIII restriction endonucleases demonstrated that the specialized transducing phages carried part of the genome from the E. coli K-12 chromosome.     

Isolation and characterization of specialized transducing lambda phages carrying ribosomal protein genes of Escherichia coli

Summary Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54°C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.     

Isolation of conditional lethal mutator mutants of Escherichia coli by localized mutagenesis.

By using localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, we isolated 39 temperature-sensitive growth mutants that exhibited high mutability when the bacteria were grown at the permissive temperature. Two of the mutations, dnaQ186 and dnaQ231, were shown to be new alleles of the dnaQ gene by genetic mapping and complementation tests with the dnaQ49 mutation previously isolated. They shared common properties with the dnaQ49 strain, but their mutator activity was not temperature dependent. The dnaQ mutants exhibited increased sensitivity to inhibitors of DNA gyrase and to DNA intercalating and alkylating agents.     

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Summary Specialized transducing phages asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli, The clockwise order of genes on the chromosome is found to be: bglB, (pst, glmS), (uncA, uncB), criC, asn, trkD, rbs, rrnC, ilv.Abbreviations CCC covalently closed circular - MD 10⁶ Daltons; mw, molecular weight - R. restriction endonuclease - LFT, HFT low (high) frequency transducing - a.p.i. average phage input - m.o.i. multiplicity of infection     

Isolation and characterization of lambda transducing phages for the E. coli genes ksgA and pdxA

Summary Lambda phages carrying the Escherichia coli genes ksgA and pdxA were isolated from secondary site lysogens in araB. 1) The phage genomes were characterized by genetic complementation tests, restriction endonuclease digestion and electron microscopy. 2) A 6.3 kilobasepair (kb) EcoRI restriction fragment carrying both ksgA and pdxA was cloned in a lambda vector; this fragment has proven useful in further characterization of the ksgA gene (Andrésson and Davies, 1980a, b). The ksgA and pdxA genes are about 14 and 12–13 kb, respectively, counterclockwise of the arabinose operon and 1.5 and 2.5–3.5 kb clockwise of folA.     

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.     

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12.

Summary A strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.     

Conditional lethal amber mutations in essential Escherichia coli genes

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.     

Isolation of a lambda transducing bacteriophage carrying the relA gene of Escherichia coli.

In Escherichia coli the relA and pyrG loci are 99% cotransducible. On the basis of this knowledge, we have isolated lambdacI857S7dpyrG transducing bacteriophages carrying both the pyrG and relA genes. Single lysogens of this bacteriophage show basal levels of ppGpp that are 10-fold higher than normal. Stringent factor is present among the gene products synthesized by lambdadpyrG relA after infection of ultraviolet-killed cells, as analyzed by polyacrylamide gel electrophoresis. The intracellular content of stringent factor, as determined by enzymatic activity, rises 20-fold after induction of a single lysogen of lambdadpyrG relA. As measured by two-dimensional gel electrophoresis, the amount of stringent factor in an exponentially growing strain carrying a pyrG relA plasmid is at least 10-fold greater than in a normal strain. These data constitute strong evidence that stringent factor is the relA gene product.