Mutations Affecting Amino Sugar Metabolism in Escherichia coli K-12

The genetic loci determining N-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase have been located at min 16 on the Escherichia coli K-12 genetic map.     

Insertion mutations in the dam gene of Escherichia coli K-12

The dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable.     

Mutations Affecting the Dissimilation of Mannitol by Escherichia coli K-12

Mutants of Escherichia coli K-12 defective in the mannitol-specific enzyme II complex of the phosphoenolpyruvate phosphotransferase system (PTS) or lacking mannitol-1-phosphate dehydrogenase have been isolated. These mutants fail only to grow on mannitol. Growth of the dehydrogenase-negative mutant on casein hydrolysate can be abruptly inhibited by exposure to mannitol. A mutant with constitutive expression of both of these enzymes has also been isolated. All three mutations are clustered in a region represented at min 71 of the Taylor map. In a mutant with less than 5% of the activity of enzyme I of the PTS, both the enzyme II complex and the dehydrogenase remain inducible by mannitol. In the mutant defective in the enzyme II complex, mannitol is able to induce the dehydrogenase. Thus, mannitol, rather than its phosphorylated product, seems to be the inducer.     

Rapid Mapping of Conditional and Auxotrophic Mutations in Escherichia coli K-12

The approximate genetic map locations of auxotrophic and conditional lethal mutations of Escherichia coli can be rapidly determined with replica plating techniques. A set of patches of 15 streptomycin-sensitive (Str^S) Hfr strains with points of origin distributed around the map is replica plated onto a recombinant-selective plate with a lawn of Str^R cells which carry an unmapped mutation. The map interval defined by the Hfr points of origin which are closest to the mutant locus is seen by the presence or absence of heavy patches of recombinants produced by transfer of early wild-type genes from the Hfrs. An alternative method is to replicate patches of different mutant strains (100 per plate) onto Hfr lawns; in this case more than 1,000 different mutants can be mapped in a single experiment in a few days. In this way, many types of mutations with similar phenotypes can be grouped as to approximate location on the genetic map. For ordering mutations within groups, the same replica plating methods can be used to cross F-prime derivatives of mutants with other mutants of the same group. Relative merits of these and other mapping methods of E. coli are discussed.     

Valine-Resistant Escherichia coli K-12 Strains with Mutations in the ilvB Operon

Escherichia coli K-12 mutants resistant to growth inhibition by valine were isolated. These strains contained mutations in the ilvB operon effecting either the regulation of acetohydroxy acid synthase I or the sensitivity of the enzyme to end product inhibition by valine.     

malB Region in Escherichia coli K-12: Characterization of New Mutations

Phenotypic characterization and mapping of more than 50 Mal(-) mutations located in the malB region lead one to divide the site for Mal(-)lambdas mutations (formerly called gene malB) in that region, into two adjacent genetic segments malJ and malK. malJ and malK are both involved in maltose permeation. It is suggested that (i) malK and lamB, the only known gene specifically involved in phage lambda adsorption (20), constitute an operon of polarity malK lamB. (ii) malJ and malK correspond to two different genes, and (iii) a promoter for the malK lamB operon is located between malJ and malK. Since lambda receptors and maltose permease are inducible by maltose and absent in malT mutants, it is likely that the expression of the malK lamB operon is controlled by the product of gene malT, the positive regulatory gene of the maltose system.     

Complementation Between Different Mutations in the ilvA Gene of Escherichia coli K-12

An ilvA mutation carried by a ?80i(lambda)dilv transducing phage complemented some ilvA mutations and did not complement others. Complementation was accompanied by appearance of threonine deaminase activity in vivo. These results divided the ilvA mutations into two sets which formerly appeared to define two cistrons.     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Insertion Sequence-Related Genetic Variation in Resting Escherichia Coli K-12

Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.     

Mutations affecting the formation of acetohydroxy acid synthase II in Escherichia coli K-12.

Summary Genetic mapping experiments have established that two recently isolated valine-resistant mutants of the K-12 strain of Escherichia coli have lesions lying between ilvE and rbs. These lesions allowed expression of the ilvG gene, specifying the valine-insensitive acetohydroxy acid synthase (synthase II) and an increased expression of the ilvEDA operon. In this respect, they resembled an earlier described ilvO lesion that was reported to lie between ilvA and ilvC. All three lesions were cis-dominant in cis-trans tests. Reexamination of the earlier studied ilvO lesion revealed that it, too, lies between ilvE and rbs. Valine-sensitive derivatives with lesions presumed to be in ilvG were selected from each of the valine-resistant strains. In two of the valine-resistant strains, the ilvG mutations were on the rbs side of ilvO, indicating a gene order rbs-ilvG-ilvO-ilvE-ilvD-ilvA-ilvC. In one of the recently isolated valine-resistant stocks, however, the apparent ilvG mutation was found to be between ilvE and the aline resistance marker. This finding suggests that either ilvO and ilvG mutations are interspersed or there is another locus, ilvR, that behaves phenotypically like ilvO and which lies between ilvG and rbs.     

Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor with an Mr of 78,000. It is required for the binding and uptake of ferrichrome and is the receptor for bacteriophages T5, T1, phi 80, and UC-1 as well as for colicin M. The fhuA gene was cloned into pBR322, and the recombinant plasmid pGC01 was mutagenized by the insertion of 6-base-pair TAB (two amino acid Barany) linkers into CfoI and HpaII restriction sites distributed throughout the coding region. A library of 18 TAB linker insertions in fhuA was generated; 8 of the mutations were at CfoI sites and 10 were at HpaII sites. All mutations inserted a hexamer that encoded a unique SacI site. A large deletion in fhuA was also isolated by TAB linker mutagenesis. Except for the deletion mutant, all of the linker insertion mutant FhuA proteins were found in the outer membrane in amounts similar to those found in the wild type. Five of the linker insertion mutants were susceptible to cleavage by endogenous proteolytic activity: a second FhuA-related band that migrated at approximately 72 kilodaltons could be detected on Coomassie blue-stained gels and on Western blots (immunoblots) by using a carboxy terminus-specific anti-peptide antibody. Receptor functions were measured with the mutated genes present in a single copy on the chromosome. Some of the receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected. Several mutants were altered in their sensitivities to the lethal agents. TAB linker insertions after amino acids 69 and 128 abolished all receptor functions. Phage T5 id not bind to these mutant FhuA proteins in detergent extracts. The deletion mutant was also defective in all FhuA functions. Sensitivity to the lethal agents of cellsl that expressed mutant FhuAs with insertions after amino acids 59 and 135 was reduced by several orders of magnitude. Insertion at other selected sites decreased some or all receptor functions only slightly. An insertion after amino acid 321 selectively eliminated ferrichrome growth promotion. Finally, a strain carrying a mutant fhuA gene on the chromosome in which the linker insertion occurred after amino acid 82 showed a tonB phenotype. These subtle perturbations that were introduced into the FhuA protein resulted in changes in its stability and in the binding and uptake of its cognate ligands.     

Malate Dehydrogenase Mutants in Escherichia coli K-12

Mutants devoid of malate dehydrogenase activity have been isolated in Escherichia coli K-12. They do not possess detectable malate dehydrogenase when grown aerobically or anaerobically on glucose as sole carbon source. All mutants revert spontaneously; a few partial revertants have been found with a malate dehydrogenase exhibiting altered electrophoretic mobility. Therefore, only one such enzyme appears to exist in the strains examined. No evidence could be obtained for the presence of a malate dehydrogenase not linked to nicotinamide adenine dinucleotide. Mutants deficient in both malate dehydrogenase and phosphoenol pyruvate carboxylase activities will grow anaerobically on minimal glucose plus succinate medium; also, malate dehydrogenase mutants do not require succinate for anaerobic growth on glucose. The anaerobic pathway oxaloacetate to succinate or succinate to aspartate appears to be accomplished by aspartase. Malate dehydrogenase is coded for by a locus somewhere relatively near the histidine operon, i.e., a different chromosomal location than that known for other citric acid cycle enzymes.