Mutation affecting normal colony formation inEscherichia coli K-12

The effect of a mutation in a gene located near thepro marker and determining the normal colony formation and the cell density inEscherichia coli has been analyzed. Thencf mutation has no effect on the permeability of the cell to actinomycin D. On the other hand, a higher level of ATP and a defect in the accumulation of the reserve material in the mutant cell were detected.     

Membrane mutation affecting energy-linked functions inEscherichia coli K 12

A small-colony forming variant ofEscherichia coli with a mutation in thenof gene was analysed. The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicina indicated a membrane mutation. The effect of this mutation on some membrane-bound processes, the activity of Mg²⁺-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described. This mutation does not exert any effect on the electron transport system.     

Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.     

An unusual genetic link between vitamin B6 biosynthesis and tRNA pseudouridine modification in Escherichia coli K-12

We characterized several unusual phenotypes caused by stable insertion mutations in a gene that is located upstream in the same operon from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. Mutants containing kanamycin resistance (Kmr) cassettes in this upstream gene, which we temporarily designated usg-2, failed to grow on minimal plus glucose medium at 37 and 42 degrees C. However, usg-2::Kmr mutants did form oddly translucent, mucoid colonies at 30 degrees C or below. Microscopic examination revealed that cells from these translucent colonies were spherical and seemed to divide equatorially. Addition of D-alanine restored the shape of the mutant cells to rods and allowed the mutants to grow slowly at 37 degrees C and above. By contrast, addition of the common L-amino acids prevented growth of the usg-2::Kmr mutants, even at 30 degrees C. Furthermore, prolonged incubation of usg-2::Kmr mutants at 37 and 42 degrees C led to the appearance of several classes of temperature-resistant pseudorevertants. Other compounds also supported growth of usg-2::Kmr mutants at 37 and 42 degrees C, including glycolaldehyde and the B6 vitamers pyridoxine and pyridoxal. This observation suggested that usg-2 was pdxB, which had been mapped near hisT. Complementation experiments confirmed that usg-2 is indeed pdxB, and inspection of the pyridoxine biosynthetic pathway suggests explanations for the unusual phenotypes of pdxB::Kmr mutants. Finally, Southern hybridization experiments showed that pdxB and hisT are closely associated in several enterobacterial species. We consider reasons for grouping pdxB and hisT together in the same complex operon and speculate that these two genes play roles in the global regulation of amino acid metabolism.     

Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12

Summary We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd. X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.     

Alternative gene for biotin sulfoxide reduction inEscherichia coli K-12

A gene at 42 min on theEscherichia coli chromosome, identified as the locus of pseudoreversion of knockout mutations in the biotin sulfoxide reductase gene,bisC, has 64% base sequence identity withbisC. This makes it a member of a multigene family of molybdopterin enzymes that includes genes for anaerobic reduction of trimethylamine oxide (torA) and dimethylsulfoxide (dmsA). Disruption of this gene eliminates the background activity of biotin sulfoxide reduction observed inbisC mutants. Sequence comparison of the new gene (bisZ) withbisC indicates that certaints mutants ofbisC arise by gene conversion between the two loci.     

Control of vitamin B 6 biosynthesis in Escherichia coli

Pyridoxineless mutants of Escherichia coli B which specifically require pyridoxal or pyridoxamine for growth can be divided into classes according to their growth responses in enriched media. Members of the slowest growing class synthesize vitamin B(6) at the fastest rates when starved for pyridoxal in glycerol minimal medium. After 80 min of synthesis at 4 x 10(-10) moles of vitamin B(6) per mg of cells per hr, the rate increases four- to fivefold and continues at the new rate for several hours. The shift to the new rate is prevented by chloramphenicol, thus suggesting that a derepression mechanism exists to control vitamin B(6) synthesis in addition to the previously discovered feedback control.     

Identification of a mutation affecting an alanine-alpha-ketoisovalerate transaminase activity in Escherichia coli K-12.

Summary A mutation affecting alanine--ketoisovalerate transaminase activity has been shown to be cotransducibe with the ilv gene cluster. The transaminase deficiency results in conditional isoleucine auxotrophy in the presence of alanine.     

Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12

DNA from the marine bacteriumAlteromonas haloplanktis 214 was partially digested withSau 3A and inserted into theBam HI site of the cloning vector pBR322. The ligation mixture was used to transformEscherichia coli HB101. The gene bank plasmid preparation obtained was used to transformEscherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to containA. haloplanktis DNA and to be capable of complementing the appropriate mutation inE. coli EO2717. Restriction maps showed that each of the plasmids was different.     

Multiple dicarboxylic amino acid transport systems with different specificities inEscherichia coli K-12

Escherichia coli K-12 CS101 is unable to utilize glutamate as the sole carbon source because of its low rate of uptake. Mutants obtained by positive selection for growth on glutamate showed increased activity of a sodium ion-dependent, -methylglutamate-sensitive, membrane-bound glutamate carrier. Positive selection for growth on glutamate in the presence of -methylglutamate yielded a mutant that produced increased amounts of a glutamate-binding periplasmic protein. Glutamate transport in this latter mutant is neither dependent on sodium nor inhibited by -methylglutamate but is competitively inhibited byl-aspartate. Another glutamate/aspartate transport system independent of sodium ions and of the periplasmic glutamate-binding protein was disclosed through the isolation and analysis of -hydroxyaspartate-resistant mutants.     

Expression of a DNA methylation (dam) gene inEscherichia coli K-12

Plasmid pMQ3, carrying thedam gene ofEscherichia coli on a 6.1 Kb fragment, shows a tenfold increase in relative DNA adenine methylase activity, while plasmid pdam118, with a 1.14 Kbdam insert, shows only a twofold increase, although both plasmids were derived from plasmid pLC13–42. Since a copy number effect did not seem to be the cause of this difference, we have subcloned pMQ3 in order to determine whether the additional chromosomal DNA present in this plasmid is responsible for the enhancement of methylase activity. We show that the 346 base pairs upstream ofdam contain sequences necessary for expression. DNA sequence analysis has revealed that in pdam118 only the 118 bases 5-prime to thedam gene are present in other constructs and that the additional upstream material is pBR322 DNA. This shows that pdam118 carries a DNA duplication.     

Mapping of a mutation affecting regulation of iron uptake systems in Escherichia coli K-12.

The site of a mutation resulting in constitutive derepression of iron uptake systems has been localized at 15.7 min on the genetic map of Escherichia coli K-12.     

Genetic map position of the cistron coding for isocitrate dehydrogenase inEscherichia coli K-12

Transductional analysis indicates that theicd cistron, which codes for isocitrate dehydrogenase (E.C. 1.1.1.42), is located between thepurB anddadR cistrons.     

Mutant of Escherichia coli K-12 defective in D-glucosamine biosynthesis

A mutant was isolated from a derivative of Escherichia coli K-12 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. This mutant contained normal levels of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10), but no detectable activity of l-glutamine:d-fructose-6-phosphate amino-transferase (EC 2.6.1.16). It required either N-acetyl-d-glucosamine or d-glucosamine for growth, and went into rapid lysis when the supply of these compounds was exhausted. In medium containing 11% sucrose, the cells were converted into spheroplasts in the absence of d-glucosamine.