Interaction of RNA polymerase mutations in haploid and merodiploid cells of Escherichia coli K-12

Summary Combination in the same chromosome of tsX mutation which affects the attachment of RNA polymerase to DNA template with either of two rifampicin resistant mutations (rif-r-1 or rif-r-5) is lethal. However tsX forms viable combination with other rifampicin resistant mutation—rif-r-76. Moreover a partial restoration of rifampicin binding capacity takes place: RNA polymerase from double tsX rif-r-76 mutant binds rifampicin better than the enzyme from tsX ⁺ rif-r-76 cells. No mutual influence of rifampicin resistant and streptolydigin resistant mutations was found.Heterozygous merodiploids (rif-r/rif-s and stl-r/stl-s) demonstrate phenotypic dominance of sensitivity to each of the drugs no matter whether resistant allele is localized in chromosome or in episome. However certain chromosomal mutations which themselves have no apparent effect on RNA polymerase may cause dominance of rif-r allele.About a half of total cellular RNA polymerase in crude extracts of rif-r/rif-s and stl-r/stl-s heterogenotes was found to be drug-resistant, though rif-s allele is dominant phenotypically.The development of T2 phage is completely inhibited by rifampicin in haploid rif-s cells and is only slightly affected in rif-r mutant. A partial resistance of phage development to rifampicin was observed in rif-r/rif-s heterogenotes which confirms that both rif-s and rif-r enzymes are simultaneously present in such cells.Sensitive and resistant RNA polymerase function independently when mixture of the two enzymes was incubated with the excess of DNA template. However a competition between the two enzymes for the DNA was observed if the limiting amount of the template is available. The result of this competition to major extent depends on which of the enzymes was added first. It is supposed that in certain conditions normal RNA polymerase may act as a repressor of the mutant enzyme: drug-sensitive RNA polymerase may bind to the template in the presence of the drug and thus prevent the function of drug-resistant enzyme. This hypothesis explains phenotypic dominance of sensitive alleles to resistant alleles which leads to inability of heterogenote cells to multiply in the presence of corresponding drugs.     

Isolation and characterization of mutants deleted for the sulA-ompA region of the Escherichia coli K-12 chromosome

Abstract We have isolated mutants deleted for different segments of the sulA-ompA region of the Escherichia coli K-12 chromosome using gene fusion techniques. Genetic and physical analysis showed that the deletions ranged from 500 to more than 4000 base pairs (bp) Strains were found in which all, or part, of the sulA and ompA genes had been deleted.     

Isolation of Temperature-Sensitive Membrane Mutants of Escherichia coli K-12

Mutants with impaired biosynthesis of unsaturated fatty acids or altered metabolism of the phospholipids were isolated at a rather high frequency from a set of temperature-sensitive lysis mutants. It is suggested that preselection for the lysis phenotype makes it possible to isolate several kinds of mutants affected in the integrity of the cytoplasmic membrane.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

Rapid and accurate identification of Escherichia coli K-12 strains.

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.     

Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.     

Isolation and properties of Escherichia coli K-12 mutants impaired in the utilization of gamma-aminobutyrate.

We have isolated mutants of Escherichia coli K-12 CS101B that have lost the ability to utilize gamma-aminobutyrate as a source of nitrogen. One class of mutants, which were not affected in the utilization of other nitrogen sources (proline, arginine, glycine), included many isolates with lesions in gamma-aminobutyrate transport or in its transamination and one mutant completely devoid of succinic semialdehyde dehydrogenase activity and exhibiting low gamma-aminobutyrate transport and transamination. gamma-Aminobutyrate-utilizing revertants of the latter recovered full transport and transamination capacities but remained dehydrogenaseless. Another class of mutants showed pleiotropic defects in nitrogen metabolism. One such mutant was lacking glutamate synthase activity. The genes specifying the synthesis of gamma-aminobutyrate permease, gabP, gamma-aminobutyrate transaminase, gabT, and succinic semialdehyde dehydrogenase, gabD, and the control gene, gabC, that coordinately regulates their expression all form a cluster on the E. coli chromosome, linked to the srl and recA loci (at 57.5 min). The mutations with pleiotropic effects on the metabolism of nitrogenous compounds are not linked to the gab cluster.     

Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome

Summary A recombinant bacteriophage lambda, argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome. The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined. In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene. The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels. The clockwise gene order was found to be argG-nusA-pnp.     

Isolation of the nonviable cells produced during normal growth of recombination-deficient strains of Escherichia coli K-12

During normal growth, cultures of recombination-deficient (Rec(-)) strains contain a population of cells that do not form colonies. Such cells are not present in a culture of an isogenic Rec(+) strain. We present a procedure for isolating and studying this defective population of cells. Exposure of a growing Rec(-) or Rec(+) culture to low levels of penicillin causes the dividing cells to elongate. The size of the nonviable cells present in the Rec(-) cultures is unaffected. The nonviable cells are then separated from the elongated cells by velocity sedimentation. This isolation technique provides a convenient way of analyzing the composition, biosynthetic capacity, and enzymatic function of the nonviable cells before isolation. In this paper we present data showing that before fractionation the nonviable cells in the Rec(-) culture are defective in their ability to synthesize beta-galactosidase, whereas the Rec(-) viable cells behave like the Rec(+) cells in this regard. This observation confirms the existence of at least two classes of cells in liquid cultures of Rec(-) strains grown under normal conditions. That class of cells which is unable to synthesize beta-galactosidase is the same class that cannot form colonies when plated on solid medium.     

Preferred secondary attachment site of prophage phi80 on Escherichia coli K-12 chromosome]

The integration frequency of phage att80 immlambdac1857 into the chromosome of a mutant strain H47 Escherichia coli K-12 deleted for the normal prophage insertion site is found to be about 20-fold decreased as compared with its integration into the wild type strain. The most of the resulting lysogens contain the prophage at the secondary attachment site of the mutant bacterial chromosome which is preferentially utilized for prophage insertion. This attachment site (att80-II) is located close to his-genes on the chromosome of H47 strain. Prophage curing procedure of such abnormal lysogens results in the appearance of rare auxotrophic heat-resistant survivors with the His- phenotype. In some cases the prophage insertion can induce an inversion of a neighbouring genetic region. Such lysogens contain the purC gene near prophage located at the att80-II site, and after curing they segregate the heat-resistant His- and Pur- colonies.     

Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.     

Polymorphism in the dgt-dapD-tsf region of Escherichia coli K-12 strains.

Restriction analysis of the dapD region cloned from several strains of Escherichia coli, revealed a restriction-fragment length polymorphism (RFLP). This RFLP, which includes the BamHI, EcoRI and SalI sites, may be useful in classification of various E. coli strains.     

Sequence organization of replication origin of the Escherichia coli K-12 chromosome

A sequence of 245 base-pairs (oriC) in the replication origin of the Escherichia coli K-12 chromosome has been shown to provide all the information essential for initiation of bidirectional replication. In order to elucidate the sequence organization of oriC, numerous mutants carrying a single-to-multiple transitions from G X C to A X T base-pair were constructed by localized mutagenesis in vitro, which uses sodium bisulfite, and the correlation between the mutation sites and replicating ability (Ori function) was systematically analyzed. By isolating non-defective (Ori+) mutants with multiple base changes, transitions at 71 positions among 101 G X C pairs in oriC were found to have no effect on Ori function. Investigation of defective (Ori-) mutants, on the other hand, showed that individual replacements at 18 positions were detrimental to Ori function to some extent. These irreplaceable G X C pairs fell in the positions where no substitution was detected in the Ori+ mutants. The defect of the Ori- mutants with a single base substitution was generally weaker than that of the previously constructed Ori- mutants lacking a part of oriC. The addition of two or more base changes each giving a faint Ori- phenotype, however, resulted in a more intensive Ori- phenotype. We have previously demonstrated that oriC contains several regions where deletion or insertion of oligonucleotides leads to strong Ori- phenotypes. Transitions in those areas did not cause any defect of Ori function. Combining present results on base substitution mutants with the previous observations together, we assumed that the oriC sequence provides multiple interaction sites with replication initiation factors, and the precise arrangement of these sites are required for Ori function.     

Synthesis of ribosomal proteins in merodiploid strains of Escherichia coli

Summary The regulation of the synthesis of r-proteins in Escherichia coli was investigated by increasing the dosage of the genes for a limited number of ribosomal proteins (r-proteins) using either transducing phage fus 3 (Lindahl et al. 1977) or rif ^d18 (Kirschbaum and Konrad 1973). During exponential growth the presence in the cell of either lysogenised transducing phage did not increase the rate of synthesis or degradation of any of the 31 r-proteins whose genes are duplicated. Experiments were also performed to determine whether r-protein synthesis during the period of unbalanced r-protein synthesis that follows nutritional enrichment was sensitive to an increase in gene dosage. Duplication of the 27 r-protein genes on fus 3 did not alter the rate of synthesis of any of the r-proteins after enrichment. However, gene dosage effects were detected for at least 3 of the r-proteins whose genes were duplicated of rif ^d18.     

Integration of plasmid RP1 with the chromosome of Escherichia coli K-12 recA. 2 classes of Hfr strains

Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied. Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains. Properties of these Hfrs have been examined. From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1. The other class of Hfr strains is formed due to a stable integration of RP1. In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E. coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids). On the basis of the latter results, a new simple method for R' plasmids selection has been proposed. Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated. Probable mechanisms of the stable Hfr strains formation are discussed.     

Isolation and Characterization of a Phosphonomycin-Resistant Mutant of Escherichia coli K-12

A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.     

Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome.

Between 59 and 60 min on the Escherichia coli genetic map, there is a highly conserved sequence of 29 base pairs, containing an inverted repeat of seven base pairs that appears 14 times, 32 or 33 base pairs apart, downstream of the iap gene coding region. About 24 kilobase pairs downstream of the 14 repeats, a similar 29-base-pair sequence with a spacing of 32 base pairs appears seven times. Nucleotide sequences hybridizing with the 29-base-pair fragment were also detected in Shigella dysenteriae and Salmonella typhimurium but not in Klebsiella pneumoniae or Pseudomonas aeruginosa.     

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.