ESCHERICHIA COLI K-12 Auxotrophs Induced by Insertion of the Transposable Element Tn5

The sites of insertion of the transposable kanamycin-neomycin resistance-determining element, Tn5, in the E. coli K-12 chromosome were assessed in a collection of over 300 auxotrophs. Although mutations in at least 45 different cistrons were obtained, the distribution of insertion sites was not completely random: proA or proB; cysG; and cysH, cysD or cysC mutants were found in excess.     

Formation of F''TRP Plasmids in ESCHERICHIA COLI K12

From strains carrying two different F-prime factors, we recovered F' derivatives that acquired the trp chromosomal region. These F'trp plasmids can be isolated at a frequency of 10^-5 to 10^-6. They were characterized genetically by looking at the size of the trp segment they acquired and at the location of that segment in the parental F' plasmid. Results are discussed in relationship to possible transposition mechanisms.     

Mismatch Repair Mutations of ESCHERICHIA COLI K12 Enhance Transposon Excision

Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.--Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision. Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.     

Physiological Characterization of Polar Tn5-Induced Isoleucine-Valine Auxotrophs in ESCHERICHIA COLI K-12: Evidence for an Internal Promoter in the ilvOGEDA OPERON

The properties of 22 isoleucine-valine auxotrophs induced in Escherichia coli K-12 by the transposable element, Tn5, were characterized on the basis of growth requirements, cross-feeding behavior, and enzyme activity. Mutants defective in ilvA, ilvC, ilvD and ilvE were found. Mutation in ilvE were not completely polar on ilvD and ilvA enzyme activities (that is, ilvE mutants possessed a low constitutive level of expression of the enzymes coded by ilvD and ilvA), while mutations in ilvD were completely polar on ilvA enzyme activity. The data suggest that there is an internal promoter between the sites of Tn5 insertion in ilvE and ilvD.     

Frequency-Dependent Selection for Plasmid-Containing Cells of ESCHERICHIA COLI

Colicin-producing plasmid-containing cells of E. coli exhibit frequency-dependent selection when grown in glucose-limited continuous culture with the corresponding plasmid-free strain. The bases of this frequency-dependent effect are shown to be (1) the lower growth rate of the plasmid-containing strain under these conditions, and (2) the production of colicin, which attenuates the growth rate of the plasmid-free strain. These results are discussed in relationship to the maintenance of genetic variation in prokaryotes.     

Genetic Analysis of a Double Male Strain of ESCHERICHIA COLI K12

The behavior of a double male strain of Escherichia coli K12 has been compared to that of its parents and a primary F' strain carrying F14 in order to determine whether the genome of the double male, responsible for the double origin mode of gene transmission, is composed of one or two linkage groups. F-prime plasmids carrying leu and pyrB and argG and metC have been detected following mating an F(-)recA(-) recipient with the double male. Their existence strongly supports the contention that the double male is composed of a single linkage group with two integrated sex factors. Data from acridine orange curing experiments places the frequency of double male cells in the population of growing cells in the two-chromosome configuration as less than one percent. Evidence for a unique origin and terminus of DNA replication deduced by the density labelling and transduction of double male DNA supports the contention that the double male is best considered a cell with a single chromosome carrying two integrated F plasmids.     

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.     

Mapping of the polA Locus of ESCHERICHIA COLI K12: Genetic Fine Structure of the Cistron

The close linkage of the glnA gene with polA was exploited to construct a fine structure map of polA by means of generalized transduction with phage P1. Nine different polA^- alleles were mapped by recombinational crosses. The results indicate a gene order consistent with previous observations (Kelley and Grindley 1976a; Murray and Kelley 1979). Three mutations, polA5, polA6 and polA12 map within the "carboxy-terminal" or "large-fragment" portion of the gene in unambiguous order. Four alleles, known to affect the "aminoterminal" portion of the gene, polA107, polA214, polA480ex and polA4113, appear to be closely linked with certain ambiguities in their exact order. All four of these mutations are known to alter the 5''→3'' exonuclease activity of DNA polymerase I and three of them result in the conditional lethal polA^- phenotype. The polA1 nonsense mutation maps between these two groups in a position consistent with its known effect, production of an amber fragment that includes the 5''→3'' exonuclease. The final allele, resA1, is another nonsense mutation that maps at the extreme "amino-terminus" of the cistron.——A number of control experiments were conducted to determine the effects of polA^- mutations on the P1-mediated recombinational event. These experiments indicated that abortive transduction occurs quite frequently, but the formation of abortive transductants and segregation of unselected transduced markers among daughter progeny is like that observed by other investigators. There was no evidence that any individual polA^- allele behaved in an exceptional fashion during recombination.     

High Frequency of Genetic Duplications in the dnaB Region of the ESCHERICHIA COLI K12 Chromosome

The region that includes the dnaB locus on the E. coli K12 chromosome was shown to be duplicated at high frequency in cell populations. The duplications were shown to be arranged in tandem and segregated at various frequencies. Segregation was dependent on the recA recombination system, but independent of recB,C. Though most of the data was obtained with dnaB::Tn10 insertion mutants, the duplications were shown to occur in the absence of Tn10.