Evolution of Transposons: Natural Selection for Tn5 in ESCHERICHIA COLI K12

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.     

Dominant Mutators in ESCHERICHIA COLI

In this paper we report on the isolation and genetic analysis of a series of strong mutators mapping at five minutes on the E. coli chromosome. These mutations are dominant and show no evidence of interaction in merodiploids. Cultures grown in broth medium exhibit mutant frequencies five to six orders of magnitude higher than mut⁺ strains. Cultures propagated in minimal salts media mutate at rates one to three orders higher than wild-type. Three-factor crosses have been used to order these mutators relative to metD, proA, and a Tn10 insertion near five minutes.     

ESCHERICHIA COLI Rho Factor Is Involved in Lysis of Bacteriophage T4-Infected Cells

A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho+ hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho+ allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.--Rid394 X 4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394 X 4 showed strong lysis inhibition (a known t- phenotype) but no prolonged phage production (another well-known t- phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effect on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394 X 4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.     

IMPROVEMENT OF SELECTIVITY FOR ISOLATION OF ESCHERICHIA COLI O157:H7 FROM GENERIC ESCHERICHIA COLI

A modified selective medium was developed to increase selectivity for isolation of Escherichia coli O157 from generic E. coli based on the knowledge that E. coli O157:H7 has more resistance against HCl condition than E. coli. As a preliminary experiment, four strains of E. coli O157:H7 (ATCC 35150, 43889, 43890, and 43894) were tested to determine the maximum concentration of 6N HCl (from 0 to 250 μL) added to 50 mL of MacConkey agar medium (MAC). The maximum level was 125 μL/50 mL (6N HCl/MAC), which E. coli O157:H7 strains could tolerate against the HCl concentration. After determination, comparative growth of 15 isolates of E. coli O157 and generic E. coli were evaluated on modified selective medium (HCl-MAC; with the addition of 125 μL/50 mL) and conventional MAC, respectively. All tested strains of E. coli O157 were grown on both media, whereas 9 out of 15 generic E. coli (60% of tested strains) were strongly inhibited on HCl-MAC. For selective isolation of E. coli O157 from generic E. coli, HCl-MAC has an effective potential for an implemental use. This information can extend as a baseline for use of HCl to conventional medium for successful isolation E. coli O157 from generic E. coli.     

ESCHERICHIA COLI Gene Induction by Alkylation Treatment

Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.     

STUDIES ON THE LACTASE OF ESCHERICHIA COLI

A "lactase solution" was prepared from Escherichia coli. The mechanism of its action has been studied and changes in the rate of hydrolysis under various conditions investigated. The hydrolysis of lactose by the enzyme approximates the course of reaction of the integrated Michaelis-Menten equation. One molecule of enzyme combines with one molecule of substrate. E. coli lactase is readily inactivated at pH 5.0, and its optimal activity at 36°C. is reached between pH 7.0 and pH 7.5. The optimal temperature for its action was found to be 46°C. when determinations were carried out after an incubation period of 30 minutes. Its inactivation by heat follows the course of a first order reaction, and the critical thermal increment between the temperatures of 45°C. and 53°C. was calculated to be 56,400 calories per mol. The enzyme is activated by potassium cyanide, sodium sulfide, and cysteine, and irreversibly inactivated by mercuric chloride, silver nitrate, and iodine. After inactivation with copper sulfate partial reactivation is possible, while the slight inhibition brought about by hydrogen peroxide is completely reversible. The possible structure of the active groups of E. coli lactase as compared with other enzymes has been discussed.