Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis.

This paper describes a novel genetic method used to isolate mutations that alter proper assembly of OmpF in the outer membrane. The thermolabile nature of assembly intermediates allowed selection of temperature-sensitive mutations within the ompF gene. A variant allele of ompF (ompF-Dex) was used because it provided a convenient selectable phenotype (Dex+). Assembly mutants were isolated in two steps. First, amber mutations were obtained that mapped in ompF-Dex. This resulted in a Dex- phenotype. Starting with these Dex- strains, Dex+ revertants were isolated. Mutants that displayed a temperature-sensitive Dex+ phenotype were further characterized. Three such mutants possessed a single substitution within ompF that reverted the nonsense codon to a sense codon which replaced W214 with either an E or Q and Y231 with a Q residue in the mature OmpF protein. All three mutant OmpF proteins showed an assembly defect. This defect led to a substantial reduction in the amount of stable OmpF trimers with the concomitant increase of a high-molecular-weight form of OmpF which migrated at the top of the gel. Suppressor mutations were sought that corrected the assembly defect of OmpF. These extragenic suppressor mutations were mapped at 45 min on the Escherichia coli chromosome. The suppressor mutations displayed no allele specificity and were recessive to the wild-type allele. In the presence of a suppressor, mutant stable trimers appeared in an almost normal manner. The appearance of stable trimers concurred with a substantial loss of the high-molecular-weight OmpF species. At this stage, it is not clear whether the high-molecular-weight species of OmpF is a normal assembly intermediate or a dead-end assembly product. The results presented in this study raise the intriguing possibility of a chaperone-like activity for the wild-type suppressor gene product.     

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.     

Purification and preliminary characterization of the Escherichia coli K-12 recF protein.

The recF gene of Escherichia coli is known to encode an Mr-40,000 protein that is involved in DNA recombinationa nd postreplication DNA repair. To characterize the role of the recF gene product in these processes, the recF gene was cloned downstream of a tac promoter to facilitate overproduction of the recF gene product. The RecF protein was overproduced and purified to apparent homogeneity. N-terminal protein sequence analysis demonstrated that the purified protein had the sequence that was predicted from the DNA sequence of the recF gene, except that the predicted N-terminal Met was not present. The RecF protein bound to single-stranded oligonucleotides in filter binding and gel filtration assays. Maximal binding required 2 to 3 min of incubation at 37 degrees C; the binding reaction had a pH optimum of 7.0, did not require divalent cations, and was inhibited by NaCl concentrations of greater than 250 mM. The Kd of RecF protein binding to a 59-base single-stranded oligonucleotide was on the order of 1.3 X 10(-7) M, and the reaction did not show cooperativity. Experiments measuring the binding to various DNA substrates and competition binding experiments with different DNA molecules demonstrated that RecF protein binds preferentially to single-stranded, linear DNA molecules.     

Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation.

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.     

Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase.

Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase (EC 2.3.2.2) were isolated after mutagenesis of cells with ethyl methanesulfonate. They lost the enzyme activity to different extents. The mutations of two mutants that had lost the enzyme activity completely were mapped at 76 min of the E. coli K-12 linkage map. These mutations made the cells neither nutrient requiring nor cold sensitive. The mutants leaked much more glutathione into the medium than the wild type. We propose the symbol ggt for these mutations.     

Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene.

Mutants defective in utilization of uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding uracil phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated Mr of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids.     

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.     

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.     

Study of a pts-gene-linked pleiotropic mutation influencing expression of catabolyte-sensitive genes of Escherichia coli K-12

Properties of the pleiotropic mutation pts17 are described. This mutation is liked to pts1 gene, which specifies the synthesis of the enzyme I of phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Escherichia coli K-12. Genetic analysis has shown that pts17 mutation is located between purC and pts1 markers and that the wild type allele pts17+ has transdominant character over the mutant allele pts17. The mutant strain J6217, isogenic to parent J62, shows normal growth properties in the minimal salt media with a number of carbohydrates used as a single carbon source. The pts17 mutations does not affect the enzyme I activity, but significantly suppresses the total PTS activity in the bacterial cell extracts. The intact mutant cells reveal the enhanced rate of accumulation and phosphorylation of alpha-methylglucoside. The pts17 bacteria show 3-fold enhanced phosphohydrolase activity with glucose-6-phosphate as substrate. It is established that pts17 mutation decreases the differential rate of the L-tryptophanase synthesis and makes the process of unductions resistant to glucose catabolite repression. It is suggested that this mutation affects the activity of the PTS factor III. One can suppose that the latter mediates the influence of ptsI and ptsH mutations upon the expression of catabolite-sensitive operons in E. coli.     

A novel pleiotropic mutation in Escherichia coli K12 which affects transduction, transformation and rates of mutation.

A mutant strain of Escherichia coli K12, R2721, has been shown to differ from its parent strain, S491, in four associated phenotypic characters as a result of a single mutation. This strain did not give recombinants with DNA transduced by bacteriophage PI or bacteriophage Mu, nor transformats after exposure to R factor DNA: lysates of bacteriophage PI grown on this strain did not appear to contain any transducing particles when tested on normal recipients. Moreover, the reversion rates, both spontaneous and ultraviolet-induced, for two auxotrophic markers were reduced. The frequency of revertants was at least two orders of magnitude lower in cultures of R2721 than in cultures of S491I. Many of the rare revertants for one or other of the auxotrophic markers were found to have regained normal reversion frequencies for the other marker and for the capacity to be transduced. In all other respects, recombination in R2721 appeared normal, the frequency of chromosomal mobilization by and F' factor was unaffected and normal yields of recombinants were obtained from matings with Hfr strains. The only circumstance in which transduction of R2721 was observed was when the capacity to ferment galactose was selected and PI had been grown on a strain carrying lambdadgal when, presumably, integration was effected by the phage-coded gene products. The mutation has been located on the E. coli chromosone map between tonA and pro and has been given the symbol tdi (transduction inhibition). Double mutants, (tdi recA) and (tdi recB), have been isolated and show no unexpected properties.     

Isolation and characterization of an Escherichia coli K-12 mutant defective in tyrosine- and phenylalanine-specific transport systems.

A mutant strain of Escherichia coli K-12 that is defective in both the tyrosine-specific and phenylalanine-specific transport systems was isolated. The defects in these systems were shown to be due to mutations in two distinct loci, tyrP and pheP, respectively.     

Regulation of tyrosine biosynthesis in Escherichia coli K-12: isolation and characterization of operator mutants

Mutant strains of Escherichia coli have been isolated in which the synthesis of two of the enzymes involved in tyrosine biosynthesis, 3-deoxy-d-arabinoheptulosonic acid-7 phosphate synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, is partially constitutive. The mutations involved are closely linked to aroF and tyrA, the structural genes of these enzymes. The gene in which the mutations occur has been designated aroK, and the gene sequence is aroK, aroF, tyrA. In aroK(+)/aroK diploids, the aroK allele only affects the structural genes in the cis position. The mutant allele aroK is not recessive to aroK(+) and aroK/aroK(+) strains exhibit the aroK phenotype of resistance to 4-aminophenylalanine. It is proposed that aroK is an operator locus for an aroF tyrA operon.     

Isolation and characterization of Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor.

Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor (ConI-) were isolated and characterized. These mutants are specific in that they are conjugation proficient with other types of donor strains. They have an altered susceptibility to phages and detergents. Chemical analysis of the cell envelopes of mutant strains has shown that the lipopolysaccharide (LPS) is altered and that one major outer-membrane protein is absent. Conjugation experiments in which LPS from wild-type cells was added to a mating mixture, made up with wild-type donor and recipient cells, showed inhibition in transconjugant formation when an I-type donor, but not an F-type donor, was used. This strongly suggests that LPS of the recipient cell is directly involved in the ability to mate with an I-type donor but not with an F-type donor. The mutations are located in the 78- to 82-min region of the E. coli map, with one exception where the mutation maps near or in the galactose operon.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.