Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase.

A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA.     

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.     

Peptidoglycan association of bacteriophage T5 receptor in Escherichia coli K-12.

A total of 50% of the FhuA proteins (also called TonA proteins) present in Escherichia coli cells were associated with the peptidoglycan and 50% were free, whether or not this protein was overproduced. This FhuA-peptidoglycan association was made via the lipoprotein.     

Chloramphenicol causes fusion of separated nucleoids in Escherichia coli K-12 cells and filaments.

Chloramphenicol is frequently used for better visualization of the Escherichia coli nucleoid. Here, we show that chloramphenicol causes not only rounding off of the nucleoid but also fusion of as many as four separated nucleoids. Nucleoid fusion occurred in fast-growing cells and in filaments obtained by dicF antisense RNA induction or in ftsZ84(Ts) and pbpB(Ts) mutants. Thus, treatment with chloramphenicol erroneously suggests that DNA segregation is inhibited.     

Nonrandom F-plasmid replication in Escherichia coli K-12.

Both the autonomous and chromosomally integrated F plasmids were found to replicate in a nonrandom fashion after a density transfer from heavy medium ([13C]glucose, 15NH4) to light medium ([12C]glucose, 14NH4). The data are consistent with the hypothesis that both the chromosome and the F plasmid are replicated in a cell cycle-specific manner. Thus, these data support the proposal (J. D. Keasling, B. O. Palsson, and S. Cooper, J. Bacteriol. 173:2673-2680, 1991) that plasmids replicate in a cell cycle-specific manner.     

Uroporphyrin-accumulating mutant of Escherichia coli K-12.

An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.     

L-arabinose transport systems in Escherichia coli K-12.

Mutations in the arabinose transport operons of Escherichia coli K-12 were isolated with the Mu lac phage by screening for cells in which beta-galactosidase is induced in the presence of L-arabinose. Standard genetic techniques were then used to isolate numerous mutations in either of the two transport systems. Complementation tests revealed only one gene, araE, in the low-affinity arabinose uptake system. P1 transduction placed araE between lysA (60.9 min) and thyA (60.5 min) and closer to lysA. The operon of the high-affinity transport system was found to contain two genes: araF, which codes for the arabinose-binding protein, and a new gene, araG. The newly identified gene, araG, was shown by two-dimensional gel electrophoresis to encode a protein which is located in the membrane. Only defects in araG could abolish uptake by the high-affinity system under the conditions we used.     

Nonrandom minichromosome replication in Escherichia coli K-12.

The intervals between rounds of chromosome and minichromosome replication were measured by density shift experiments and found to be similar. Thus the minichromosome, a lambda asnA oriC bacteriophage, mostly replicates once each division cycle rather than randomly, despite its high copy number. Slight differences between the chromosome and the oriC plasmid are explained.     

Kinetics of methylation in Escherichia coli K-12.

Newly synthesized DNA is undermethylated in E. coli K-12. The amount of N6-methyl deoxyadenylic acid in labeled DNA varied from 0.3 mol% of total adenine for a 2-min pulse to 1.7 mol% for DNA that was labeled for more than two generations.     

Genome-wide expression profiling in Escherichia coli K-12.

We have established high resolution methods for global monitoring of gene expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655). Glass- and nylon-based arrays of PCR products were prepared and used to assess global changes in gene expression. Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-beta-D-thiogalactopyranoside (IPTG). Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon. Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Genetic analysis of the 5-azacytidine sensitivity of Escherichia coli K-12.

DNA containing 5-azacytidine (5-azaC) has been shown to form stable detergent-resistant complexes with cytosine methylases. We reasoned that if 5-azaC treatment causes protein-DNA cross-links in vivo, then mutations in DNA repair and recombination genes may increase the sensitivity of a cell to 5-azaC. We found that although recA (defective) and lexA (induction-negative) mutants of Escherichia coli were very sensitive to the drug, mutations in uvrA and ung genes had little effect on cell lethality. The sensitivity of recA strains to 5-azaC was dose dependent and was enhanced by the overproduction of a DNA cytosine methylase in the cell. Unexpectedly, a strain of E. coli carrying a recA mutation and a deletion of the DNA cytosine methylase gene (dcm) was found to be significantly sensitive to 5-azaC. Study of mutations in the pyrimidine salvage pathway of E. coli suggests that direct phosphorylation of 5-azaC, rather than phosphorylation of its degradation products, is largely responsible for the lethal effects of the drug. The addition of uracil to the growth medium has little effect on cell lethality of recA mutants, but it partially reversed the inhibition of cell growth caused by 5-azaC. This reversal of the bacteriostatic effects of the drug could not be achieved by adding cytosine or orotic acid to the growth medium and required the presence of functional UMP-pyrophosphorylase (gene upp) in the cell.