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.     

Coordinate initiation of chromosome and minichromosome replication in Escherichia coli.

Escherichia coli minichromosomes harboring as little as 327 base pairs of DNA from the chromosomal origin of replication (oriC) were found to replicate in a discrete burst during the division cycle of cells growing with generation times between 25 and 60 min at 37 degrees C. The mean cell age at minichromosome replication coincided with the mean age at initiation of chromosome replication at all growth rates, and furthermore, the age distributions of the two events were indistinguishable. It is concluded that initiation of replication from oriC is controlled in the same manner on minichromosomes and chromosomes over the entire range of growth rates and that the timing mechanism acts within the minimal oriC nucleotide sequence required for replication.     

Kinetics of minichromosome replication in Escherichia coli B/r.

Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed.     

Random replication of the stringent plasmid R1 in Escherichia coli K-12.

The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication). The replication of R1 was studied in a density-shift experiment. One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice. The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication. This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication). Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication. However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle. The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed.     

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.     

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.     

Characteristics of cold-sensitive mutants of Escherichia coli K-12 defective in deoxyribonucleic acid replication.

Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.     

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.     

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.     

Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12

In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3'-5' helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.     

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.