Metabolism of D-arabinose: origin of a D-ribulokinase activity in Escherichia coli

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical K(m) values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose.     

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin.

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.     

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin.

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.     

Adhesive properties associated with the Vir plasmid: a transmissible pathogenic characteristic associated with strains of invasive Escherichia coli

Escherichia coli strain S5 (O15:K+:H21) isolated from a septicaemic lamb and previously shown to possess a virulence plasmid, Vir, attached in vitro to calf epithelial tissue from the ileum, oesophagus and trachea in the presence of 0.5% (w/v) D-mannose. The Vir+ recombinant strains 711v and H209av, which had received the Vir plasmid(s) from strain S5, also attached to these epithelia but the parent strains 711 and H209a without the Vir plasmid were non-adhesive. The attachment of the Vir+ strain 711v to intestinal brush borders was inhibited by antiserum to live Vir+ strain H209av but not by antiserum to strain H209a lacking Vir. No adherence occurred with Vir+ organisms grown at 18 degrees C or after heating at 65 degrees C. Adhesion was unaffected by 0.5% (w/v) formaldehyde. Glucosamine, mannosamine, their N-acetyl derivatives and wheat germ lectin each inhibited attachment of Vir+ strain 711v to brush border epithelia.     

Replication initiation at the Escherichia coli chromosomal origin

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.     

Isolation and characterization of plasmids carrying a partially defective Escherichia coli replication origin

The replication origin (oriC) of the Escherichia coli chromosome has been cloned and the region essential for chromosomal replication has been delimited to 245 base pairs. In previous studies the ability of recombinants between oriC and ColE1-type vectors, to transform E. coli polA- strains was used to determine which nucleotides in oriC are essential for replication. In this paper we have used a different approach by isolating partial defective replication mutants of a minichromosome (pCM959) that contains oriC as the single replication origin. Our results demonstrate that many mutations are allowed within oriC that do not affect oriC function as measured by the ability to transform E. coli polA- strains. In the minimal oriC region we detected 8 mutations at positions that are conserved in the sequence of six bacterial origins. The implications of these results on previous work will be discussed. Our data also demonstrate that a mutation producing an oriC- phenotype may be suppressed by secondary mutations. An E. coli strain was found that facilitates the isolation of partially defective minichromosomes. The results with this strain indicate a specific function of the sequence surrounding the base pair at position 138.     

Microcalorimetric study of Escherichia coli aerobic growth: kinetics and experimental enthalpy associated with growth on succinic acid.

A new batch calorimeter was used to study the aerobic growth of Escherichia coli in a minimal containing a growth-limiting concentration of succinic acid. The shape of the thermograms obtained was simple when the energy source was the only growth-limiting factor and showed a stationary phase when the oxygen transfer was limiting. The experimentally determined value of the heat production during growth was found to be significantly lower than the heat of combustion of the succinic acid corrected for the assimilated-substrate fraction. An interpretation has been given to explain the difference between the experimental and the theoretical values.     

The replication origin region of Escherichia coli: nucleotide sequence and functional units

The minichromosome pCM959 contains the DNA segment from bp -677 (left) to bp + 3335 (right) of the Escherichia coli replication origin, oriC. The nucleotide sequence of this plasmid was determined. The coding regions for proteins were identified, and the possible function of those proteins is discussed. Within oriC two extended systems of dyad symmetry were found, and their possible significance is considered.     

Expression of a Thiobacillus ferrooxidans origin of replication in Escherichia coli.

A cryptic plasmid from an autotrophically grown arsenic-resistant strain of Thiobacillus ferrooxidans was isolated and cloned into pBR325. The origin of replication of pBR325 was deleted, and the recombinant plasmid was shown to replicate in Escherichia coli, using an origin of replication located on the Thiobacillus plasmid.     

Proteins associated with rRNA in the Escherichia coli ribosome.

Ribosomal proteins located near the rRNA have been identified by cross linking to [14C]spermine with 1,5-difluoro-2,4-dinitrobenzene. The polyamine binds to double-stranded rRNA; those proteins showing radioactivity covalently bound after treatment with the bifunctional reagent should therefore be located in the vicinity of these regions of rRNA. Six proteins from the small subunit, S4, S5, S9, S18, S19 and S20 and ten proteins from the large subunit L2, L6, L13, L14, L16, L17, L18, L19, L22 and L27 preferentially take up the label. The results obtained with three proteins from the large subunit, L6, L16 and L27, show a high degree of variability that could reflect differences of conformation in the subunit population. Several proteins were drastically modified by the cross-linking agent but were not detected in the two-dimensional gel electrophoresis (e.g., S1, S11, S21, L7, L8 and L12) and therefore could not be studied.     

Escherichia coli with a linear genome

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.     

On the origin of replication of Escherichia coli chromosome

DNA labelled with [³H]thymine near the beginning or near the terminus of chromosomal replication was hybridized with isolated F′13, F′14 and F′15 DNA's. The presented results show that the ilv marker replicates earlier than the lac or thy markers in the cycle of chromosomal replication of Escherichia coli strains, K12F⁻ AY5 and 15T⁻ 557.