Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport.

A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine. Thymidine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Conferral of transposable properties to a chromosomal gene in Escherichia coli

A normally stable gene of Escherichia coli was converted into a transposable element. A bacterial strain was constructed in which the malK gene was flanked on each side by the transposable element Tn5. The resulting Tn5-malK⁺-Tn5 structure (Tn651) became a transposable element with properties very similar to those of Tn5 itself. Tn651 transposes into regions of both the E. coli chromosome and bacteriophage lambda and is able to induce mutations. Transposition of Tn651 does not require the product of recA. Based on a physical analysis of lambda Tn651 DNA it is shown that the two Tn5s flanking the malK gene are in inverted orientation. In these experiments a new derivative of bacteriophage lambda is used that can accept a 14 kilobase insertion in vivo and still yield a plaque-forming transducing particle.     

Identification of entero-aggregative Escherichia coli based on surface properties

Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a'stacked brick' pattern (EAggEC), and four nonadherent control strains, wereexamined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA)HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outer- membrane protein (MAP), to agglutinate erythrocytes, and toproduce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outermembrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did notexpress an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cellswith a stacked brick pattern, these strains exhibit a diverse range of physical and biochemicalproperties. From the results of this study, it was concluded that currently, the possession ofEAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain theonly reliable means of identifying EAggEC.     

Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli

Mutants of Escherichia coli K-12 have been isolated in which several enzymes concerned with tyrosine biosynthesis are derepressed. These mutants were obtained from a parent strain possessing only a single 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase isoenzyme, DAHP synthetase (tyr), by selecting for resistance to the tyrosine analogue, 4-aminophenylalanine. The mutation responsible for this derepression has been mapped and the gene, which is not closely linked to aroF and tyrA, has been designated tyrR.     

The dnaB gene product of Escherichia coli. I. Purification, homogeneity, and physical properties.

The dnaB gene product was purified to homogeneity and its physical properties were characterized. Purification was aided by the use of the Escherichia coli strain. MV12/28, which overproduced the dnaB gene product 10-fold (Wickner, S. H., Wickner, R. B., and Raetz, C. R. H. (1976) Biochem. Biophys. Res. Commun. 70, 389-396) and by taking advantage of the enzyme's high affinity for both DEAE-cellulose and phosphocellulose. The most highly purified fractions gave a single stained band on native, polyacrylamide gels and dnaB enzymatic activity was coincident with this band. On denaturing sodium dodecyl sulfate-polyacrylamide gels, a single band was observed corresponding to a molecular weight of 48,000 +/- 2,000. The native molecular weight of 290,000 +/- 12,000 was calculated from determinations of the sedimentation coefficient, which was 11.3 S, and the Stokes radius, which was 60 A. Cross-linking the protein with dimethyl suberimidate yielded six bands. We conclude that the enzyme consists of six identical subunits. The apparent pI was 4.9 and the amino acid composition was typical except for the absence of cysteine.     

Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12.

In this study, we examined cell surface properties of mutants of Escherichia coli for which organic solvent tolerance levels were elevated. The cell surface of each mutant was less hydrophobic than that of the parent, probably due to an increase in lipopolysaccharide content. OmpF synthesis was repressed in the mutants. Organic solvent bound readily to viable E. coli cells in response to the polarity of the solvent. The mutants were bound less abundantly with the organic solvent than was the parent.     

Alkaline induction of a novel gene locus, alx, in Escherichia coli.

A novel pH-regulated locus inducible over 100-fold in alkaline media was identified in Escherichia coli through screening of 93,000 Mu dI1734 (lacZ Kmr) operon fusions at pH 6.5 and pH 8.5. Four lacZ fusions that showed expression only at the higher pH were mapped at 67.5 min by P1 transduction crosses. The locus was designated alx.     

Cloning of trg, a gene for a sensory transducer in Escherichia coli.

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.     

Growth of the Escherichia coli cell surface.

Phage T6 was used as a label to follow the growth of the outer membrane in a strain of Escherichia coli temperature sensitive for the production of the T6 receptor. Extension of the surface takes place at the cell poles. Small cells extend at only one pole, whereas larger cells grow from both poles. The change from unipolar to bipolar growth appears to depend on the attainment of a particular cell size and not on completion of chromosome replication.     

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli.

We have isolated an Escherichia coli gene which, when overexpressed, is able to complement the permeability defects of a vancomycin-susceptible mutant. This gene, designated sanA, is located at min 47 of the E. coli chromosome and codes for a 20-kDa protein with a highly hydrophobic amino-terminal segment. A strain carrying a null mutation of the sanA gene, transferred to the E. coli chromosome by homologous recombination, is perfectly viable, but after two generations at high temperature (43 degrees C), the barrier function of its envelope towards vancomycin is defective.     

Influence of surface cell structures on physicochemical properties of Escherichia coli

The partition of cells in a polyethylene glycol-dextran two phase system was used to compare the relative hydrophobicity of E. coli strains expressing different surface structures. The role of fimbriae and surface antigens on the behavior partition was investigated. The strains expressing PAP fimbriae and/or O-antigen showed a higher surface hydrophobicity than strains which express only type 1 fimbriae and/or R-antigen. No relation was found between K and H antigen and hydrophobicity measurements. The influence of surface structures on electrophoretic mobility has been evaluated. The polysaccharide capsules of AL 213 and AL 499 strains generated a high EPM. For non capsulated E. coli the EPM of rough strains (AL 46, 382) is higher than smooth strain (AL52).     

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.     

A new Escherichia coli cell division gene, ftsK.

A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer.     

Cyanobacterial metallothionein gene expressed in Escherichia coli. Metal-binding properties of the expressed protein.

The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT). To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase. The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT). E. coli expressing this gene showed enhanced (ca. 3-fold) accumulation of Zn.     

osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli.

A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.