Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.     

High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12

Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [³H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells. The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.     

Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome

Summary Two-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 by oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome. Bending was strongly enhanced by Dam methylation. In DNA from a Dam^– strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro. Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam^– strand with the Dam⁺ strand. Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 by per turn. We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis.     

The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE

Summary PhoE protein of Escherichia coli K12 is an outer membrane protein which is supposed to span the membrane sixteen times. By creating a deletion which removes the last membrane-spanning fragment and studying the localization of the truncated PhoE, we show that this fragment is indispensable for trimerization and outer membrane localization. In addition, circumstantial evidence for the proposed topology model of the protein was obtained. An insertion mutation in a region supposed to be cell surface-exposed, interferes with the binding of a monoclonal antibody which recognizes a cell surface-exposed epitope of the protein.     

Acute phase response to recombinant interleukin-6 and macrophage- and fibroblast-derived crude cytokine preparations in primary cultures of mouse hepatocytes

A number of acute phase proteins were determined by electroimmunoassay in media from CBA mouse hepatocytes cultured for 2 days with human recombinant IFN beta 2/IL-6, as well as with conditioned media from LPS-stimulated rat macrophages, and of murine L fibroblasts. It was found that human recombinant IL-6 caused three-fold increase in secretion of fibrinogen, while haptoglobin, complement C3 and transferrin were increased respectively, to 168 per cent, 151 per cent, and 145 per cent of the control. DEX(10(-7) M) in DMEM supplemented with 5 per cent FCS, enhanced the IL-6 effect on the three positive acute phase proteins. IL-6 elevated haptoglobin mRNA in mouse hepatocytes to a degree comparable with the concentration of the protein in the culture medium. The effect of conditioned media from murine fibroblasts and peritoneal rat macrophages was generally similar to that of recombinant IL-6. However, both natural preparations of the cytokines caused decrease in albumin and alpha-1-proteinase inhibitor secretion.     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Thiolutin inhibits utilization of glucose and other carbon sources in cells of Escherichia coli

Thiolutin was found to inhibit the utilization of glucose and other growth substrates in Escherichia coli. The inhibition was detected by a sharp drop of the respiration rate after addition of the antibiotic. The actual function affected was allocated to the cytoplasmic membrane of the bacterial cells by the following evidence: