The role of lipid in the energy-dependent transhydrogenase systems ofEscherichia coli

The energy-dependent and independent transhydrogenase activities and the NADH oxidase of membrane particles ofEscherichia coli WS1 were inactivated by phospholipase A fromCrotalus terrificus. Ca²⁺-activated ATPase was stimulated by this treatment. Although these results suggest that phospholipid is involved in the transhydrogenase systems, trypsin treatment produced similar results. Proteolytic activity was not detected in the phospholipase preparation but its presence could not be ruled out. Membranes containing different unsaturated fatty-acid components were obtained by growing the fatty-acid auxotroph,E. coli K1060, on linoleic, oleic, or elaidic acids. Discontinuities in the Arrhenius plots of the activities of NADH oxidase, Ca²⁺-activated ATPase, energy-dependent and independent transhydrogenases, were observed at definite temperatures (transition temperatures). With the exception of NADH oxidase, the transition temperatures could not be correlated with those expected for phase changes in the phospholipids of the membranes. Transition temperatures were also found when a lipid-free, purified ATPase was used. It is concluded that phase changes in the bulk of the phospholipids do not effect transhydrogenase and ATPase activities, and that there is no evidence that the bulk of the phospholipid is involved in the activity of these enzymes. However, we cannot exclude the possibility that a limited amount of lipid in immediate contact with the enzyme protein is essential for its activity.     

The effect of liquid holding on chemical induced lethality and mitotic gene conversion in Saccharomyces cerevisiae

Summary Mitotic gene conversion was induced with a variety of chemical mutagens in a double heteroallelic strain of Saccharomyces cerevisiae. Cells were treated with various mutagens and plated immediately onto selective and nonselective growth medium or else they were subject before plating to liquid holding in buffer for various lengths of time. In respiratory competent cells liquid holding caused a decrease in lethality and in conversion frequencies. Respiratory deficient cells, unable to use a non-fermentable substrate as an energy source, behaved different. Untreated cells started to die in buffer after two days of storage, and moreover, there was a considerable increase in potential convertants i.e. cells giving rise to gene convertants when plated on selective growth media. Respiratory deficient cells treated with various chemical mutagens were still more sensitive to liquid holding. After low, sublethal doses cells started to die after one day of liquid holding already and when plated on media selective for convertants, showed an increasing frequency of gene convertants. Addition of very low concentrations of glucose to the liquid holding buffer post-poned the lethal and convertogenic effects. Higher concentrations of glucose completely abolished sensitivity to liquid holding-induced lethality and genetic alterations. The results are interpreted to mean that in respiratory deficient cells no repair activities are possible to an accumulation of spontaneous lethal damage and genetic alterations which are expressed as gene conversion when an energy source becomes available. Such a repairless condition causes an increased sensitivity to genetically active agents, and provides a useful system to detect genetic effects of slowly reacting agents.     

Multiresistant strains ofEscherichia coli isolated from the rumen of young calves

Five multiresistant strains ofEscherichia coli were isolated from the rumen fluid of young calves. Resistance to tetracycline and ampicillin was found to be associated with the transfer of a 6.4 kbp plasmid present in two investigated strains.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

Production of vanillin from ferulic acid using recombinant strains ofEscherichia coli

Vanillin is one of the world's principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with P_BAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.     

Simplified fluctuation test to distinguish liquid holding recovery from cell multiplication in ultraviolet-irradiated Escherichia coli.

Improved plating techniques and a fluctuation test have been used to distinguish between liquid holding recovery of ultraviolet-irradiated cells and cell multiplication in Escherichia coli. Among tested strains, only recA mutants showed true recovery.     

Isolation and characterization ofEscherichia coli O157:H7 strains in China

Five strains ofEscherichia coli O157:H7 were isolated from 486 stool specimens collected in 1986, 1987, and 1988 from patients with diarrhea in Xuzhou City, Jiangsu Province, China; 21 of the specimens were from patients with bloody diarrhea. The biochemical reactions of all five strains were almost identical with those of the well-knownE. coli O157:H7 strain 933. All of the strains were found to carry a 60 Md plasmid and two small plasmids. The plasmid DNA Hind 111 restriction patterns were identical. The strains were lysed byE. coli typing phage E1, E2, and E3, but not by E4 or E5. Data suggested that it might belong to a single phage or plasmid group. All strains produced vero toxin and caused diarrhea and death in infant rabbits and mice.     

The opsonic activity of complement to rough strains ofEscherichia coli in vivo in newborn precolostral pigs

The participation of complement of newborn precolostral piglets (in the absence of antibody) in the opsonization of roughEscherichia coli was investigated using blood clearance test. It was found that the complement inhibitor-yeast mannan inhibits thein vivo blood clearance when injected i. venously into newborn precolostral piglets. This inhibition takes place at the serum level and not by saturation of RES cells by mannan: sera from mannan-treated piglets are not bactericidal in anin vitro bactericidal assay against roughEscherichia coli; secondly, the presence of antibody removes the blockade of phagocytosis by mannan indicating that the RES cells are functionally capable to perform phagocytosis. On the basis of our findings we postulate that in newborn precolostral piglets in which antibody is not present in their sera, complement alone (or with cooperation with other unknown serum component) acts as an opsonin on roughEscherichia coli and is responsible for the blood clearance rate of these bacteria.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.     

The hydrogenases and formate dehydrogenases ofEscherichia coli

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.     

Influence of salts and sodium chloride on the recovery ofEscherichia coli from seawater

The objective of this study was to evaluate the influence of seawater salts, and more specially sodium chloride, on the recovery ofEscherichia coli cells after exposure to natural seawater in laboratory microcosms, and the possible adaptation in this bacterium to high salinity. The recovery efficiency of a complex organic medium supplemented with sodium chloride largely depended on the strain and varied with starvation time and salinity. Moreover, cells previously grown on salted medium appeared more able to survive after exposure to seawater. It is assumed that, withinE. coli populations, some cells are able to adapt to seawater in the presence of both salts and organic matter.     

Stimulation ofEscherichia coli adenylate cyclase by lactose in strains carrying mutations in lactose permease

When a wild-type strain ofEscherichia coli contains lactose permease, the accumulation of cyclic AMP (cAMP) by intact cells isinhibited by lactose. This inhibitory effect of lactose is observed in a strain with a mutant cAMP phosphodiesterase and therefore involves a regulation of adenylate cyctase activity. Some E. coli strains carrying mutations in lactose permease show an effect opposite to that of the wild-type strain; the accumulation of cAMP by intact cells isstimulated by lactose, but only when the mutant permease is present. Insertion of lactose permease into the membrane of ceils can produce a change in the specific activity of adenylate cycIase; induction of the wild-type transporter is correlated with a decrease in the specific activity, while implantation of a mutant form of lactose permease can lead to an increase in the specific activity. From these data, it is suggested that the state of the lactose transporter in the cell membrane influences the activity of adenytate cyclase.     

Pyrimidine dimer excision and DNA degradation during liquid holding recovery in ultraviolet-irradiated Escherichia coli K12 uvr+ rec.

The survival of ultraviolet-irradiated Escherichia coli K12 uvr+re was increased by post-irradiation incubation in phosphate buffer. During this incubation both dimer excision and DNA breakdown were inhibited. It is suggested that the bacteria coped with the remaining dimers in a manner which did not involve excision.