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Biomedical subjects

N J Russell

Publications and source records attributed to N J Russell.

At least 19 recordsLinked to original sources
PubMed2001

Alkalibacterium olivoapovliticus gen. nov., sp. nov., a new obligately alkaliphilic bacterium isolated from edible-olive wash-waters.

A novel Gram-positive, obligately alkaliphilic, non-sporulating, rod-shaped, flagellated bacterium is described. Three different strains of the bacterium were isolated from the wash-waters of edible-olive production. The strains are motile, psychrotolerant, halotolerant, facultatively anaerobic bacteria with a pH optimum of 9.0-9.4 for two strains and 9.8-10.2 for the third. They are catalase- and oxidase-negative. A range of hexoses and some disaccharides composed of hexoses, but not pentoses are metabolized by the bacterial strains: D(+)-glucose, D(+)-glucose 6-phosphate, D(+)-cellobiose, starch or sucrose are the carbohydrates best utilized. No common amino acids are utilized by the three alkaliphilic strains, but yeast extract can serve as sole carbon and energy source. The major membrane phospholipids are diphosphatidylglycerol, phosphatidylglycerol and an unknown phospholipid, all containing saturated and unsaturated, even-carbon-numbered fatty acyl chains with hexadecanoic and hexadecen(7)oic as the predominant components. The G+C content of the DNA in all three strains is 39.7+/-1.0 mol% and the DNA relatedness by hybridization is >88% for all pairings of the three strains. The results of 16S rRNA sequence comparisons revealed that the strains represent a new alkaliphilic linkage in the order Bacillales, belonging to the Carnobacterium/Aerococcus-like spectrum. It is proposed that the strains should be assigned to a new genus and species, Alkalibacterium olivoapovliticus. The three strains, designated WW2-SN4aT, WW2-SN4c and WW2-SN5, have been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) as DSM 13175T, DSM 12937 and DSM 12938 respectively, and in the National Collection of Industrial and Marine Bacteria as NCIMB 13710T, NCIMB 13711 and NCIMB 13712, respectively. The type species of this genus is Alkalibacterium olivoapovliticus and the type strain is WW2-SN4aT.

Anaerobiosis↗
PubMed2000

Mechanism of action of pulsed high electric field (PHEF) on the membranes of food-poisoning bacteria is an 'all-or-nothing' effect.

Salmonella typhimurium (CRA 1005) was more sensitive than Listeria monocytogenes (NCTC 11994) to pulsed high electric field (PHEF) treatment in distilled water (10, 15 and 20 kV/cm), 10 mM tris-maleate buffer pH 7.4 (15 kV/cm) and model beef broth (0.75% w/v: 15 kV/cm). Sublethal injury could not be detected using a selective medium plating technique, indicating that bacterial inactivation by PHEF may be an 'all-or-nothing' event. PHEF-induced membrane permeabilization resulted in increased UV-leakage and a decreased ability of L. monocytogenes to maintain a pH gradient.

Cell Membrane↗
PubMed2000

Bacillus sp. WW3-SN6, a novel facultatively alkaliphilic bacterium isolated from the washwaters of edible olives.

A novel Gram-positive facultatively alkaliphilic, sporulating, rod-shaped bacterium, designated as WW3-SN6, has been isolated from the alkaline washwaters derived from the preparation of edible olives. The bacterium is nonmotile, and flagella are not observed. It is oxidase positive and catalase negative. The facultative alkaliphile grows from pH 7.0 to 10.5, with a broad optimum from pH 8.0 to 9.0. It could grow in up to 15% (w/v) NaCl, and over the temperature range from 4 degrees to 37 degrees C, with an optimum between 27 degrees and 32 degrees C: therefore, it is both halotolerant and psychrotolerant. The bacterium is sensitive to a range of beta-lactam, sulfonamide, and aminoglycoside antibiotics, but resistant to trimethoprim. The range of amino acids, sugars, and polyols utilized as growth substrates indicates that this alkaliphile is a heterotrophic bacterium. D(+)-glucose, D(+)-glucose-6-phosphate, D(+)-cellobiose, starch, or sucrose are the substrates best utilized. The major membrane lipids are phosphatidylglycerol and diphosphatidylglycerol, with smaller amounts of phosphatidylethanolamine and an unknown phospholipid. During growth at high pH, the proportion of phosphatidylglycerol is increased relative to phosphatidylethanolamine. The fatty acyl components in the membrane phospholipids are mainly branched chain, with 13-methyl tetradecanoic and 12-methyl tetradecanoic acids as the predominant components. The G + C content of the genomic DNA is 41.1 +/- 1.0 mol%. The results of 16S ribosomal RNA sequence analysis place this alkaliphilic bacterium in a cluster, together with an unnamed alkaliphilic Bacillus species (98.2% similarity).

Alkalies↗
PubMed2000

Toward a molecular understanding of cold activity of enzymes from psychrophiles.

Despite the fact that a much greater proportion of the earth environment is cold rather than hot, much less is known about psychrophilic, cold-adapted microorganisms compared with thermophiles living at high temperatures. In particular, investigation of the molecular basis of cold-active enzymes from psychrophiles has only recently received concerted research attention, in measure as a result of the EC-funded project COLDZYME. This research effort has been stimulated by the realization that such cold-active enzymes offer novel opportunities for biotechnological exploitation. Only very recently has the first cold-active enzyme, alpha-amylase, been crystallized, and this success was followed rapidly by others. This effort has facilitated a direct approach to solving the three-dimensional structure of cold-active enzymes to complement the gene homology modeling that had been performed previously. Recently studies have highlighted how different adaptations are used by different enzymes to achieve conformational flexibility at low temperatures, and how such adaptations are not necessarily the opposite of those that confer thermostability to proteins in thermophilic counterparts. This review also highlights initial successes in engineering genetically improved thermal stability in cold-active enzymes to give improved catalysts for low-temperature biotechnology.

Adaptation, Physiological↗
PubMed2000

Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55.

The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented. EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms. To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced. The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa. A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins. Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis. These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions. Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature.

Amino Acid Sequence↗
PubMed1999

Pulsed high electric field causes 'all or nothing' membrane damage in Listeria monocytogenes and Salmonella typhimurium, but membrane H+-ATPase is not a primary target.

Salmonella typhimurium (CRA 1005) was more sensitive than Listeria monocytogenes (NCTC 11994) to pulsed high electric field (PHEF) treatment in distilled water (10, 15 and 20 kV/cm), 10 mM Tris-maleate buffer, pH 7.4 (15 kV/cm) and model beef broth (0.75%, w/v; 15 kV/cm). Sublethal injury could not be detected using a selective medium plating technique, indicating that bacterial inactivation by PHEF may be an 'all or nothing' event. PHEF-induced membrane permeabilisation resulted in an increase in the leakage of UV-absorbing material from the bacteria (UV-leakage) and a decreased ability of L. monocytogenes to maintain a pH gradient. A lack of correlation between the inhibition of H+-ATPase activity and PHEF treatment, cell viability or UV-leakage indicates that this enzyme is probably not a primary site of bacterial inactivation despite its role in the maintenance of internal pH.

Cell Membrane↗
PubMed1998

Preliminary crystallographic studies of citrate synthase from an Antarctic psychrotolerant bacterium.

Recombinant citrate synthase from a psychrotolerant bacterium, DS2-3R, recently isolated in Antarctica, has been crystallized. The crystals belong to space group P6122 or P6522, with cell dimensions a = b = 70.8, c = 307.8 A. Diffraction data collected on a synchrotron from a cryoprotected crystal extends to at least 2.0 A. Knowledge of the structure of this enzyme will add to the understanding of cold activity and thermolability, and will be of biotechnological interest. Previously, the structure of citrate synthase from Archaea inhabiting environments at 328 and 373 K, has been reported. This present study will extend our understanding of the structural integrity and activity of proteins at the temperature extremes of life.

Adaptation, Physiological↗
PubMed1998

The effect of growth temperature on the phospholipid and fatty acyl compositions of non-proteolytic Clostridium botulinum.

A non-proteolytic strain of Clostridium botulinum (NCIB 4270) was found to have a complex lipid composition, comprising five major phosphorus-containing lipids: phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylserine (PS) and a glycophospholipid of unknown structure (GPL), in order of abundance. Changing the growth temperature did not alter the lipid composition either qualitatively or quantitatively. The main fatty acyl components of the lipids are 14:0, 16:0 and 16:1. When the growth temperature was lowered from 37 to 8 degrees C, there was an increase in 14:0 from 16.4 to 37.5%, an increase in 16:1 from 10.5 to 22.5%, and a decrease in the proportion of 16:0 from 40.3 to 19.1%. There was also a decrease in the proportion of cyclopropane fatty acids (15:0cyc and 17:0cyc) from 7.3 to 0.5%, and in the equivalent chain length of the total fatty acids from 15.9 to 15.3 as the temperature was lowered. The same temperature-dependent changes occurred in the five major lipid classes examined. Despite reports of the presence of plasmalogenic forms of phospholipids (i.e. those lipids which have the acyl chain in the sn-1 position replaced by an alk-1-enyl group) in some Clostridium spp., none were detected in C. botulinum NCIB 4270 using either commercially available spray reagents or by gas-liquid chromatographic analysis of the products or acid methanolysis of total lipid extracts. It is concluded that non-proteolytic C. botulinum lacks plasmalogens, typical of other clostridia, in its membranes and instead modulates its fatty acid composition in response to temperature changes in a manner that is typical of other (non-clostridial) bacteria.

Chromatography, Gas↗
PubMed1998

Molecular adaptations in psychrophilic bacteria: potential for biotechnological applications.

Bacteria which live in cold conditions are known as psychrophiles. Since so much of our planet is generally cold, i.e. below 5 degrees C, it is not surprising that they are very common amongst a wide variety of habitats. To enable them to survive and grow in cold environments, psychrophilic bacteria have evolved a complex range of adaptations to all of their cellular components, including their membranes, energy-generating systems, protein synthesis machinery, biodegradative enzymes and the components responsible for nutrient uptake. Whilst such a systems approach to the topic has its advantages, all of the changes can be described in terms of adaptive alterations in the proteins and lipids of the bacterial cell. The present review adopts the latter approach and, following a brief consideration of the definition of psychrophiles and description of their habitats, focuses on those adaptive changes in proteins and lipids, especially those which are either currently being explored for their biotechnological potential or might be so in the future. Such applications for proteins range from the use of cold-active enzymes in the detergent and food industries, in specific biotransformations and environmental bioremediations, to specialised uses in contact lens cleaning fluids and reducing the lactose content of milk; ice-nucleating proteins have potential uses in the manufacture of ice cream or artificial snow; for lipids, the uses include dietary supplements in the form of polyunsaturated fatty acids from some Antarctic marine psychrophiles.

Bacteria↗
PubMed1997

Polyunsaturated fatty acids in the psychrophilic bacterium Shewanella gelidimarina ACAM 456T: molecular species analysis of major phospholipids and biosynthesis of eicosapentaenoic acid.

The production of eicosapentaenoic acid [20:5omega3; EPA] from Shewanella gelidimarina (ACAM 456T) was investigated with respect to growth temperature and growth on sole carbon sources. The percentage and quantitative yield of EPA remained relatively constant at all growth temperatures within or below the optimal growth temperature region. At higher growth temperatures, these values decreased greatly. Growth on differing sole carbon sources also influenced the percentage and amount of EPA produced, with the fatty acid composition influenced by provision of potential acyl chain primers as sole carbon sources. The highest amounts of EPA occurred from growth on propionic acid and L-leucine respectively, while the highest percentage of EPA occurred from growth on L-proline. Monounsaturated fatty acid components and EPA were concentrated in phosphatidylglycerol (PG), while the proportion of branched-chain fatty acids was elevated in phosphatidylethanolamine (PE); the two major phospholipid classes. Specific associations of EPA with other acyl chains were identified within cellular phospholipid classes. The association of EPA with 17:1 and 18:0 acyl chains in phospholipid species was specific to PG, whereas the association of EPA with i13:0/13:0 and 14:0/i14:0 was specific to PE. Such acyl chain 'tailoring' is indicative of the important role of EPA in bacterial membrane adaptive responses. EPA was also a large component (22%) of a non-esterified fatty acid (NEFA) fraction within the total lipid extract of the bacterium. This may point toward a particular role of NEFA in polyunsaturated fatty acid (PUFA) metabolism. The formation of EPA was investigated by labelling with L-[U-14C]serine and sodium [1-14C]acetate. The accumulation of radiolabel within unsaturated intermediates (di-, tri- and tetraunsaturated fractions) was low, indicating a rapid formation and derivatisation of these components. Similar results were found for the unsaturated fatty acid fractions of both PE and PG using sodium [1-14C]acetate radiolabel. The regulation of triunsaturated fatty acid components may be a potential control site in PUFA biosynthesis.

Carbon Radioisotopes↗
PubMed1997

Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3R.

The gene encoding citrate synthase from a novel bacterial isolate (DS2-3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degrees C, and show significant activity at 10 degrees C and below. Comparison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The amino acid sequence of the DS2-3R enzyme shows several features previously recognised in other cold-active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.

Amino Acid Sequence↗
PubMed1997

Inhibition of ergosterol biosynthesis is not accompanied by a change in fatty acid composition in Saccharomyces cerevisiae treated with the antifungal agent 6-amino-2-n-pentylthiobenzothiazole.

The antifungal agent 6-amino-2-n-pentylthiobenzothiazole at a concentration of 40 microM lowered the specific growth rate of exponentially growing cultures of Saccharomyces cerevisiae by 36%. Treatment with 6-amino-2-n-pentylthiobenzothiazole inhibited the biosynthesis of ergosterol and caused an accumulation of the methylated sterol precursors ergosta-5,7-dienol and squalene, but had no significant effect on the composition and the rate of biosynthesis of fatty acids. The results indicate that neither the inhibition of ergosterol biosynthesis, nor the slowing-down of culture growth by this antifungal agent, led to a compensatory alteration in the pattern of fatty-acyl chains in membrane lipids. This finding contradicts the accepted wisdom for the action of a number of antifungal agents.

Acetates↗
PubMed1997

The germinability of spores of a psychrotolerant, non-proteolytic strain of Clostridium botulinum is influenced by their formation and storage temperature.

The formation and storage temperatures of Clostridium botulinum spores are shown to influence their subsequent ability to germinate. Spores were formed at 10 degrees, 20 degrees, 30 degrees and 37 degrees C and following harvest were stored as aqueous suspensions at 20 degrees C (ambient temperature), 4 degrees C (refrigerated) or -20 degrees C (frozen) for periods of up to 1 month. The spores formed at 20 degrees C germinated most rapidly and to the greatest extent. When the spores were germinated immediately after harvest (fresh), there was no difference in the germinability of those spores formed at 20 degrees or 30 degrees C, whether or not they had been heat-shocked before use. However, following storage overnight or longer, differences in the relative germinabilities of the different spore samples were seen. Spores which had been stored at ambient temperature overnight germinated significantly faster and to a greater extent than did those which had been stored for up to 1 month. Similar differences were also observed between spores germinated fresh and those stored overnight, when the spores were stored refrigerated or frozen. Germinability was also influenced by the temperature of storage, since there were differences between spores formed at the same temperature but stored at different temperatures for the same period of time: for example, when spores which had been formed at 20 degrees C were germinated at 10 degrees C following a heat-shock, those which had been stored at ambient temperature germinated faster and to a greater extent than did those which had been stored refrigerated or frozen. It is concluded that there is a complex interaction between formation, storage and germination temperatures, which determines spore germinability. The fact that the changes are time-dependent and can occur in the frozen state is taken to mean that they are physico-chemical rather than metabolic. It is also significant in relation to refrigerated foods which are at risk from Cl. botulinum in that changes which occur during cool or frozen storage can enhance the germinability of spores if the temperature rises above that of chill cabinets.

Clostridium botulinum↗
PubMed1997

Modifications of membrane phospholipid composition in nisin-resistant Listeria monocytogenes Scott A.

A nisin-resistant (NISr) variant of Listeria monocytogenes Scott A was isolated by stepwise exposure to increasing concentrations of nisin in brain heart infusion (BHI) broth. The NISr strain was about 12 times more resistant to nisin than was the wild-type (WT) strain. Accordingly, higher nisin concentrations were required to dissipate both components of the proton motive force in the NISr strain than in the WT strain. Comparison of the membrane fatty acyl composition of the sensitive strain with that of its NISr derivative revealed no significant differences. From phospholipid head group composition analysis and phospholipid biosynthesis measurements during growth in the absence and presence of nisin, it could be inferred that the NISr strain produces relatively more phosphatidylglycerol (PG) and less diphosphatidylglycerol (DPG) than the parent strain does. Monolayer studies with pure lipid extracts from both strains showed that nisin interacted more efficiently with lipids derived from the WT strain than with those derived from the NISr strain, reflecting qualitative differences in nisin sensitivity. Involvement of the cell wall in acquisition of nisin resistance was excluded, since the WT and NISr strains showed a comparable sensitivity to lysozyme. Recently, it has been demonstrated that nisin penetrates more deeply into lipid monolayers of DPG than those of other lipids including PG, phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol (R.A. Demel, T. Peelen, R.J. Siezen, B. de Kruijff, and O.P. Kuipers, Eur. J.Biochem. 235:267-274, 1996). Collectively, the mechanism of nisin resistance in this L. monocytogenes NISr strain is attributed to a reduction in the DPG content of the cytoplasmic membrane.

Drug Resistance, Microbial↗