Gentle lysis of mucous producing cold-adapted bacteria by surfactant treatment combined with mechanical disruption.

A procedure for the enhanced lysis of mucous producing psychrotrophic gram positive bacteria for subsequent enzyme studies is described. An initial washing of bacterial cells with Tween 80 was found to improve the degree of cell disruption in subsequent sonication or grinding with glass beads, resulting in about 20-200% increase in total soluble protein content. However, in terms of lactate dehydrogenase (LDH) activity present in the lysate, pretreatment with Tween 80 was more effective in combination with grinding, especially in the highly mucous producing strain GY11. The type of surfactant used in the pretreatment procedure before grinding strongly influenced the percentage lysis of tested strains, both in terms of released soluble protein and enzyme activity. Zymograms of LDH and glutamate dehydrogenase (GDH) activity present in the lysates also very well supported the results obtained by total protein and enzyme activity measurements.

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.

Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria.

Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.

Relationship between effect of ethanol on proton flux across plasma membrane and ethanol tolerance, in Pichia stipitis.

Pichia stipitis efficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. In Saccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. In P. stipitis the passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H(+)-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H(+)-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H(+)-extrusion. Comparison of the rates of H(+)-flux with the related in vitro H(+)-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) of P. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H(+)-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grown P. stipitis cells.

Exposure to enteroviruses and hepatitis A virus among divers in environmental waters in France, first biological and serological survey of a controlled cohort.

An epidemiological study of hepatitis A and enteroviruses was conducted in a military diving training school, by evaluating the viral contamination of water using an ultrafiltration concentration technique, and assessing seroconversion and the presence of virus in stool specimens obtained from 109 divers and 48 controls. Three of 29 water specimens were positive for enterovirus by cell culture and 9 by molecular hybridization. There was little or no risk of virus infection during the training course (49 h exposure) because there was no significant difference between divers and controls for both viral isolation and seroconversion. However, a higher percentage of coxsackievirus B4 and B5 seropositive divers suggests that these were more exposed during previous water training. No hepatitis A virus (HAV) detection and no seroconversion to HAV was observed. The rate of HAV seropositive subjects was 17% in this 24.5-year-old population.

Microbial oxidation and reduction of manganese: consequences in groundwater and applications.

In the natural environment, manganese is found as reduced soluble or adsorbed Mn(II) and insoluble Mn(III) and Mn(IV) oxides. Mn oxidation has been reported in various microorganisms. Several possible pathways, indirect or direct, have been proposed. A wider variety of Mn-reducing microorganisms, from highly aerobic to strictly anaerobic, has been described. The mechanisms of Mn reduction can be either an indirect process resulting from interactions with organic or inorganic compounds, or a direct enzymatic (electron-transfer) reaction. The role of microorganisms in Mn cycle is now well demonstrated by various methods in superficial natural environments, and research has been initiated on subsurface sediments. Observations in vivo (Rhône valley) and under in vitro suggested that bacterial activities are the main processes that promote manganese evolution and migration in shallow aquifers. After the building of hydroelectric dams, the stream of the Rhône was modified, giving rise to mud deposition on the bank. In the mud, bacteria are stimulated by the high organic content and consume oxygen. The redox potential drops. The manganese oxides previously formed under aerobic conditions are reduced and soluble manganese (Mn(II)) migrates into the aquifer. If the subsurface sediments are coarse-grained, the aquifer is well aerated, allowing the re-oxidation of Mn(II) by the oligotrophic attached bacteria in aquifer sediments. If the aquifer is confined, aeration is not sufficient for Mn-reoxidation. Mn(II) remains in a reduced state and migrates to the wells. Furthermore, the presence of organic matter in subsurface sediments results in the reduction of previously formed Mn oxides. Pseudo-amorphous manganese oxides, which were probably recently formed by bacteria, are more readily reduced than old crystalline manganese oxides. Although the concentrations of soluble manganese found in groundwaters are not toxic, it still is a problem since its oxidation results in darkening of water and plugging of pipes in drinking or industrial water systems. Soluble manganese can be removed from water by biological processes involving manganese-oxidizing bacteria, either in situ, or in sand filters after pumping. Various procedures are mentioned.

Microbial manganese reduction mediated by bacterial strains isolated from aquifer sediments.

One hundred and five strains isolated from aquifer sediments andEscherichia coli ML30S were tested for their ability to reduce manganese oxides. Eighty-two strains, includingE. coli, reduced manganese. In most cases the bacterial activity decreased the pH and Eh below 6.75 and 350 mV, respectively, enhancing a spontaneous and nonspecific reduction of manganese. However, for 12 strains the reduction was specifically catalyzed by bacteria; the high pH and Eh values would not permit a spontaneous reduction of manganese. Some of the most active strains were identified as genera common in soils and waters, i.e.,Pseudomonas, Bacillus, Corynebacterium, andAcinetobacter. Two strains were studied in detail. One of the strains, identified asPseudomonas fluorescens, required contact between the cells and the manganese oxides for reduction to occur. The reduction was inhibited by 15 mM of sodium azide. The other strain, identified asAcinetobacter johnsonii, catalyzed manganese reduction by an inductive and dialyzable substance which was excreted by the bacteria. The mechanism involved has not been previously demonstrated.

Psychrophilic and psychrotrophic microorganisms.

Psychrophilic and psychrotrophic microorganisms have the ability to grow at 0 degree C. Psychrotrophic microorganisms have a maximum temperature for growth above 20 degrees C and are widespread in natural environments and in foods. Psychrophilic microorganisms have a maximum temperature for growth at 20 degrees C or below and are restricted to permanently cold habitats. This ability to grow at low temperature may be correlated with a lower temperature characteristic than that of the mesophiles, an increasing proportion of unsaturated fatty acids in the lipid phase of the cell membrane, which makes it more fluid, and a protein conformation functional at low temperature. The relatively low maximum temperature of growth for these microorganisms is often considered to be due to the thermolability of one or more essential cellular components, particularly enzymes, while some degradative activities are enhanced, resulting in an exhaustion of cell energy, a leakage of intracellular substances or complete lysis. Psychrotrophic microorganisms are well-known for their degradative activities in foods. Some are pathogenic or toxinogenic for man, animals or plants. However in natural microbial ecosystems psychrotrophic and psychrophilic microorganisms can play a large role in the biodegradation of organic matter during cold seasons.

Screening of microorganisms for nitrosation catalysis at pH 7 and kinetic studies on nitrosamine formation from secondary amines by E. coli strains.

Thirty-eight strains of microorganisms isolated from infected human trachea, urine, blood and faeces were examined for their ability to form N-nitrosomorpholine from morpholine and nitrite at pH 7.25. Twenty-five bacterial strains exhibited nitrosation activity, including 18 out of 19 strains of Escherichia coli and three out of nine Pseudomonas aeruginosa, Proteus morganii, Proteus mirabilis, Klebsiella pneumoniae and Neisseria strains; E. coli A10 strain showed the highest activity. Formation of N-nitrosomorpholine was proportional to the incubation time up to 2 h and to the number of resting E. coli A10 cells; the reaction followed Michaelis-Menten kinetics. Nitrosation rate appeared to be dependent on the pKa value of several amines studied. As the nitrosation catalysis was heat-labile, our data suggest that N-nitrosation is catalysed by a bacterial enzyme(s). This reaction may lead to enhanced endogenous nitrosation in subjects suffering from an achlorhydric stomach or from chronic urinary tract infections.

Effects of temperature on the macromolecular composition and find structure of psychrophilic Arthrobacter species.

A facultatively psychrophilic bacterium, Arthrobacter SI 55, grows at 20 degrees C, but growth, as measured by increase of viable cell count, is inhibited at 32 degrees C. Corresponding temperatures for an obligate psychrophile, Arthrobacter glacialis SI 137, are 10 degrees C and 19-20 degrees C. At the higher temperatures for each organism increases of cell mass, as measured by turbidity and of DNA, RNA, and protein, were not inhibited. At the upper temperatures, fewer septa were formed in Arthrobacter SI 55, and cells appeared as distorted filaments with irregular brancehs. Arthrobacter glacilis grew as single cells at the lower temperature, but as clumps of coccoid cells with well marked septa at the higher temperature. It appears that in Arthrobacter SI 55 septum formation may be inhibited at the higher temperature. In contrast, in A. glacialis septation occurs but the cells do not separate.

Effects of temperature on the growth of psychrophilic bacteria from glaciers.

Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.