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Involvement of the spore coat in germination of Bacillus cereus T spores.

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.

Alkanes↗

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments.

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.

Dicarboxylic Acids↗

Thermophilic sulfate reduction in hydrothermal sediment of lake tanganyika, East Africa.

In environments with temperatures above 60 degrees C, thermophilic prokaryotes are the only metabolically active life-forms. By using the SO(4) tracer technique, we studied the activity of sulfate-reducing microorganisms (SRM) in hot sediment from a hydrothermal vent site in the northern part of freshwater Lake Tanganyika (East Africa). Incubation of slurry samples at 8 to 90 degrees C demonstrated meso- and thermophilic sulfate reduction with optimum temperatures of 34 to 45 degrees C and 56 to 65 degrees C, respectively, and with an upper temperature limit of 80 degrees C. Sulfate reduction was stimulated at all temperatures by the addition of short-chain fatty acids and benzoate or complex substrates (yeast extract and peptone). A time course experiment showed that linear thermophilic sulfate consumption occurred after a lag phase (12 h) and indicated the presence of a large population of SRM in the hydrothermal sediment. Thermophilic sulfate reduction had a pH optimum of about 7 and was completely inhibited at pH 8.8 to 9.2. SRM could be enriched from hydrothermal chimney and sediment samples at 60 and 75 degrees C. In lactate-grown enrichments, sulfide production occurred at up to 70 and 75 degrees C, with optima at 63 and 71 degrees C, respectively. Several sporulating thermophilic enrichments were morphologically similar to Desulfotomaculum spp. Dissimilatory sulfate reduction in the studied hydrothermal area of Lake Tanganyika apparently has an upper temperature limit of 80 degrees C.

Journal Article↗

Sensitive in situ monitoring of a recombinant bioluminescent Yersinia enterocolitica reporter mutant in real time on Camembert cheese.

Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10 degrees C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm(2). Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a "real-product" status, and at a low temperature.

Cheese↗

Molecular characterization of the nonphotosynthetic partner bacterium in the consortium "Chlorochromatium aggregatum".

Phototrophic consortia represent valuable model systems for the study of signal transduction and coevolution between different bacteria. The phototrophic consortium "Chlorochromatium aggregatum" consists of a colorless central rod-shaped bacterium surrounded by about 20 green-pigmented epibionts. Although the epibiont was identified as a member of the green sulfur bacteria, and recently isolated and characterized in pure culture, the central colorless bacterium has been identified as a member of the beta-Proteobacteria but so far could not be characterized further. In the present study, "C. aggregatum" was enriched chemotactically, and the 16S rRNA gene sequence of the central bacterium was elucidated. Based on the sequence information, fluorescence in situ hybridization probes targeting four different regions of the 16S rRNA were designed and shown to hybridize exclusively to cells of the central bacterium. Phylogenetic analyses of the 1,437-bp-long sequence revealed that the central bacterium of "C. aggregatum" represents a so far isolated phylogenetic lineage related to Rhodoferax spp., Polaromonas vacuolata, and Variovorax paradoxus within the family Comamonadaceae. The majority of relatives of this lineage are not yet cultured and were found in low-temperature aquatic environments or aquatic environments containing xenobiotica or hydrocarbons. In CsCl-bisbenzimidazole equilibrium density gradients, genomic DNA of the central bacterium of "Chlorochromatium aggregatum" formed a distinct band which could be detected by quantitative PCR using specific primers. Using this method, the G+C content of the central bacterium was determined to be 55.6 mol%.

Base Composition↗

Quantitative analysis of group II intron expression and splicing in Lactococcus lactis.

The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

Bacterial Proteins↗

Microbiology of ensiled high-moisture corn.

The effects of gaseous environment and temperature on the microbial populations of ensiled high-moisture corn were investigated. Molds, coliform bacteria, mesophilic aerobic bacteria, and yeasts were enumerated at intervals during ensiling. The numbers of aerobic bacteria were similar in structures containing different concentrations of gas and held at different temperatures. Coliform bacteria could not be detected after 10 days of ensiling. Mold numbers were relatively low, but were important in the deterioration of corn at the surface of the silos. Yeast and bacterial numbers increased rapidly following an initial aeration period, but no increase was observed after a second aeration series. Lack of a growth response to the second aeration series is believed related to the depletion of an assimilable carbon source.

Animal Feed↗

Microbial life at 90 C: the sulfur bacteria of Boulder Spring.

The physiology of the bacteria living in Boulder Spring (Yellowstone National Park) at 90 to 93 C was studied with radioactive isotope techniques under conditions approximating natural ones. Cover slips were immersed in the spring; after a fairly even, dense coating of bacteria had developed, these cover slips were incubated with radioactive isotopes under various conditions and then counted in a gas flow or liquid scintillation counter. Uptake of labeled compounds was virtually completely inhibited by formaldehyde, hydrochloric acid, and mercuric bichloride, and inhibition was also found with streptomycin and sodium azide. The water of Boulder Spring contains about 3 mug of sulfide per ml. Uptake of labeled compounds occurs only if sulfide or another reduced sulfur compound is present during incubation. The pH optimum for uptake of radioactive compounds by Boulder Spring bacteria is 9.2, a value near that of the natural spring water (8.9). Many experiments with a variety of compounds were performed to determine the temperature optimum for uptake of labeled compounds. The results with all the compounds were generally similar, with broad temperature optima between 80 and 90 C, and with significant uptake in boiling (93 C) but not in superheated water (97 C). The results show that the bacteria of Boulder Spring are able to function at the temperature of their environment, although they function better at temperatures somewhat lower. The fine structure of these bacteria has been studied by allowing bacteria in the spring to colonize glass slides or Mylar strips which were immediately fixed, and the bacteria were then embedded and sectioned. The cell envelope structure of these bacteria is quite different from that of other mesophilic or thermophilic bacteria. There is a very distinct plasma membrane, but no morphologically distinct peptidoglycan layer was seen outside of the plasma membrane. Instead, a rather thick diffuse layer was seen, within which a subunit structure was often distinctly visible, and connections frequently occurred between this outer layer and the plasma membrane. The thick outer layer usually consisted of two parts, the outer part of which was sometimes missing. Within the cells, structures resembling ribosomes were seen, and regions lacking electron density which probably contained deoxyribonucleic acid were also visible.

Acetates↗

Hypothermia.

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Cold Temperature↗

Seasonal variation in coronary heart disease in Scotland.

STUDY OBJECTIVE: Seasonality of coronary heart disease (CHD) was examined to determine whether fatal and non-fatal disease have the same annual rhythm. DESIGN: Time series analysis was carried out on retrospective data over a 10 year period and analysed by age groups ( < 45 to > 75 years) and gender. SETTING: Data by month were obtained for the years 1962-71. The Registrar General provided information on deaths and the Research and Intelligence Unit of the Scottish Home and Health Department on hospital admissions. SUBJECTS: In Scotland, between 1962 and 1971, 123 000 patients were admitted to hospital for CHD, of whom 29 000 died. There were a further 97 000 CHD deaths outside hospital. These two groups were also examined as one (coronary incidence) - that is, all coronary deaths and coronary admissions discharged alive. STATISTICAL ANALYSIS AND MAIN RESULTS: Where there was a single annual peak, the sine curve was analysed by cosinor analysis. When there were two peaks the analysis was by normal approximation to Poisson distribution. In younger men (under 45 years) admitted to hospital there was a dominant spring peak and an autumn trough. A bimodal pattern of spring and winter peaks was evident for hospital admissions in older male age groups: with increasing age the spring peak diminished and the winter peak increased. In contrast, female hospital admissions showed a dominant winter/summer pattern of seasonal variation. In male and female CHD deaths seasonal variation showed a dominant pattern of winter peaks and summer troughs, with the winter peak spreading into spring in the two youngest male age groups. CHD incidence in women showed a winter/summer rhythm, but in men the spring peak was dominant up to the age of 55. CONCLUSION: The male, age related spring peak in CHD hospital admissions suggests there is an androgenic risk factor for myocardial infarction operating through an unknown effector mechanism. As age advances and reproduction becomes less important, the well defined winter/summer pattern of seasonal variation of CHD is superimposed, and shows a close relationship with the environment, especially temperature, or the autumn and early winter fall in temperature.

Adult↗

Effects of menstrual cycle and physical training on heat loss responses during dynamic exercise at moderate intensity in a temperate environment.

We evaluated the effects of the menstrual cycle and physical training on heat loss (sweating and cutaneous vasodilation) responses during moderate exercise in a temperate environment. Ten untrained (group U) and seven endurance-trained (group T) women (maximal O2 uptake of 36.7+/-1.1 vs. 49.4+/-1.7 ml.kg-1.min-1, respectively; P<0.05) performed a cycling exercise at 50% maximal O2 uptake for 30 min during both the midfollicular and midluteal menstrual phase in a temperate environment (ambient temperature of 25 degrees C, relative humidity of 45%). In group U, plasma levels of estrone, estradiol, and progesterone at rest and esophageal temperature (Tes) during exercise were significantly higher during the midluteal than during the midfollicular phase (P<0.05). Sweating rate and cutaneous blood flow (measured via laser-Doppler flowmetry) on the chest, back, forearm, and thigh were lower during the midluteal than during the midfollicular phase during exercise. Tes threshold for heat loss responses was significantly higher and sensitivity of the heat loss responses was significantly lower in the midluteal than in the midfollicular phase, regardless of body site. These effects of the menstrual cycle in group U were not observed in group T. The sweating rate and cutaneous blood flow were significantly higher in group T than in group U, regardless of menstrual phase or body site. Tes threshold for heat loss responses was significantly lower and sensitivity of heat loss responses was significantly greater in group T than in group U in the midluteal phase; however, sensitivity of the sweating response was significantly greater in the midfollicular phase. These results suggest that heat loss responses in group U were inhibited in the midluteal phase compared with in the midfollicular phase. Menstrual cycle had no remarkable effects in group T. Physical training improved heat loss responses, which was more marked in the midluteal than in the midfollicular phase.

Adult↗