Bacterial growth rate in the sea: direct analysis by thymidine autoradiography.

Autoradiography with tritiated thymidine was used to study microbial growth directly in nature. The epiphyte Leucothrix mucor was used since it is a large filamentous bacterium with a characteristic morphology making it recognizable in natural collections. The technique was developed initially with pure cultures. The relation between growth rate and the rate of accumulation of radioactive cells permitted derivation of a constant for use in calculating growth rate in natural material and in two-membered cultures of L. mucor growing epiphytically on pure cultures of marine algae. The growth rate (generation time) in two-membered cultures with the red alga Antithamnion sarniense was 94 minutes under the conditions used. In nature the growth rate of a sample from Iceland was 685 minutes; that of a sample from Long Island Sound was 660 minutes. There was no evidence of preferential growth in the basal portion of bacterial filaments nearest the algal surface. However, filamentous growth in nature, but not in pure or two-membered culture, was nonrandom, growth being clustered in some regions.

Life at high temperatures.

Water environments with temperatures up to and above boiling are commonly found in association with geothermal activity. At temperatures above 60 degrees C, only bacteria are found. Bacteria with temperature optima over the range 65 degrees to 105 degrees C have been obtained in pure culture and are the object of many research projects. The upper temperature limit for life in liquid water has not yet been defined, but is likely to be somewhere between 110 degrees and 200 degrees C, since amino acids and nucleotides are destroyed at temperatures over 200 degrees C. Because bacteria capable of growth at high temperatures are found in many phylogenetic groups, it is likely that the ability to grow at high temperature had a polyphyletic origin. The macromolecules of these organisms are inherently more stable to heat than those of conventional organisms, but only small changes in sequence can lead to increases in thermostability. Because of their unique properties, thermophilic organisms and their enzymes have many potential biotechnological uses, and extensive research on industrial applications is under way.

Lower pH limit for the existence of blue-green algae: evolutionary and ecological implications.

Observations on a wide variety of acidic environments, both natural and man-made, reveal that blue-green algae (Cyanophyta) are completely absent from habitats in which the pH is less than 4 or 5, whereas eukaryotic algae flourish. By using enrichment cultures with inocula from habitats of various pH values, the absence of blue-green algae at low pH was confirmed.

Life at high temperatures. Evolutionary, ecological, and biochemical significance of organisms living in hot springs is discussed.

The time is now ripe for a concerted attack on the evolutionary, ecological, and molecular aspects of life at high temperatures. Hot springs provide nearly ideal ecosystems for such study, since they are natural environments of great antiquity and relative constancy, where organisms have evolved to meet the environmental challenges of high temperatures. Even from our present limited knowledge, we can draw a number of conclusions.

Molecular aspects of specific cell contact.

Complementary macromolecules were isolated from yeasts of opposite mating type. These cell-surface molecules neutralize each other as do antibodies and antigens. Both yeast factors are glycoproteins of low molecular weight. Other specific cell associations may be due to the interaction of such complementary macromolecules.

Bacterial stromatolites: origin of laminations.

Laminated mats composed of motile filamentous photosynthetic bacteria and nonmotile unicellular blue-green algae occur in a large number of Yellowstone hot springs at temperatures between 55 degrees and 70 degrees C. Field studies indicate that the bacteria are the predominant mat-forming component. Under low light intensities, mats composed exclusively of bacteria can be formed. The bacteria undergo a diurnal migration, moving on top of the algae dnring the night and becolming mixed again with the algae during the day of differential migration of the bacteria in daily response to reduced light intensities. This response to light is exactly opposite to that previously reported for filamentous stromatolite-forming, blue-green algae, but the net result is the same-formation of a laminated mat.

Siliceous algal and bacterial stromatolites in hot spring and geyser effluents of yellowstone national park.

Growing algal and bacterial stromatolites composed of nearly amorphous silica occur around hot springs and geysers in Yellowstone National Park, Wyoming. Some Precambrian stromatolites may be bacterial rather than algal, which has important implications in atmospheric evolution, since bacterial photo-synthesis does not release oxygen. Conophyton stromatolites were thought to have become extinct at the end of the Precambrian, but are still growing in hot spring effluents.

Bacterial growth rates above 90 degrees C in Yellowstone hot springs.

Growth rates of bacteria living in boiling springs have been measured by determining rate of increase in cell numbers on microscope slides immersed in the springs. Distinction between growth and passive attachment was made with ultraviolet radiation. In all cases, slides irradiated at intervals had significantly fewer bacteria than controls. Estimated generation times ranged from 2 to 7 hours, values which are comparable to those of aquatic bacteria living in less extreme environments.

Limits of microbial existence: temperature and pH.

A microscopic survey made to detect the presence of bacteria in hot springs of varying temperature and pH characteristics revealed that in neutral and alkaline hot springs bacteria are found at temperatures up to the boiling point of water (92 degrees to 100 degrees C, depending on the altitude). In hot springs of increasing acidity the upper temperature limit at which bacteria are found decreases; at pH 2 to 3 the upper temperature limit is 75 degrees to 80 degrees C. Bacteria have thus been able to evolve with the ability to grow at either high temperature or high acidity, but not at both high temperature and high acidity. These results suggest that there are physicochemical limitations of the environment beyond which life is impossible.

Bacterial origin of sulfuric Acid in geothermal habitats.

Natural populations of Sulfolobus, a new genus of bacteria occurring in sulfur-rich, acid hot springs and soils, were found to oxidize large amounts of sulfur to sulfuric acid at temperatures up to 85 degrees C. These bacteria are important high-temperature geochemical agents in solfatara soils.

A thermophilic, acidophilic mycoplasma isolated from a coal refuse pile.

A thermophilic, acidophilic procaryote lacking a cell wall has been isolated from a coal refuse pile which had undergone self-heating. Electron micrographs, chemical assays for hexosamine, and the inability of vancomycin to inhibit growth confirm the lack of a cell wall. The apparent ability of the organism to reproduce by budding and the low guanine plus cytosine content of its DNA indicate a relation to the mycoplasmas. The temperature optimum of the organism is 59 degrees C, and growth occurs over a range of 45 degrees to 62 degrees C. No growth occurs at 37 degrees C or at 65 degrees C. The optimum pH for growth is between 1 and 2, and growth occurs between pH 0.96 and 3.5 but does not occur at pH 0.35 and only poorly at pH 4.0. We propose to call this organism Thermoplasma acidophila. The existence of this organism extends considerably the range of habitats in which mycoplasma may occur.

TAXONOMIC CONFUSION CONCERNING CERTAIN FILAMENTOUS BLUE-GREEN ALGAE(1).

Throughout a long history many filamentous bacteria may have been identified in natural collections as blue-green algae. This problem has been especially acute regarding the thermophilic species of hot springs, especially at the higher temperatures. It is suggested that in the absence of pure cultures, the minimal criteria for distinguishing filamentous bacteria from blue-green algae microscopically should be: (1) observation of the chlorophyll fluorescence with a fluorescent microscope and (2) demonstration of light-dependent (14) CO2 fixation autoradiographically. Pure cultures of a number of filamentous thermophiles have been obtained from habitats at temperatures above 60 C. These cultures resemble microscopically the natural material, grow only heterot rophically, and do not contain chlorophyll.