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Showing papers by "Thomas D. Brock published in 1971"


Journal ArticleDOI
TL;DR: The objective was to establish an experimental procedure and show that rate of appearance of Unlabeled Cells in Labeling Experiments was positively correlated with the number of labeled Cells.
Abstract: INTRODUCTION ........................................................... Commitment to Nature..................................................... What is Nature? ............................................................ POPULATION PROBLEMS .............................................. ASSESSMENT OF MICROBIAL NUMBERS OR MASS......................... DEFINING BOUNDARIES OF THE HABITAT ............................... ARTIFICIAL SUBSTRATES ............................................. USE OF THE MICROSCOPE IN NATURE ................................ MEASUREMENT OF GROWTH RATE OF SINGLE ORGANISMS............ GROWTH RATES OF DEVELOPING MICROCOLONIES........ GROWTH RATES OF FILAMENTOUS ORGANISMS ..................... METHODS BASED ON ANALYSIS OF THE CELL-DIVISION CYCLE........ Labeled Thymidine Methods............................................... GROWTH RATES OF PROCARYOTES AS MEASURED BY THYMIDINE AUTORADIOGRAPHY Ecological Approximations for Labeling Experiments.......................... Rate of Appearance of Unlabeled Cells......................................... GENETIC METHODS .................................................... METHODS ESPECIALLY SUITED TO STEADY-STATE POPULATIONS...... INOCULUM SIZE: RATE OF EFFECT..................................... CONCLUDING STATEMENT ............................................ LITERATURE CITED.....................................................

164 citations


Journal ArticleDOI
TL;DR: 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.
Abstract: 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 μg 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. Images

102 citations


Journal ArticleDOI
TL;DR: Metabolic studies indicated turnover of the diacyl phospholipids during pulse-chase experiments at rates comparable with mesophilic bacteria and the majorospholipid and the carotenoids did not turn over.
Abstract: The complex lipids of Thermus aquaticus include phospholipids, glucolipids, carotenoids, and vitamin K(2) isoprenologues. The phospholipids account for 30% of the total lipids and have been identified as phosphatidylethanolamine (4%), phosphatidylglycerol (3%), phosphatidylinositol (10%), cardiolipin (3%), and phosphatidic acid (1%). The major phospholipid contained three fatty acids, a long-chain unsaturated amine, and one glycerol per phosphate and accounted for 80% of the lipid phosphate. The carotenoids accounted for 60% of the membrane lipid. The majority of the carotenoids were very polar. Mono- and diglucosyldiglyceride and the 35-, 40-, and 45-carbon vitamin K(2) isoprenologues were also identified. All these lipids were localized in the membrane of T. aquaticus. When the growth temperature was increased from 50 to 75 C and measured at 5 C intervals, there was a progressive increase in the total lipid content. The phospholipids increased 2-fold, the carotenoids increased 1.8-fold, and the glucolipids increased 4-fold between cells grown at 50 C and 75 C. The vitamin K(2) level did not change. The proportions of the individual lipids within each lipid class remained constant as the temperature of growth was raised. Metabolic studies indicated turnover of the diacyl phospholipids during pulse-chase experiments at rates comparable with mesophilic bacteria. The major phospholipid and the carotenoids did not turn over.

80 citations


Journal ArticleDOI
TL;DR: When cells were grown on glutamate instead of yeast-extract and tryptone at 70 C, the total fatty acid content remained constant, but there was an increase in the proportions of iso-C(16) and normal-C (16) fatty acids concomitant with a decrease in the proportion of the iso- C(15) and iso-D(17) fatty acid.
Abstract: Thermus aquaticus contains four major fatty acids, iso-C15 (28%), iso-C16 (9%), normal-C16 (13%), and iso-C17 (48%), when grown at 70 C, as determined by gas chromatography and mass spectrometry. Small amounts of iso-C12, normal-C12:1, iso-C13, normal-C14, iso-C14, and normal-C15:1 were also detected. A change in growth temperature (50 to 75 C at 5-C intervals) affects a shift in the proportions of some of the fatty acids. The proportions of the monoenoic and branched-C17 fatty acids decreased and the proportions of the higher-melting iso-C16 and normal-C16 fatty acids increased. Cells grown at 75 C contained 70% more total fatty acids than cells grown at 50 C. The largest increases, in absolute amounts, were in the content of iso-C16 and normal-C16 fatty acids, with only a 1.6-fold increase in the major iso-C15 and iso-C17 fatty acids. There was a 2.5-fold decrease in normal-C15:1 and at least a 24-fold decrease in anteiso-C17, which is present at 50 and 55 C but not at higher temperatures. There was no difference in proportion or amount of fatty acids between exponential and stationary-phase cells grown at 70 C. When cells were grown on glutamate instead of yeast-extract and tryptone at 70 C, the total fatty acid content remained constant, but there was an increase in the proportions of iso-C16 and normal-C16 fatty acids concomitant with a decrease in the proportions of the iso-C15 and iso-C17 fatty acids.

79 citations


Journal ArticleDOI
TL;DR: The upper temperature limit for blue green algae in New Zealand is 60-65°c, and the species living at the thermal limit is generally Mastigocladus laminosus, although in some cases Phormidium sp. or Synechococcus sp. was found as discussed by the authors.
Abstract: This account of studies on the algae and bacteria of North Island thermal areas records temperature, pH and species found in these microbial habitats, with special attention to organisms living at the highest temperatures. Thermal features were studied at Rotorua (Whakarewarewa and Ohinemutu), Waiotapu (Tourist Reserve and Lady Knox Geyser), Orakei Korako, Taupo Spa, Waikite Springs, Wairakei thermal valley, Wairakei geothermal field, Tikitere, Ketetahi, Lake Rotokawa (Taupo region), Waimangu, De Brett Thermal Hotel (Taupo). The upper temperature limit for blue‐green algae in New Zealand is 60–65°c, and the species living at the thermal limit is generally Mastigocladus laminosus, although in some cases Phormidium sp. or Synechococcus sp. was found. The Synechococcus sp. characteristic of high temperatures (73–74°c) present in North America was not found in New Zealand. In virtually all boiling pools (99–101°c) with pH values in the neutral and alkaline range bacteria were found, but in acidic boi...

39 citations


Journal ArticleDOI
TL;DR: In this paper, a bimodal distribution curve was obtained for many springs with pH values of 2-4 and 7-9 but relatively few in between, providing quantitative evidence for the existence of two main kinds of thermal springs: acid sulfate and neutral bicarbonate.
Abstract: Frequencies of thermal springs of various pH values are given for the main thermal areas in Yellowstone Park, other parts of the western United States, New Zealand, and Iceland. A bimodal distribution curve was obtained for many springs with pH values of 2-4 and 7-9 but relatively few in between. These data provide quantitative evidence for the existence of two main kinds of thermal springs: acid sulfate and neutral bicarbonate.

28 citations


Journal ArticleDOI
TL;DR: Protoplasts of Sarcina lutea and Streptococcus faecalis underwent thermal lysis when heated to 60° and above, and spermine, spermidine, cadaverine and Mg2+ partially protected protoplasts against thermalLysis.
Abstract: SUMMARY: Protoplasts of Sarcina lutea and Streptococcus faecalis underwent thermal lysis when heated to 60° and above. [14C]Glycine was released from the internal pool of Strep, faecalis at 50°. Spermine, spermidine, cadaverine and Mg2+ partially protected protoplasts against thermal lysis.

26 citations


Journal ArticleDOI
TL;DR: Data from Thermus aquaticus provides further evidence that thermophily is achieved by virtue of inherently thermal stable constituents which have high temperature optima for biological function.

21 citations


Journal ArticleDOI
19 Feb 1971-Nature
TL;DR: The purpose of this note is to call attention to the fact that usual methods of measuring soil pH seriously underestimate the pH of very acid soils, such as those of coal mine spoils, cat clays3 and solfataras4, in which acidity is due to the presence of free sulphuric acid.
Abstract: THE purpose of this note is to call attention to the fact that usual methods of measuring soil pH1 seriously underestimate the pH of very acid soils, such as those of coal mine spoils2, cat clays3 and solfataras4, in which acidity is due to the presence of free sulphuric acid. Standard methods for measuring soil pH involve making a slurry of soil with (usually) distilled water at a 1 : 2, 1 : 5, or 1 : 10 dilution, and reading the pH of this slurry. Implicit in this procedure is the assumption that the soil is buffered and hence that pH does not change with dilution. This assumption is erroneous in the case of the sulphuric acid soils.

16 citations


Journal ArticleDOI
15 Oct 1971-Nature
TL;DR: This work on Boulder Spring, a superheated spring with a relatively large sulphide content, revealed that the bacteria in that spring incorporated radioactively labelled compounds only when sulphide was present, yet the uptake by bacteria from pool A was inhibited by sulphide.
Abstract: IT is well established that bacteria live in hot springs at temperatures greater than 90° C depending on altitude1–4. We have studied the physiology and temperature optima of these organisms by growing them on coverslips and measuring directly the uptake of various radioactively labelled compounds5. The bacteria were obtained from an unnamed spring, known to us as “pool A”, which is in the Lower Geyser Basin of Yellowstone National Park4, and which has a temperature of 90°–91.5° C and is therefore just below the boiling point for this altitude. It has a pH of 8.6–8.9 and a low sulphide content (0.096 µg/ml.). Our work on Boulder Spring, a superheated spring with a relatively large sulphide content (3.1 µg/ml.), revealed that the bacteria in that spring incorporated radioactively labelled compounds only when sulphide was present6, yet the uptake by bacteria from pool A was inhibited by sulphide.

15 citations


Journal ArticleDOI
TL;DR: In this article, the optimal temperatures for photosynthesis and growth were determined for thermal bluegreen algae inhabiting the drainway of a small geyser in Yellowstone National Park, where the sparse algal mat was exposed to high temperatures only during the brief, highly periodic eruptions of the geysers.
Abstract: Optimal temperatures for photosynthesis and growth were determined for thermal bluegreen algae inhabiting the drainway of a small geyser in Yellowstone National Park. The sparse algal mat was exposed to high temperatures only during the brief, highly periodic eruptions of the geyser. Optimal temperatures for the algae were higher than the mean environmental temperatures; as a consequence the algae experienced temperatures optimal for photosynthesis and growth only during a small fraction of the time. A heat-treatment experiment indicated that the algae of this mat were resistant to the high temperatures that occurred during eruption, whereas organisms having the same optimal temperatures but inhabiting a thermally constant environment were more heat sensitive. The thermally fluctuating environment appears to limit the extent of algal growth.

01 Jan 1971
TL;DR: A comparison of thefatty acid differences between thermophilic andmesophilic strains ofsporeforming bacilli indicated that higher temperatures ofgrowthcorrelate with higher proportions ofiso-branched-chain saturated fatty acids thanmesophiles.
Abstract: Thermus aquaticus contains fourmajorfatty acids, iso-C15 (28%), iso-C16 (9%), normal-C16 (13%), andiso-C17 (48%), whengrown at70C,as determined bygas chromatography andmass spectrometry. Smallamountsofiso-C12, normal-C12:1, iso-C,1, normal-C14, iso-C14, andnormal-C,,:, were alsodetected. A changein growth temperature (50to75C at5-Cintervals) affects a shift intheproportions of some ofthefatty acids. Theproportions ofthemonoenoic andbranched-C17 fatty acids decreased andtheproportions ofthehigher-melting iso-C16 andnormal-C16 fatty acids increased. Cells grown at75Ccontained 70%more total fatty acids than cells grown at50C.Thelargest increases, inabsolute amounts,were inthecontent ofiso-C16 andnormal-C,1 fatty acids, withonly a 1.6-fold increase inthemajorisoC,1andiso-C1, fatty acids. There was a 2.5-fold decrease innormal-C,1, andat least a 24-fold decrease inanteiso-C17, whichispresentat50and55C butnotat higher temperatures. There was nodifference inproportion or amountoffatty acids between exponential andstationary-phase cells grown at70C.Whencells were grown on glutamate instead ofyeast-extract andtryptone at70C,thetotal fatty acid content remained constant, butthere was an increase intheproportions ofisoC1,andnormal-C,1 fatty acids concomitant with a decrease intheproportions of theiso-C,5 andiso-C17 fatty acids. Themolecular mechanism ofthermophily has beenattributed to(i) thestability ofthelipids in themembrane, (ii) ahighrate ofbreakdown and synthesis ofthemacromolecules, and(iii) aphysicochemical difference between themacromolecules ofthermophiles andmesophiles (17). Brock (3)hypothesized thattheintegrity ofthecell membrane maybethelimiting factor inthermal death, thussuggesting that themembrane componentsofmesophiles andthermophiles should differ. Thefatty acidcomposition ofsomemesophiles hasaprogressively higher melting point as thetemperature ofgrowth increases (14). Inthese organisms, theprogressively higher melting point corresponds todecreasing proportions ofmonounsaturated fatty acids inthelipids. Recent workhasshownthatthermophiles containlarger proportions ofbranched-chain saturated fatty acids thanmesophiles. Bacillus stearothermophilus presumably grownathigh temperature contains 64%branched-chain fatty acids (7). Otherthermophilic sporeforming bacilli containhigher proportions ofbranched-chain and straight-chain saturated fatty acidsandlower proportions ofunsaturated fatty acids thanmesophilic strains (8). A comparison ofthefatty acid differences between thermophilic andmesophilic strains ofsporeforming bacilli indicated that higher temperatures ofgrowthcorrelate with higher proportions ofiso-branched-chain

Book ChapterDOI
01 Jan 1971
TL;DR: In this article, the authors discuss the fundamental physical and chemical limitations on the evolution of organisms able to grow under extreme environmental conditions such as we find in polluted situations, such as high temperature and low pH.
Abstract: The question I would like to raise in this brief article is whether there are fundamental physical and chemical limitations on the evolution of organisms able to grow under extreme environmental conditions such as we find in polluted situations. The specific environmental factors I would like to consider are high temperature and low pH, and high temperature and low pH taken together. Temperature and hydrogen ion concentration are probably the most basic environmental factors which organisms must cope with, and both natural and polluted environments exist in which these factors exist in the extreme. The attitude and approach here is quite different than that of the physiologist or biochemist, who looks at how a given organism responds to environmental change. We are interested in how living organisms, taken as totality, respond. This means we must look at stable natural environments which have been available for colonization for millions of years, so that we know there has been time for evolution to reach an equilibrium. We thus study natural thermal and acidic springs, since these provide relatively constant environments of types which have probably been available for colonization as long as life has been present on earth. Much of our own work has been reviewed recently (Brock, 1967, 1969, 1970) so that in the present paper I will give only an outline of past studies and concentrate on recent studies which have not yet been published. To simplify the discussion, I will consider high temperature and low pH separately, and then consider the two factors together.

01 Jan 1971
TL;DR: It was shown thatthermophilic bacilli increase the proportions ofbranched-chain fatty acids and decrease theproportions ofmonoenoic andheptanoic fatty acids asthetemperature ofgrowth is increased, and it was shown by Shenet al.(20) that thermophily is more closely associated with theinherent thermostability of thecellular membrane thantothe specific macromolecules.
Abstract: Thecomplexlipids ofThermus aquaticus include phospholipids, glucolipids, carotenoids, andvitamin K2isoprenologues. Thephospholipids accountfor30%of thetotal lipids andhavebeenidentified as phosphatidylethanolamine (4%), phosphatidylglycerol (3%), phosphatidylinositol (10%), cardiolipin (3%), andphosphatidic acid(1%). Themajorphospholipid contained three fatty acids, a long-chain unsaturated amine, andoneglycerol perphosphate andaccounted for80%ofthe lipid phosphate. Thecarotenoids accounted for60%ofthemembrane lipid. The majority ofthecarotenoids were verypolar. Mono-anddiglucosyldiglyceride and the35-, 40-,and45-carbon vitamin K2isoprenologues were alsoidentified. All these lipids were localized inthemembrane ofT.aquaticus. Whenthegrowth temperature was increased from50to75C andmeasured at5C intervals, there was a progressive increase inthetotal lipid content. Thephospholipids increased 2fold, thecarotenoids increased 1.8-fold, andtheglucolipids increased 4-fold betweencells grown at50C and75C.Thevitamin K2level didnotchange. The proportions oftheindividual lipids within eachlipid class remained constant asthe temperature ofgrowth was raised. Metabolic studies indicated turnoverofthe diacyl phospholipids during pulse-chase experiments atratescomparable with mesophilic bacteria. Themajor phospholipid andthecarotenoids didnotturnover. Attempts toexplain thermophily havegenerally beencentered aroundproteins andproteinsynthesizing systems(10). Brock(5)proposed thatthemolecular mechanism ofthermophily is more closely associated withtheinherent thermostability ofthecellular membrane thantothe specific macromolecules. Thistheory issupported bytheworkofBodmanandWelker(4) concerning thestability ofBacillus stearothermophilus protoplasts whenexposed totemperaturesup to65C,andour work(unpublished data) concerning thestability ofThermus aquaticus spheroplasts toincreased temperatures (90 C).Inconsidering thestability ofa membrane, one can either investigate themembrane as a whole or study individual components, like proteins or lipids. Inthisstudy, thelipids ofthe membrane ofT.aquaticus wereexamined. Mostoftheinvestigations on thelipids ofthermophilic bacteria havebeenconcerned withthe fatty acidcomposition. Itwas shownbyShenet al.(20)thatthermophilic bacilli increase the proportions ofbranched-chain fatty acidsand decrease theproportions ofmonoenoic andheptanoic fatty acids asthetemperature ofgrowth is