“ 微生物学 ” 考试时间地点 § 时间： 2000 年 1 月 9 日上午 8:00-10:00 § 地点：四教 4206

Detailed phylogenetic tree of the Archaea based on 16S ribosomal RNA sequence Comparisons

Archaeal Membranes and Cell Wall §Archaea lack fatty acids, instead have hydrocarbon moieties bonded to glycerol by ether (instead of ester) linkages §Glycerol diethers and diglycerol tetraethers are the major classes of lipids present in Archaea §Archaea do not contain muramic acid and D-amino acids, as in Bacteria §A pseudopeptidoglycan is found in some archaea, it consists of two amino sugars: N-acetylglucosamine and N- acetyltalosaminuronic acid, with only L-amino acids linkages §Some contain a thick wall consists only polysaccharide §Some contain cell walls made of glycoprotein §Some lack carbohydrate in their cell walls and have walls consisting of only protein.

Chapter 20 Prokaryotic Diversity: Archaea §Extremely Halophilic ArchaeaExtremely Halophilic Archaea §Methane-Producing Archaea: MethanogenesMethane-Producing Archaea: Methanogenes §Hyperthermophilic ArchaeaHyperthermophilic Archaea §Thermoplasma: A Cell-Wall-Less ArchaeanThermoplasma: A Cell-Wall-Less Archaean §Limits of Microbial Existence: TemperatureLimits of Microbial Existence: Temperature §Archaea: Earliest Life Forms?Archaea: Earliest Life Forms?

Extremely Halophilic Archaea: inhabitants of highly saline environments such as solar salt evaporation ponds and natural salt lakes Hypersaline habitats: Great Salt Lake in Utah Seawater evaporating ponds: the red-purple Color is due to bacterioruberins and bacterio- rhodopsin of halobacterium

Environments for extremely halophile §Solar salt evaporation ponds §Natural salt lakes §Artificial saline habitats (surfaces of heavily salted food such as certain fish and meats) §Require at least 1.5 M (9%) NaCl for growth §Most species require 2-4 M (12-23%) NaCl for growth §Some can grow at pH of §No harmful to human and animals

Physiology of Extremely Halophilic Archaea §All are chemoorganotrophs §Most are obligate aerobes §All require large amount of sodium for growth §All stain gram negatively, binary fission growth §Most are nonmotile §Halobacterium and Halococcus contain large plasmids §Peptidoglycan replaced by glycoportein in the cell wall §Cellular components exposed to the external environment require high Na + for stability §Cellular internal components require high K + for stability §Na + stabilize the cell walls.

Bacteriorhodopsin and Light-mediated ATP Synthesis Bacteriorhodopsin

Methane-Producing Archaea: Methanogens §Methane formation occurs under strictly anoxic conditions. §CO 2 -type substrates (CO 2, HCOO - and CO) can be used as carbon sources. §Methyl substrates (CH 3 OH, CH 3 NH 2 +, (CH 3 ) 2 NH +, (CH 3 ) 3 NH +, CH 3 SH, (CH 3 ) 2 S) are methanogenic carbon sources. §Acetotrophic substrates such as acetate can also be used to produce methane. l Three classes of methanogenic substrates are known and all release free energy suitable for ATP synthesis

Diversity and Physiology of Methanogenic Archaea §16S ribosomal RNA sequence analyses classify methanogen into seven major groups §All methanogens use NH 4 + as a nitrogen source §A few species can fix molecular nitrogen §Nickel is a trace metal required by all methanogens, it is a component of coenzyme Factor 430 §Iron and Cobalt are also important for methanogens. Pictures on the left: morphological diversity of methanogens

Diversity and Physiology of Methanogenic Archaea §Picture on the left are hyperthermophilic and thermophilic methanogens §Methanococcus jannaschii (85 o C optimal) §Methanococcus igneus (88 o C optimal) §Methanothermus fervidus (85 o C optimal) §Methanothrix thermophila §60 o C optimal) Picture on the right: thin section of methanogenic Archaea: Methanobrevibacter ruminantium Methanosarcina barkeri

Unique Methanogenic Coenzymes §Methanofuran (MF): a low-molecular-weight coenzyme that interacts in the first step of methanogenesis from CO 2. §Methanopterin (reduced form tetrahydro-methanopterin or MF): a methanogenetic coenzyme containing a substituted pterin ( 蝶呤 ) ring, a C 1 carrier during the reduction of CO 2 to CH 4. §Coenzyme M: involved in the final step in methane formation, is the carrier of the methyl group that is reduced to methane by the F 430 -methyl reductase enzyme complex in the final step of methanogenesis. §Coenzyme F 430 : a yellow, soluble, nickel-containing tetrapyrrole that plays an intimate role in the terminal step of methanogenesis as part of the methyl reductase system.

Unique Methanogenic Coenzymes Coenzymes involved in redox reactions §Coenzyme F 420 : an electron donor in methanogenesis. §7-mercaptoheptanoylthreonine phosphate (HS- HTP): an electron donor in methanogenesis, is the final unique coenzyme of the methanogens to be considered.

Coenzymes unique to methanogenic Archaea

Coenzymes Unique to Methanogenic Archaea §The oxidized form of F 420 absorbs light at 420 nm and fluresces blue-green. On reduction, the coenzyme becomes colorless. §The fluorescence of F 420 is a useful tool for preliminary identification of an organism as a methanogen Autofluorescence of the methanogen Methanosarcina barkeri due to the presence of the unique electron carrier F 420.

Pathway of methanogenesis from CO 2

Autotrophy in Methanogens How autotrophic methanogens combine aspects of biosynthesis and bioenergetics. Note how half of the acetyl-CoA molecule produced comes from reactions leading to methanogenesis. C 1 -carrying corrinoid- containing enzyme

Methanogenesis from methyl compounds and acetate Utilization of reactions of the acetyl-CoA pathway during growth on methanol (a) acetate (b)

Energetic of Methanogenesis ATP synthesis linked to a proton motive force established during the terminal step of methanogenesis

Hyperthermophilic Archaea Temperature Optima above 80 o C §Most isolated from geothermally heated soils or waters containing sulfur an sulfides §Most are obligate anaerobes §Many grow chemolithotrophically, with H 2 as energy source

Hyperthermophilic from Volcanic Habitats Acidophilic Hyperthermophilic Archaea Sulfolobus acidocaldarius Acidianus infernus The first such organism discovered, Sulfolobus, grows in sulfur-rich hot acid springs at temperature up to 90 o C and at pH values of 1-5. Acidianus, a facultative aerobe resembling Sulfolobus is also present in acidic solfataric springs, it can also grow anaerobically.

Hyperthermophilic from Volcanic Habitats Acidophilic Hyperthermophilic Archaea §Spherical, obligately anaerobic, S 0 -respiring organism. §Grows best at neutral pH and o C Desulfurococcus saccharovorans

Hyperthermophilic from Volcanic Habitats Acidophilic Hyperthermophilic Archaea §Thermoproteus and Thermofilum inhabit neutral or slightly acidic hot springs, are highly variable in length, ranging from 1-80 microns. §Both are strict anaerobes that carry out a S 0 -based anaerobic respiration. §Most can grow chemolithotrophically. Thermoproteus neutrophilus Thermofilum librum

Hyperthermophilic from Submarine Volcanic Areas §Boiling points increase with water depth. §Pyrodium has a growth optimum of 105 o C, has higher GC(62%). §Cells are irregularly disc- and dish-shaped, grow in culture as a moldlike layer on sulfur crystals suspended in the medium. §Strict anaerobe that grows chemolithotrophically at neutral pH on H 2 with S 0 as electron acceptor. §Growth occur between o C. Pyrodium occultum (optima 105 o C)

Hyperthermophilic from Submarine Volcanic Areas §Pyrobaculum is capable of both aerobic respiration and denitrification (NO 3 - N 2 ). §Organic or inorganic substrates can be used as electron donors §Maxima T=103 o C §H 2, as well as various complex nutrients but not sugars support its growth. §Elemental S o is not used by this organism, even inhibits its growth. Pyrobaculum aerophilum (optima 100 o C)

Hyperthermophilic from Submarine Volcanic Areas §Thermococcus, a spherical hyperthermophilic archaean indigenous to anoxic submarine thermal waters in various location worldwide. §Contains a tuft of polar flagella, highly motile. §Obligately anaerobic chemoorganotroph that grows on proteins and other complex organic mixtures (including some sugars) with Hyperthermococcus celer Dividing cells of Pyrococcus furiosus S 0 as electron acceptor. Optima T=88 o C Pyrococcus grows at between o C with an optimum of 100 o C. Metabolic requirement similar to Hyperthermococcus.

Hyperthermophilic from Submarine Volcanic Areas §Staphylothermus consists of spherical cells about 1 micron in diameter that form aggregates of up to 100 cells. §Strictly anaerobic hyperthermophile growing optimally at 92 o C. §Capable of growth between 65 and 98 o C. §S 0 is required for growth, yet oxidation of complex organic compounds is not tightly coupled to S 0 reduction. Staphylothermus marinus

Hyperthermophilic from Submarine Volcanic Areas §Most Archaea use S 0 as an electron acceptor for anoxic growth, most are unable to use sulfate as an electron acceptor. §Archaeoglobus, is a true sulfate-reducing hyper- thermophile. §Grow at between 64 and 92 o C with T optima=83 o C §Share some metabolic features with methanogens. Archaeoglobus lithotrophicus Methanopyrus kandleri Methanopyrus: gram-positive rod-shaped methanogen grown above 100 o C. The most ancient hyper- thermophile Share phenotypical properties with both the hyperthermophiles and methanogens.

Hyperthermophilic from Submarine Volcanic Areas §Aquifex and Thermotoga are not Archaea but hyperthermophilic bacteria that otherwise strongly resemble hyperthermophilic Archaea. Thermotoga maritima (80 o C) Aquifex pyrophilus (85 o C) Chemoorganotrophic and anaerobic Obligate chemolithotrophic, micro- aerobically or anaerobically growth with only H 2, S 0 or S 2 O 3 - as electron donor and O 2 or NO 3 - as electron acceptor.

Thermoplasma: A Cell-Wall-Less Archaea §Thermoplasma acidophilum is a cell-wall-less prokaryote resembling the mycoplasmas. §Acidophilic, aerobic chemoorganotroph, thermophilic Archaea (pH=2 and To=55oC). §All strains of Thermoplasma have been isolated from self-heating coal refuse piles. Thermoplasma acidophilum an acidophilic, thermophilic mycoplasma-like archaea Thermoplasma volcanium shadowed preparation Thermoplasma volcanium has been isolated from Solfatara fields throughout the world.

Thermoplasma: A Cell-Wall -Less Archaea §Thermoplasma has evolved a cell membrane of chemically unique structure. §It contains lipopolysaccharide consisting of a tetraether lipid with mannose and glucose units. Self-heating coal refuse pile habitat of Thermoplasma The membrane also contains glyco- proteins but not sterol, the overall structure render the thermoplasma membrane stable to hot acid conditions

Limits of Microbial Existence: Temperature §Laboratory experiments on the heat stability of biomolecules suggest that living processes could be maintained at temperature as high as o C. Structure of the tetraether lipoglycan of Thermoplasma acidophilum Pyrodictium occultum (optima 105 o C, maxima 110 o C)

Archaea: Earliest Life Forms? §Early geochemical conditions: l High temperature l High salt l Low pH l Strict anoxic conditions §Only Archaea can stand such environmental extrems. §Do you agree with the argument: l Archaea are the Earliest Life Forms