bacteria = procaryotae (原核生物) + monera (= mychota, anucleobionta)

Endosphere: all the endophytes of a plant
= arbuscular mycorrhizal and other endophytic fungi (dominant) + bacteria and archaea
Phyllosphere (葉圏): all the endophytes of an aboveground part of plant

Gram stain (グラム染色)

Classified into gram-positive and gram-negative bacteria by staining ⇒ the chemical and physical (morphological) properties of cell envelopes and walls

Morphology (形態)

1. Capsules and slime layer

pl. -a

2. Flagellum

Terminology: acronematic (adj.) referring to a flagellum that is smooth and terminated with a fine fibril, thus tapered at its distal end

P ring, L ring, S ring, M ring + rod

the diameter of hook is larger than that of filament
= 900 nm long, E. coli, Salmonella = 42000 dalton, B. subtilis = 33000 dalton

flagellin (フラジェリン) = subunits, consisting of proteins, that form filaments - recovered by centrifugation

Species ______________ Subunit MW
Pseudomonas rhodos ___ 37000
Vibrio cholerae _________ 45000
Bacilus subtilis _________ 33000-40000

B. subtilis flagellin: lacking four amino acids, Try, Cys, Asn and Glu, and consisting of 16 kinds of amino acids (Rhizobium meliloti, the same with B. subtilis)
Caulobacter crescentus: stalk = 25 kD (transfer to the daughter cell) or 27.5 kD (cessation of cell division) – probably each protein originating from different structural genes
Sequence of amino acids at N-terminal

C. aecsentus 25 kD, 27.5 kD
B. subtilis non-N-side
S. typhimurum N-side (flagella antibody = H-antibody)
0 type antigen filament – one phase
1, 2 type antigen filament – two phase

strain → flagellin → [differentiation] → one phase / two phase

Ex. put two types of flagella into a test tube → [plate culture] → obtaining two types of colonies
probably because of phase mutation (相変異)

3. Pili (繊毛)

  Type Size Red blood      Response  Examples
       (nm) -------------  to
            attach agglu-  mannose
                   tinate

    1    7     +      +       +      Most Enterobaceriaceae
    2    7     -      -       -      Some Salmonella
    3    5    (+)    (+)     (+)     Seratia marescens*,
                                     Klebsiella neumoniae*
    4    4     +      +       -      Proteus
    5    5     +      -       +      Pseudomonus echinoides
    6   10     +      -      +/-     Klebsiella ozuenae

Note that the responses differ when the red blood originatig from cow and chiken

affected by differences in the sequence of amino acids

male straing - female strain

4. nucleus (in prokaryotes) (核)

5. cytoplasmic granules (細胞内顆粒)

6. cytoplasm (サイトプラスム)

7. mesosome (メソゾーム)

1-3 mesosomes/cell - changing with culture condition (< 40/cell)

from gram-positive bacteria

H1: analog of mitochondria in eukaryotes
→ not accepted by research conducted by gram-negative bacteria

P1: most gram-negative bacteria do not form mesosome. Mesosomes are reported from only three taxa (Caulobacter aresceutus, Flexibacter columnaris, Chromobacterium violaceum)

mesosome cell
membrane mesozome

Fixed chemically__Frozen______Fixed then frozen
Fig. Mesosomes form in bacterial cells prepared for electron microscopy by chemical fixation, but not by freeze-fracture fixation
By this experiment, mesosome is now recognized as artifacts

[Gram stain]

8. (cell) envelopes (細胞外皮)

Basic structure of cell envelopes

Fig. 9. The complete primary structure of the lipoprotein bound to the peptidoglycan. Amino-terminal lipid consists of glycerylcysteine [S-(propane0-2', 3'-diol)-3-thioamonopropainc acid], to which two fatty acids are attached by two ester linkages and one fatty acid by an amide linkage. Arrows indicate the bonds which are cleaved by lysozyme. DAP: diaminopimelic acid; NAM: N-acetylglucosamine.

cf. mitochondria → nucleus →chlorophyll

the cells treataed by toluene (C6H5CH3) that uncouples lipoprotein → mRNA (LP) is synthesized in vitro
chains longer than 58 amino acids

L-form bacteria (L型菌)

discovered strains of bacteria that lack cell walls

Fig. Phylogeny

Morphology (at microscope level)

coccus (球菌), bacillus (棹菌), coccobacillus (球桿菌), vibrio (コンマ菌), spirillum (螺旋菌), spirochaete (スピロヘータ), streptomyces (ストレプトミセス), crenothrix (クレノスリクス)

IMViC test (インビック試験)

→ p-Dimethylaminobenzaldehyde → Red Indole

D-dimethyaminobenyaldehyde = 10 g, conc = 50 ml

Medium: Glucose  K₂HPO₄  Peptone  Distilled water
                  0.5 g      0.5 g        0.5 g          100 ml

→ autoclave at 115°C for 15 min.
→ Methyl red (0.1 g methyl red + 300 ml 95% Et-OH = final 500 ml by adding water)
→ positive (+): red, negative (–): yellow

Glucose
Acetylmethylcarbinol (= acetone)
→ Diacethyl CH₃COCOCH₃ + creatine (arginine) or peptone
→ + Barritt indicator (6% α-naphtinol in Et-OH + 16% KOH) → + red

Mineral salts (無機塩類)

• metal: enzyme
• Zn: Alchohol DH
• Mg: Phosphohydrase
• Mn: Arginase, Isocitrate DH
• Fe: Various enzymes
• Cu: Tyrosinase, Cytochrome oxydase
• K: Pyruvate phosphokinase
• Na: Membrane ATPase
• Se: Hydrogenase, Gly reductase

Kingdom Prokaryote 原核生物界

Domain Bacteria 細菌 (Eubacteria 真正細菌)

Phylum Aquificae

Phylum Thermotogae

Phylum Thermodesulfobacteria

Phylum Deinococcus-Thermus

Order Deinococcales

Order Thermales

Phylum Chrysiogenetes

Phylum Chloroflexi (緑色滑走細菌)

Class Chloroflexi

Order Chloroflexales

Order Herpetosiphonales

Phylum Thermomicrobia

Phylum Nitrospira

Phylum Deferribacteres

Phylum Cyanobacteria

Phylum Chlorobi (クロロビウム)

Class Chlorobia

Order Chlorobiales

Phylum Proteobacteria (プロテオバクテリア)

Class α-proteobacteria

Order Rhodospirillales

Order Rickettsiales (リケッチア)

sex-ratio distorters (male-killing, parthenogenesis, feminization, etc.)

Order Rhodobacterales

Order Sphingomonadales

Order Caulobacteriales

Order Rhizobiales (リゾビウム)

rod, gram-negative, aerobic, not sporulated

Nitrobacter: NO₂ + 1/2O₂ → NO₃ (oxidation of NO₂ - N cycle)

Class β-proteobacteria (ベータプロテオバクテリア)

Order Burkholderiales

Order Hydrogenophilales

Order Methylophiales

Order Neisseriales

Order Nitrosomonadales

Nitrosomonas: NH₃ + 1·1/2O₂ → NO₂ + H₂O + 2H (oxidation of NH₄)

Order Rhodocyclales

Class γ-proteobacteria

Order Chromatiales

Order Actithiobacillales

Order Xanthomonadales

Order Cardiobacteriales

Order Thiotrichales

Order Legionellales

Order Methylococcales

Order Oceanspirillales

Order Pseudomonadales (シュードモナス)

Order Alteromonadales

Order Vibrionales

Order Aeromonadales

Order Enterobacteriales

Order Pasteurellales

Class δ-proteobacteria, nom. nud. (δ-プロテオバクテリア)

Order Bdellovibrionales

Order Desulfarculales

Order Desulfobacterales

Order Desulfovibrionales

Order Desulfurellales

Order Desulfuromonadales

related to the diffusion of uranium - bioremediation

fermentative metabolism

Order Myxobacteriales (slime bacteria, 粘液細菌)

Suborder Cystobacterineae

Suborder Sorangiineae

Suborder Nannocystineae

Order Syntrophobacterales

Class ζ-proteobacteria

Phylum Firmicutes (グラム陽性細菌門)

Class Bacilli (バシラス)

Order Bacillales (バシラス)

Order Lactobacillales (ラクトバシラス)

Class Clostridia (クロストリジウム)

Order Clostridiales (クロストリジウム)

Order Halanaerobiales

Order Natranaerobiales

Order Thermoanaerobacteriales

Class Erysipelotrichi (エリュシペロトリクス)

Order Erysipelotrichales

Class Thermolithobacteria Sokolova et al. 2007

Order Thermolithobacterales

Class Limnochordia Watanabe et al. 2015 (リムノコルダ)

Class Tissierellia ティッセレッラ

Phylum Tenericutes or Firmicutes (テネリクテス)

Class Mollicutes (モリクテス)

not stained by Gram staining

Order Mycoplasmatales (マイコプラズマ)

Order Entomoplasmatales (エントモプラズマ)

Order Acholeplasmatales (アコレプラズマ)

Order Anaeroplasmatales (アナエロプラズマ)

Order Haloplasmatales (ハロプラズマ)

Phylum Actinobacteria (アクチノバクテリア)

Class Actinobacteria

Subclass Actinobacteridae

actinorrhizal: adj. 放線菌の

Order Actinomycetales (放線菌)

Mycelium = vegetative (or substrate) mycelium + aerial mycelium

Suborder Actinomycineae

Suborder Corynebacterineae

Mycobacteroides
Mycolicibacterium

Mycolicibacter
Mycolicibacillus
Mycobacterium, acid fast bacteria (抗酸菌): M. tuberculosum (結核菌), M. leplae (癩菌), M. marinum - pathogenic (tuberculosis-like infections in fish → skin lesions in humans)

Subclass Rubrobacteridae

Order Rubrobacteriales

Suborder Rubrobacterineae

Subclass Coriobacteridae

Order Coriobacteriales

Suborder Coriobacterineae

Subclass Sphaerobacteridae

Order Sphaerobacterales

Suborder Sphaerobacterineae

Suborder Frankineae

Suborder Micrococcineae

Suborder Micromonosporineae

Suborder Propionibacterineae

Suborder Pseudonocardineae

Suborder Streptomycinae

Suborder Streptosporangineae

Order Bifidobacteriales (ビフィドバクテリウム)

Phylum Planctomycetes (プランクトミケス)

Class Chlamydiae

Order Chlamydiales

Phylum Spirochaetes

Class Spirochaetes

Order Spirochaetales (螺旋菌/スピロヘータ)

Phylum Fibrobacteres Garrity et Holt 2012 (フィブロバクテル)

Class Chitinispirillia

Class Chitinivibrionia

Class Fibrobacteria

Phylum Acidobacteria (アシドバクテリウム)

Phylum Bacteroidetes (バクテロイデス)

Bacteroidetes/Chlorobi group = CFB group + Chlorobi

(Class Bacteroidia)

Order Bacteroidales

Order Marinilabiliales

Class Flavobacteriia (フラボバクテリウム)

Order Flavobacteriales

Class Sphingobacteriia

Order Sphingobacteriales

Class Cytophagia Nakagawa 2012 emend. Hahnke et al. 2016

Phylum Fusobacteria (フソバクテリウム)

Class Fusobacteriia

Order Fusobacteriales

Phylum Verrucomicrobia (ウェルコミクロビウム)

Class Verrucomicrobiae

Class Opitutae

Phylum Dictyoglomi Saiki et al. 1985 (ディクチオグロムス)

Order Dictyoglomales

Microbial community (微生物群集)

Analytical methods

Pre-analysis (remove primers, chimera, etc.)

Gene function abundance
Pathway abundance
Pathway reconstruction
Taxonomic abundance

Pipeline

Microbiota (微生物叢)

• periphyton: associated with submersed surfaces of all kinds, often used only to micro-algae
• epilithon: attached to rocks
• epipelon: algae that migrate up and down at the sediment-water interface
• plocon: non-migratory algae growing on the sediment surface
• epiphyton: growing on submersed living plants, often used only to micro-algae
• neuston: found at the water-air interface
• plankton: suspended or entrained in the water column, phytoplankon = plants of the plankton
• metaphyton: masses of filamentous algae that float in the water column or plocon, all kinds of microbiota are found in the metaphyton

Quarum sensing (quorum signalling, QS, 集団感知)

Symbiosome

Domain Archaea (始原菌, 古細菌, アーケア)

Phylum Crenarchaeota

Class Thermoprotei Reysenbach 2002

Order Thermoproteales Zillig et Stetter 1982

Order Desulfurococcales

Order Sulfolobales

Phylum Euryarchaeota Garrity et Holt 2002

Class Methanobacteria Boone 2002

Order Methanobacteriales

Class Methanococci

Class Halobacteria

Class Thermoplasmata

Class Thermococci

Class Archaeoglobi Garrity et Holt 2002

Class Methanopyri

Environments (環境)

1) Temperature

2) pH

3) Salt (halophile)

4) Pressure

Methanogen (メタン菌)

4H₂ + CO₂ → CH₄ + 2H₂O

⊂ Domain Eukaryota (Eukaryotes)

Kingdom (or unranked) Amoebozoa

Infraphylum Mycetozoa de Bary 1873 (粘菌類)

Class Protostelea (原生粘菌), protosteloid amoebae

Order Protosteliida

Class Myxogastria Macbride 1899 (変形菌), myxomycetes

Order Liceida (コホコリ)

Order Echinosteliida (ハリホコリ)

Order Trichiida or Trichiales (ケホコリ)

Order Stemonitida (ムラサキホコリ)

Order Physarida (モジホコリ)

Class Dictyostelia Lister 1909, emend. Olive 1970 (タマホコリ), dictyostelids

Order Dictyosteliida or Dictyosteliales (タマホコリ)

D. discoideum Raper (キイロタマホコリ): model organism

Gram stain or Gram staining (グラム染色)

Four basic steps:

1. Primary dye: apply a primary stain (crystal violet*) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they do not rinse out during the staining procedure

   * carbal gentian violet can be used (1-2 min)

   → fixation
2. Trapping agent: Add iodide (Gram's iodine solution), which binds to crystal violet and traps it in the cell

   = tip out the solution and then the remainder is blotted by a filter paper

3. Decolorizer: Decolorize rapidly with ethanol or acetone

   = pour 95% ethanol adn shake lightly for 5-15. seconds

   → rinse
4. Counter stain (対比染色): Counterstain with safranin. Carbol fuchsin* is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain

   * dilute fuchsin can be used

   → rinse → air-dry → visualize microscopically

Extraction procedure

at 20°C for 21 hr
at 37°C for 15 hr (Optimal temperature for P. aeruginosa)
use a rotary shaker (400 ml/3000 ml)

↓ add 50 ml methanol
↓ add 25 ml chloroform
↓ add N₂-gas for oxidation of C=C bond until use
↓↓ use 300-ml flask

pipett top
is immersed
at the bottom

↓ add 25 ml chloroform

→ add 15 ml 10mM NaCl
Mixing*
→ centrifuge with 3000 rpm for 5 min