Microbiology

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Microbiology (微生物学)

Mount Usu / Sarobetsu post-mined peatland
From left: Crater basin in 1986 and 2006. Cottongrass / Daylily

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microbe < 100 μm = bacteria + archaea (+ fungi + eukaroyotes)

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

Endophyte (エンドファイト): endo = within + phyte = plant (L.)
bacteria and fungi living symbiotically within a plant for at least part of their life cycles

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)

= Gram staining and Gram's method

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

Gram-positive
Gram-negative
+ Gram-variable and Gram-indeterminate bacteria

Morphology (形態)

Fig. 1: capsule, slime layer, 2: microcapsule, 3: pili, fimbriae, 4: flagellum, 5: cell wall, 6: cytoplasmic membrane (cell membrane, plasma membrane), 7: periplasmic space, 8: cytoplasm, 9: ribosome, 10: bacterial chromosome (double-strand attached with PM), 11: mesosome, 12: vacuole, 13: granules, 14: plasmid

1. Capsules and slime layer

pl. -a

2. Flagellum

Polarity (極性): single polar, monotrichous, monopolar, dipolar, several polar, polytrichous polar, multitrichous, subpolar, lateral, peritrichous

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

Structure: three components
Basal body

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

Hook (フック) = function as universal joint (自由継手)

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

Filament (フィラメント)

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)

→ analyzing the operon in this part

3. Pili (繊毛)

a) bacterial fimbria or common pili
Table. Types and characteristics of pili. *: after tannic-acid treatment

  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

Response to mannose: MS type: mannose sensitive → MR type: mannose resistant

affected by differences in the sequence of amino acids

pili → [heat treatment] → subunits = subunit of finbice???
b) sex pili (or fimbriae): firstly discovered from E. coli

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)

= chondrioid
Invaginations in the plasma membrane of bacteria - concentrical multi-layred membrane

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

1953 Chapman J & Hillier J: reported peripheral bodies

from gram-positive bacteria

1959 Fitz-James JD: termed mesosome
After discovering mesosome, the function was discussed

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)

1971 Nanninga: experiment that demonstrates mesosomes are artifact

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)

= cell wall + periplasm (including periplasmic space) + peptidoglycan + cell membrane OCM

Basic structure of cell envelopes

A) Membrane model
B) Cytoplasmic membrane
C) Outer membrane
Molecular shield (分子盾)

shield
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.

cell membrane (IM), periplasm, outer membrane, extracellular membrane → protein synthesis in cytoplasm

cf. mitochondria → nucleus →chlorophyll

(N-end) Cys₁-Ser₂-Ser₃- … -Lys₅₈ (C-end)

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

Met [20 bases larger] → Cys-Ser-…
Extra-peptide = leader peptide or signal peptide
D) Periplasmic space or periplasm
E) Adhesion zone
phage

Fig. A: Bacteriophage T2 adsorbed to a plasmolized E. coli B cell. The virus part with their tail tips (= cores) to the adhesion sites. B: Ultrathin section through E. coli HfrH, revealing the F-pilus insertion at the membrane adhesion zone. The pilus is labeled with bacteriophage MS2. C: Ultrathin section of E. coli showing a flagellum insertion area. Note the apparent organization of the inner membrane at the insertion point.

L-form bacteria (L型菌)

= L-phase bacteria, L-phase variants and cell wall-deficient (CWD) bacteria
1935 Klieneberger-Nobel E (1892-1985)

discovered strains of bacteria that lack cell walls

[ nomenclature ]

Taxonomoy (分類)

s.s. prokaryotes = archaea (archaebacteria) + eubacteria (bacteria)

Fig. Phylogeny

s.l. prokaryotes (s.s.) + viroids + viruses

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)

= Indole test + Methyl red test + Voges-Proskauer test + Citrate test
1. Indole reaction
Trypthane → Tryptophanase → Indole

→ p-Dimethylaminobenzaldehyde → Red Indole

Medium: 1-2% Peptone, 0.5% NaCl (pH = 7.2)
↓ Autoclave, Inoculation at 24-37°C, Incubation for 2-7 days
↓ + Kamacs indole regent
Kamacs indole regent: amylalhol (cis-amylalchol) = 150 ml,

D-dimethyaminobenyaldehyde = 10 g, conc = 50 ml

2. methyl red reaction

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

3. Voges-Proskauer test

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

4. Citrate test

Growth (成長・増殖)

Mineral salts (無機塩類)

preserve the structure of bacteria

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

Ecology (生態)

Microbial community (微生物群集)

Analytical methods

16S/18S rRNA gene amplicon sequencing analysis
↓ DNA extraction
↓ PCR amplification ↓ DNA sequencing

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

↓ Sequene clustering at a taxon level by CD-HIT-EST, UCLUST, etc.
Taxonomical assignment and analysis
Metagenomic sequencing analysis
↓ DNA extraction
↓ DNA sequencing ↓ Metagenomic reads
↓ Assemble: MEGAHIT, MetaPlatanus, IDBA-UD, etc.
↓ Contig sets
↓ Gene finding: MGS, MetaGeneMark
↓ Gene sets
↓ BLASTP → Comparative metagenomics (metadata)

Gene function abundance
Pathway abundance
Pathway reconstruction
Taxonomic abundance

Pipeline

QIIME 2: free, open source
= Quantitative Insights Into Microbial Ecology
Mothur
USEARCH

Microbiota (微生物叢)

= formerly microbiol flora, because bacteria were grouped into plants
Table. Major microbiota assemblages found in wetlands.

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)

the ability to detect and respond to cell population density by gene regulation

Symbiosome

vacuole symbiotically infected by bacteria (s.s., bacteriology)
specialized compartment in a host cell that houses an endosymbiont (s.l.)

Taxa (分類群)

1970s' Woose Carl (1928-2012): proposed classification based on rRNA

Kingdom Prokaryote 原核生物界

Domain Bacteria 細菌 (Eubacteria 真正細菌)

coccus - bacillus

Phylum Aquificae

Aquificaceae
Desulfurobacteriaceae
Hydrogenothermaceae

Phylum Thermotogae

Thermotogaceae: Thermotoga maritima, optimal temeprature for the reproduction = 80°C (reproduced at 90°C)

Phylum Thermodesulfobacteria

Thermodesulfobacteriaceae

Phylum Deinococcus-Thermus

Order Deinococcales

Deinococcaceae: Deinococcus, Deinobacterium
Trueperaceae: Truepera

Order Thermales

Thermaceae: Thermus, Marinithermus, Meiothermus, Oceanithermus, Vulcanithermus

Phylum Chrysiogenetes

Chrysiogenes arsenatis, Desulfurispirillum alkaliphilum

Phylum Chloroflexi (緑色滑走細菌)

Class Chloroflexi

Order Chloroflexales

Chloroflexaceae (緑色糸状細菌): Chloroflexus, Chloronema, Heliothrix - photosynthesis
Oscillochloridaceae: Oscillochloris

Order Herpetosiphonales

Herpetosihpnaceae: Herpetosihpon

Phylum Thermomicrobia

Phylum Nitrospira

Phylum Deferribacteres

Phylum Cyanobacteria

Phylum Chlorobi (クロロビウム)

Class Chlorobia

Order Chlorobiales

Chlorobiaceae
Chlorobium (green sulfur bacteria, 緑色硫黄細菌), Ancalochloris, Chlorobaculum, Chloroperpeton, Pelodictyon, Prosthecochloris

Phylum Proteobacteria (プロテオバクテリア)

Class α-proteobacteria

Order Rhodospirillales

Rhodospirillaceae (formerly Athiorhodaceae) 紅色非(無)硫黄細菌
Azospirillum, Rhodospirillum, Rhodopseudomonas, Rhodomicrobium
Acetobactaceae
Acetobacter (A. aceti 酢酸菌)

Order Rickettsiales (リケッチア)

Rickettsiaceae: Rickettsia
Ehrlichiaceae
Holosporaceae
Anaplasmataceae Philip 1957 (アナプラズマ)
Anaplasma Theiler 1910
Ehrlichia
Neorickettsia
Wolbachia Hertig 1936: symbiont with invertebrates (arthropods) and filariae

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

Order Rhodobacterales

Order Sphingomonadales

Sphingomonadaceae: Sphingomonas

Order Caulobacteriales

Caulobacteraceae Henrici et Johnson 1935
Asticcacaulis, Brevundimonas, Caulobacter, Phenylobacterium

Order Rhizobiales (リゾビウム)

(Hyphomicrobiales, discarded)
Rhizobiaceae
Rhizobium Frank 1989: nodule, nitrate bacteria

rod, gram-negative, aerobic, not sporulated

Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Sinorhizobium
Bartonellaceae
Brucellaceae
Phyllobacteriaceae
Methylocystaceae
Beijerinckiaceae: Beijerinckia, Camelimonas, Chelatococcus, Methylocapsa, Methylocella, Methyloferula, Methylorosula, Methylovirgula, Pseudochelatococcus
Nitrobacteraceae, corrig. Buchanan 1917 (incl. Bradyrhizobiaceae)
Variibacter, Pseudorhodoplanes, Pseudolabrys, Afipia, Rhodopseudomonas, Bradyrhizobium

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

Hyphomicrobicaceae
Methylobacteriaceae
Rhodobiaceae

Class β-proteobacteria (ベータプロテオバクテリア)

gram-negative

Order Burkholderiales

Burkholderiaceae: Burkholderia, Cupriavidus, Lautropia, Pandoraea, Thermothrix
Ralstoniaceae: Ralstonia
Oxalobacteraceae: Janthinobacterium De Ley et al. 1978, emend. Lincoln et al. 1999: soil bacteria (J. agaricidamnosum Lincoln et al. 1999, J. lividum De Ley et al. 1978)
Alcaligenaceae De Ley et al. 1986: found in water, soil and animals
Alcaligenes Castellani et Chalmers 1919
Achromobacter Yabuuchi et Yano 1981, aerobic in water: A. fisheri
Bordetella Moreno-López 1952: B. pertussis
Comamonadaceae: Comamonas, Acidovorax, Aquabacterium, Brachymonas, Delftia, Hydrogenophaga, Ideonella, Leptothrix, Polaromonas, Rhodoferax, Roseateles, Rubrivivax, Sphaerotilus (S. nataus), Tepidimonas, Thiomonas, Variovorax

Order Hydrogenophilales

Order Methylophiales

Order Neisseriales

Neisseriaceae: Neisseria (N. grorrhoeae), Alysiella, Aquaspirillum, Catenococcus, Chromobacterium, Eikenella, Formivibrio, Iodobacter, Kingella, Microvirgula, Prolinobarus, Simonsiella, Vitreoscilla, Vogesella

Order Nitrosomonadales

Nitrosomonadaceae: Nitrosolobus, Nitrosospira

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

Methylophilaceae: Methylophilus, Methylobacillus, Methylovorus
Spirillaceae: Spirillum
Thiobacillaceae: Thiobacillus, Annwoodia, Sulfuritortus
Gallionellaceae: Gallionolla (G. ferginea), Ferriphaselus, Sulfuriferula, Sulfurirhabdus, Sulfuricella
Sterolibacteraceae: Sterolibacterium, Sulfurisoma, Denitratisoma, Sulfuritalea, Georgfuchsia, Sulfurisoma, Methyloversatilis

Order Rhodocyclales

Rhodocyclaceae
Azonexaceae
Zoogloeaceae

Class γ-proteobacteria

Order Chromatiales

Chromatiaceae: Chromatium, Allochoromatium, Amoebobacter, Halochromatium, Halothiobacillus, Isochromatium, Lamprobacter, Lamprocystis, Marichromatium, Nitrosococcus, Pfennigia, Rhabdochromatium, Thermochromatium, Thioalkalicoccus, Thiocapsa, Thiococcus, Thiocystis, Thiodictyon, Thiohalocapsa, Thiolamprovum, Thiopedia, Thiorhodococcus, Thiorhodovibrio, Thiospirillum
Ectothiorhodospiraceae: Ecthiorhodospira, Arhodomonas, Halohodospira, Nitrococcus, Thiorhodospira

Order Actithiobacillales

Order Xanthomonadales

Xanthomonadaceae: Xanthomonas, Stenotrophomonas

Order Cardiobacteriales

Order Thiotrichales

Thiotrichaceae: Thiothrix, Achromatium, Beggiatoa, Leucothrix, Macromonas, Thiobacterium, Thiomargarita, Thioploca, Thiospira
Piscirickettsiaceae
Francisellaceae

Order Legionellales

Legionellaceae: Legionella AL
Coxiellaceae: Coxiella, Rickettsiella

Order Methylococcales

Order Oceanspirillales

Order Pseudomonadales (シュードモナス)

Pseudomonas Migula 1894 (syn. Chlorobacterium), Azomonas, Azotobacter (A. chroocaccum), Cellvibrio, Chryseomonas, Flavimonas, Lampropedia, Mesophilobacter, Morococcus, Oligella, Rhizobacter, Rugamonas, Serpens, Thermoleophilum, Xylophilus
Moraxellaceae: Moraxella, Acinetobacter
Moraxellaceae

Order Alteromonadales

Alteromonadaceae: Alteromonas, Shewanella

Order Vibrionales

Vibrionaceae: Vibrio (Ex. Cholera group) (V. möller, V. comma, V. parahaemelyticus

Order Aeromonadales

Aeromonadaceae: Aeromonas

Order Enterobacteriales

Enterobacteriaceae (腸内細菌科)

Order Pasteurellales

Pasteurellaceae: Pasteurella, Actinobacillus, Haemophilus

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

Syntrophorhabdaceae, order not decided

Order Bdellovibrionales

Bacteriovoracaceae
Bdellovibrionaceae
Bdellovibrio Stolp and Starr

Order Desulfarculales

Desulfarculaceae

Order Desulfobacterales

Desulfobacteraceae
Desulfobulbaceae
Nitrospinaceae

Order Desulfovibrionales

Desulfohalobiaceae
Desulfomicrobiaceae
Desulfonatronaceae
Desulfovibrionaceae
Desulfovibrio Kluyver et van Niel 1936: gram-netative, nitrate-reducing

Order Desulfurellales

Desulfurellaceae

Order Desulfuromonadales

anaerobic respiration uisng various compounds as electron acceptors, including sulfur, Mn(IV), Fe(III), nitrate, Co(III), Tc(VII), U(VI) and trichloroacetic acid
Desulfuromonadaceae, corrig. Kuever et al. 2006
Geobacteraceae Holmes et al. 2004
Geoalkalibacter Zavarzina et al. 2007
Geobacter Lovley et al. 1995

related to the diffusion of uranium - bioremediation

Geopsychrobacter Holmes et al. 2005
Geothermobacter Kashefi et al. 2005
Pelobacteraceae

fermentative metabolism

Order Myxobacteriales (slime bacteria, 粘液細菌)

Suborder Cystobacterineae

Cystobacteraceae
Myxococcaceae
Myxococcus xanthus

Suborder Sorangiineae

Minicystis rosea Garcia et al. 2014, family not decided: the lrageest genome size (16,040,666 bp) in prokaryotes (by 2018)
Polyangiaceae
Phaselicystidaceae
Sandaracinaceae

Suborder Nannocystineae

Nannocystaceae
Kofleriaceae
Haliangiaceae

Order Syntrophobacterales

Syntrophaceae
Syntrophobacteraceae

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)

Bacillaceae
gram-positive, heterotrophic, rod-shaped bacteria that may produce endospores
Bacillus Cohn 1872

Order Lactobacillales (ラクトバシラス)

Aerococcaceae (アエロコックス)
gram-positive
Abiotrophia, Aerococcus (A. viridans: widespread - causative agent of gaffkaemia, a disease of lobsters), Dolosicoccus, Eremococcus, Facklamia, Globicatella, Ignavigranum
Carnobacteriaceae (カルノバクテリウム)
Granulicatella, Aerococcus, Alloiococcus, Abiotrophia, etc.
Enterococcaceae (エンテロコッカス)
Enterococcus, Melissococcus, Pilibacter, Tetragenococcus, Vagococcus
Lactobacillaceae
Pediococcus
Leuconostocaceae (ロイコノストック), formerly grouped into Lactobacillaceae
Leuconostoc: L. mesenteroides, wine fermentation
Fructobacillus, Oenococcus, Weissella, etc.
Streptococcaceae (連鎖球菌)
formerly Enterococcus (腸球菌) was categorized into this family (now Enterococcaceae)
Streptococcus Rosenbach 1884: S. pneumoniae (肺炎球菌), S. viridans (緑色連鎖球菌)
Floricoccus
Lactococcus Schleifer et al. 1986: lactic acid bacteria
Lactovum
Okadaella
Streptococcus
Genera incertae sedis: Aerosphaera, Carnococcus, Chungangia

Class Clostridia (クロストリジウム)

Order Clostridiales (クロストリジウム)

gut bacteria (gb)
Caldicoprobacteraceae
Caldicoprobacter: C. oshimai, C. algeriensis, C. guelmensis, C. faecale (syn. Acetomicrobium faecale)

Christensenellaceae (human gb)
Christensenella minuta
Clostridiaceae (クロストリジウム)
Clostridium Prazmowski 1880: C. tetani (Fl&umul;gge 1886) Bergey et al. 1923 (破傷風菌), C. botulinum, C. pasteurianum Winogradsky 1895 (syn. C. pastorianum): fixing free nitrogen from the air, C. perfringens (Veillon & Zuber 1898) Hauduroy et al. 1937 (ウェルシュ菌) (facultative human gb)
Dehalobacteriaceae
Dehalobacterium (gb)
Defluviitaleaceae
Eubacteriaceae Ludwig et al. 2010 (ユーバクテリウム)
Acetobacterium Balch et al.
Eubacterium Prévot 1938: hard cell wall
Graciibacteraceae
Heliobacteriaceae
Lachnospiraceae (ラクノスピラ) (gb)
Anaerostipes (human gb), Blautia: B. coccoides, Butyrivibrio, Coprococcus, Dorea, Lachnospira
Oscillospiraceae
Peptococcaceae (ペプトコッカス)
Desulfosporosinus Stackebrandt et al. 1997 - soil and groundwater Peptostreptococcaceae Garrity et al. 2001 (gb)
Acetoanaerobium, Clostridioides, Filifactor, Peptostreptococcus, Tepidibacter, etc.
Ruminococcaceae (ルミノコッカス)
Ethanoligenens, Faecalibacterium (F. prausnitzii, gb), Ruminococcus Sijpesteijn 1948, Oscillospira
Sulfobacillaceae
Sulfobacillus
Syntrophomonadaceae Zhao et al. emend, Jumas-Bilak et al. 2009
using carboxylic acids as a source of energy
Syntrophomonas McInerney et al. 1982
Veillonellaceae Rogosa 1971 (= formerly Acidaminococcaceae)
obligate anaerobes in rivers, lakes, and the intestines of vertebrates
Veillonella Prévot 1933 (12 spp.), Megasphaera (5), Dialister (ex Bergey et al. 1923) Moore and Moore 1994 (5), Allisonella (A. histaminiformans): ruminal, Anaeroglobus Carlier et al. 2002 (A. geminatus), Negativicoccus Marchandin et al. 2010 (N. succinicivorans)

Order Halanaerobiales

Halanaerobiaceae
Halobacteroidaceae

Order Natranaerobiales

Natranaerobiaceae

Order Thermoanaerobacteriales

Thermoanaerobacteraceae
Polyphyletic
Thermodesulfobiaceae Mori et al. 2004

Class Erysipelotrichi (エリュシペロトリクス)

Order Erysipelotrichales

Class Thermolithobacteria Sokolova et al. 2007

Order Thermolithobacterales

Thermolithobacteraceae (テルモリトバクテル)

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

Class Tissierellia ティッセレッラ

(Acidaminococcaceae)

Phylum Tenericutes or Firmicutes (テネリクテス)

Class Mollicutes (モリクテス)

lacking cell wall - small
parasites to plants and animals

not stained by Gram staining

Order Mycoplasmatales (マイコプラズマ)

cell wall-less (and virus-less)
Mycoplasmataceae Freundt 1955
Mycoplasma J Nowak 1929: over 100 species
Ureaplasma: 8 species so far, urease positive

Order Entomoplasmatales (エントモプラズマ)

Entomoplasmataceae: Entomoplasma, Mesoplasma Spiroplasmataceae (スピロプラズマ): small without cell wall, insect symbiont
Spiroplasma: S. citri - citrus stubborn disease, S. kunkelii - corn stunt disease

Order Acholeplasmatales (アコレプラズマ)

Acholeplasmataceae
Ca. Phytoplasma Firrao et al. (ファイトプラスマ): cell wall-less, mycoplasma-like organism (MLO) - plant deseases

Order Anaeroplasmatales (アナエロプラズマ)

Anaeroplasmataceae

Order Haloplasmatales (ハロプラズマ)

Haloplasmataceae: Haloplasma (H. contractile = halophilic, cell wall-less)

Phylum Actinobacteria (アクチノバクテリア)

Gram-positive, including high DNA GC content

Class Actinobacteria

Subclass Actinobacteridae

actinorrhizal: adj. 放線菌の

Order Actinomycetales (放線菌)

Formerly characterized by the development of mycelia

Mycelium = vegetative (or substrate) mycelium + aerial mycelium

Suborder Actinomycineae

Actinomycetaceae: Actinomyces, Actinobaculum, Arcanobacterium, Mabiluncus

Suborder Corynebacterineae

Corynebacteriaceae: Corynebacterium
Dietziaceae: Dietzia
Gordoniaceae: Gordonia, Skermania
Mycobacteriaceae (マイコバクテリウム)
Fast-growing mycobacteria

Mycobacteroides
Mycolicibacterium

Slow-growing mycobacteria

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)

Nocardiaceae: Nocardia, Rhodococcus
Tsukamurellaceae: Tsukamurella
Williamsiaceae: Williamsia

Subclass Rubrobacteridae

Order Rubrobacteriales

Suborder Rubrobacterineae

Rubrobacteriaceae: Rubrobacter

Subclass Coriobacteridae

Order Coriobacteriales

Suborder Coriobacterineae

Coriobacteriaceae: Coriobacterium, Atopobium, Collinsella, Cryptobacterium, Denitrobacterium, Eggerthella, Slackia

Subclass Sphaerobacteridae

Order Sphaerobacterales

Suborder Sphaerobacterineae

Sphaerobacteraceae: Sphaerobacter

Suborder Frankineae

Frankiaceae (フランキア)
Frankia Brunchorst
Hosts: all species in Alnus (Betulaceae), Elaeagnus, Shepherdia and Hippophae (Elaeagnaceae), Myrica, Morella and Comptonia (Myricaceae), Ceanothus, Colletia, Discaria, Trevoa and possibly Adolphia (Rhamnaceae), Cercocarpus, Cowania, Purshia and Chamaebatia + a few in Dryas (Rosaceae), a few species in Coriaria, Datisca cannabina and D. glomerata
F. alni (Woronin) Von Tubeuf
Geodermatophilaceae: Geodermatophilus, Blastococcus, Modestobacter
Microsphaeraceae: Microsphaera
Sporichthyaceae: Sporichthya
Acidothermaceae: Acidothermus
Kineosporiaceae: Kineosporia, Cryptosporangium, Kineococcus

Suborder Micrococcineae

Paraphyletic
Micrococcaceae: Micrococcus, Arthrobacter, Kocuria, Nesterenkonia, Renibacterium (moved), Rothia, Stomatococcus
Bogoriellaceae: Bogoriella (moved)
Rarobacteraceae: Rarobacter (moved)
Sanguibacteraceae: Sanguibacter
Brevibacteriaceae: Brevibacterium
Cellulomonadaceae: Cellumonas, Oerskovia
Dermabacteraceae: Dermabacter, Brachybacterium
Dermatophilaceae: Dermatophilus
Dermacoccaceae: Dermacoccus (moved), Demetria (moved), Kytococcus (moved)
Intrasporangiaceae: Intrasporangium, Janibacter, Ornithinicoccus, Ornithinimicrobium, Nostocoidia, Terrabacter, Terracoccus, Tetrasphaera
Jonesiaceae: Jonesia
Microbacteriaceae: Microbacterium, Agrococcus, Agromyces, Aureobacterium, Clavibacter, Cryobacterium, Curtobacterium, Frigoribacterium, Leifsonia, Leucobacter [moved], Rathayibacter, Subtercola
Beutenbergiaceae: Beutenbergia
Promicromonosporaceae: Promicromonospora

Suborder Micromonosporineae

Micromonosporaceae: Micromonospora, Actinoplanes, Catellatospora, Catenuloplanes, Couchioplanes, Dactylosporangium, Pilmelia, Spirilliplanes, Verrucosispora

Suborder Propionibacterineae

Propinibacteriaceae: Propinibacterium, Luteococcus, Microlunatus, Propioniferax, Tessaracoccus
Nocardioidaceae: Nocardioides, Aeromicrobium, Friedmanniella, Hongia, Kribbella, Micropruina, Marmoricola

Suborder Pseudonocardineae

Pseudonocardiaceae: Pseudonocardia, Actinoalloteichus, Actinopolyspora, Amycolatopsis, Kibdelosporangium, Kutzneria, Prauserella, Saccharopolyspora, Streptolloteichus, Thermobispora, Thermocrispum
Actinosynnemataceae: Actinosynnema, Actinokineospora, Lentzea, Saccharothrix

Suborder Streptomycinae

Streptomycetaceae: Streptomyces (S. griseus), Kitasatospora, Streptoverticillium

Suborder Streptosporangineae

Streptosporangiaceae: Streptosporangium, Acrocarpospora, Herbidospora, Microbispora, Microtetraspora, Nonomuraea, Planobispora, Planomonospora, Planopolyspora, Planotetraspora
Nocardiopsaceae: Nocardiopsis, Thermobifida
Thermomonosporaceae: Thermomonospora, Actinomadura, Spirillospora

Order Bifidobacteriales (ビフィドバクテリウム)

gram-positive, obligately anaerobic
Bifidobacteriaceae: Aeriscardovia, Alloscardovia,
Bifidobacterium Orla-Jensen 1924 (ビフィズス菌),
Bombiscardovia, Galliscardovia, Gardnerella, Neoscardovia, Parascardovia, Pseudoscardovia, Scardovia
A few unknown affilitation: Actinobispora, Actinocorallia, Excellospora, Pelczaria, Turicella

Phylum Planctomycetes (プランクトミケス)

Class Chlamydiae

Order Chlamydiales

Chlamydiaceae: Chlamydia
Parachlamydiaceae
Simkaniaceae
Waddliaceae

Phylum Spirochaetes

Class Spirochaetes

Order Spirochaetales (螺旋菌/スピロヘータ)

Spirochaetaceae: Spirochaeta, Borrelia (B. recurrentis), Brevinema, Clevvelandina, Cristispira, Diplocalyx, Hollandina, Pillotina, Treponema (T. pallida)
Serpulinaceae: Serpulina, Brachyspira
Leptospiraceae: Leptonema, Leptospira (L. icterohaemorrhagiae)

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

gram-negative

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)

= Cytophaga-Flavobacterium-Bacteroidetes (CFB) group

Bacteroidetes/Chlorobi group = CFB group + Chlorobi

> 350 genera

(Class Bacteroidia)

Order Bacteroidales

human gut microbiota
Bacteroidaceae: Bacteroides
Rikenellaceae: Rikenella
Porphyromonadaceae: Porphyromonas
Prevotellaceae: Prevotella
Paraprevotellaceae, formerly included in Prevotellaceae

Order Marinilabiliales

Class Flavobacteriia (フラボバクテリウム)

habitat: soil, freshwater, seawater

Order Flavobacteriales

Flavobacteriaceae: Flavobacterium (soil and freshwater/fish - disease), Capnocytophaga, Chryseobacterium

Class Sphingobacteriia

producing sphingolipids Phylum Sphingobacteria = classes Chlorobea (Chlorobi) + Fibrobacteres + Bacteroidetes + Flavobacteria (proposal - not supported)

Order Sphingobacteriales

Sphingobacteriaceae: Sphingobacterium, Pedobacter
Flexibacteraceae: Flexibacter, Cytophaga

Class Cytophagia Nakagawa 2012 emend. Hahnke et al. 2016

Phylum Fusobacteria (フソバクテリウム)

nearly all species are rod-shaped
most of them are gut bacteria

Class Fusobacteriia

Order Fusobacteriales

Fusobacteriaceae: Cetobacterium, Fusobacterium, Ilyobacter, Propionigenium, Psychrilyobacter
Leptotrichiaceae: Leptotrichia, Sebaldella, Sneathia, Streptobacillus

Phylum Verrucomicrobia (ウェルコミクロビウム)

habitats are diverse

Class Verrucomicrobiae

Class Opitutae

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

consisting a single genus that is: Dictyoglomus
gram-negative, with a triple-layered wall
survived at > 80°C

Order Dictyoglomales

Dictyoglomaceae: D. thermophilum

Archaebacteria (始原菌)

Domain Archaea (始原菌, 古細菌, アーケア)

Phylum Crenarchaeota

Class Thermoprotei Reysenbach 2002

Order Thermoproteales Zillig et Stetter 1982

Thermoproteaceae
Anaerobic thermophilic sulfur archaea (嫌気性好熱硫黄古細菌): Thermoproteus, Caldiviga, Pyrobaculum (grown well ≈ 100°C), Thermocladium
Thermofilaceae
Thermofilum

Order Desulfurococcales

Desulfurococcaceae
Pyrodictiaceae

Order Sulfolobales

Phylum Euryarchaeota Garrity et Holt 2002

Class Methanobacteria Boone 2002

methane bacteria

Order Methanobacteriales

Methanobacteriaceae: Methanobacterium, Methanobrevibacter, Methanosphaera, Methanothermobacter
Methanothermaceae: Methanothermus

Class Methanococci

methane producer

Class Halobacteria

extreme halophile, reproducing under high NaCl concentration

Class Thermoplasmata

acidophiles, growing optimally at pH below 2

Class Thermococci

grwoing in extremely hot environments

Class Archaeoglobi Garrity et Holt 2002

three genera in one family, grwoing in extremely hot environments
Archaeoglobaceae: Archaeoglobus, Ferroglobus, Geoglobus

Class Methanopyri

One species:
Methanopyrus Kurr et al. 1992: M. kandleri Kurr et al.

Environments (環境)

1) Temperature

psychrophile (好冷生物), psychrophilic bacteria (好冷菌): < 15°C
mesophile (好中温生物): 15-40°C
thermophile (好熱生物), tempmophilic bacteria (好熱菌): 45-80°C
hyperthermophile 超好熱生物: > 80°C

2) pH

acidophile (好酸性生物): < 5
neutrophile (好中性生物): 6-8
alkaliphile (好アルカリ性生物/菌): > 8

3) Salt (halophile)

haplophilic bacteria (好塩菌)

4) Pressure

Methanogen (メタン菌)

= methane-producing bacteria
Methanobacterium, etc.

4H₂ + CO₂ → CH₄ + 2H₂O

Amoebozoa (アメーボゾア)

⊂ Domain Eukaryota (Eukaryotes)

(Chatton 1925) Whittaker et Margulis 1978
Revisions to the classification, nomenclature, and diversity of eukaryotes (Adl et al. 2019)

Kingdom (or unranked) Amoebozoa

Lühe 1913 sensu Cavalier-Smith 1998
= Eumycetozoa Zopf 1884, emend Olive 1975

Infraphylum Mycetozoa de Bary 1873 (粘菌類)

= a polyphyletic grouping of slime molds
Slime mold or mould: has a life cycle that includes a free-living single-celled stage and the formation of spores

Class Protostelea (原生粘菌), protosteloid amoebae

= protosteloids, protostelids
amoeboid slime-molds that form fruiting bodies

Order Protosteliida

Class Myxogastria Macbride 1899 (変形菌), myxomycetes

= Myxogastrea, myxogastrids (ICZN) or Myxomycetes (ICBN), true slime molds
syncytial, plasmodial, or acellular slime molds

Order Liceida (コホコリ)

= Liceales

Order Echinosteliida (ハリホコリ)

Order Trichiida or Trichiales (ケホコリ)

Arcyriaceae (ウツボホコリ)
Dianemataceae (イトホコリ)
Trichiaceae (ケホコリ)
Minakataellidae (ミナカタホコリ)

Order Stemonitida (ムラサキホコリ)

Stemonitidaceae Fr. 1829
Amaurochaete, Brefeldia, Collaria, Colloderma, Comatricha, Diacheopsis, Enerthenema, Lamproderma, Leptoderma, Macbrideola, Meriderma, Paradiachea, Paradiacheopsis, Stemonaria, Stemonitis, Stemonitopsis, Symphytocarpus

Order Physarida (モジホコリ)

Physarum polycephalum Schwein 1822 (モジホコリ) many-headed slime or grape-cluster slime

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

Fig. Life cycle of Dictyostelium. Aggregate (aggregation, 細胞凝集). Fruiting body (子実体). Culmination (子実体形成). (Macro)cyst (spore, シスト)

Order Dictyosteliida or Dictyosteliales (タマホコリ)

= dictyostelids, Dictyostelea (ICZN) or Dictyosteliomycetes (ICBN)
(dictyostelid) cellular slime molds
Dictyosteliidae or Dictyosteliaceae Rostafinski (タマホコリ)
Dictyostelium Brefeld (タマホコリ)

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

Polysphondylium (ムラサキホコリモドキ)
Coenonia
Myxomitodes (fossil record)
Actyosteliidae (エツキタマホコリ)
= Acytosteliaceae Raper ex Raper et Quinlan 1958
Acytostelium Raper (エツキタマホコリ)

Experiments on microbiology (微生物学実験)

Strain observation and separation (細菌株分け)

Gram stain or Gram staining (グラム染色)

Note that young cells can not be stained well

Four basic steps:

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
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

Decolorizer: Decolorize rapidly with ethanol or acetone
= pour 95% ethanol adn shake lightly for 5-15. seconds
→ rinse
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

Gentian violet (numbers of methyls bonded with N)

methyl violet 10B = crystal violet (6)___MV 6B (5)___________MV 2B (4)

Lipid analysis methods (脂質分析法)

Extraction procedure

Preculture Pseudomonas aeruginosa at 20°C for 48 hr
Main culture (nutrient medium 1% peptone) : transfer 4 ml pre-culture 4 ml

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)

Harvest by using a centrifugation (7000 rpm for 10 min at 4°C)
Wash suspension (WW ca 2g/20 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

Rigorous mixing for 30 min
Wait for 60 min (do not forget this!)

↓ add 25 ml chloroform

Rigorous mixing for 15 min

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

Recover chloroform by a pipet
Add 20 ml chloroform to the remainder* (*: repeat these for 3 times)
Recover chloroform layer and centrifuge
Recover chloroform gently