分けたから分かった！
～アナモックス細菌Kuenenia stuttgartiensis
細胞分画から見えたこと～
石井研 雑誌会 No. 1178平成２８年２月５日
石井 正治
Did you know?
（１）That most of the air we breathe is made by
anammox bacteria?
（２）That anammox bacteria divide only once
per two weeks at maximum speed?
（３）That hydrazine (rocket fuel) and nitric
oxide are the anammox intermediates?
（４）That these intermediates are turned over
in an organelle surrounded by an impermeable
membrane made of ladderane lipids?
アンモニア酸化時のエネルギー
2NH
4++ 3O
2= 2NO
2-+ 2H
2O + 4H
+-241
NH
4++ NO
2-= N
2+ 2H
2O -335
5NH
4++ 3NO
3-= 4N
2+ 9H
2O + 2H
+-278
NH
4++ 6Fe
3+= N
2+ 6Fe
2++ 8H
+-100
8NH
4++ 3SO
42-= 4N
2+ 3H
2S + 12H
2O + 5H
+-22
NH
4++ 3HCO
3-= 2N
2+ 3CH
2O + 6H
2O + H
++94
kJ/mol ammonium
入口のアンモニア濃度
出口のアンモニア濃度
アナモックス発見のきっかけ
アナモックス菌の特徴的なエネルギー代謝
アナモキソソーム
Proposed process of nitrogen formation and ATP generation from ammonium and nitrite with nitric oxide (NO) and hydrazine (N2H2) as intermediates. Diamonds
represent putative cytochrome c proteins and the number of electrons that are transferred in between the related reactions. The hypothetical reduced cytochrome:quinone (Q)
oxidoreductase that feeds electrons from hydrazine oxidation into the Q-pool is indicated by the question mark. bc1: quinol:cytochrome c oxidoreductase (bc1, complex III), ETM: electron transfer module providing hydrazine synthase (HZS) with reductant, HDH:
hydrazine dehydrogenase, Nir: nitrite reductase. Ψ+, Ψ, the positive and negative sides of the electrochemical gradient compartments, which most likely are the anammoxosome and cytoplasm (riboplasm), respectively.
エネルギー代謝
ATP合成時の代謝
ラダラン脂質
アナモックス菌の自然界での分布例
～黒海～
パーコールグラジエントによる単離
A:アナモキソソーム
B:リボプラズム画分
C:未破壊菌体
Glass bead milling was
performed at 20 Hz for 45 s after
the addition of approximately 300
mg glass beads (Ø 0.5 mm).
Samples were then transferred
on top of a 1:1 mixture of Percoll
(GE Healthcare) and 0.1 M
HEPES buffer (pH 6.8) with a
final concentration of 0.25 M
sucrose and centrifuged at
48,000 g for 25 min at 10°C. The
separate bands from the gradient
were collected and washed with
0.1 M HEPES (pH 6.8)
単離プロセスの評価
それぞれのフラクションの電子顕微鏡写真
Molecular Microbiology
Volume 94, Issue 4, pages 794-802, 22 OCT 2014 DOI: 10.1111/mmi.12816 http://onlinelibrary.wiley.com/doi/10.1111/mmi.12816/full#mmi12816-fig-0002
The isolated anammoxosomes also frequently exhibited tubule-like protein structures and round, electrondense particles which
resembled the iron-rich particles that were previously observed in anammoxosomes (van Niftrik et al., 2004; 2008b).
アナモックス活性 (1/2)
■と□：アナモキソソーム ▲と△：Cell lysate フィルド：15NH 4+, 14NO2 -, 14N14Nヒドラジン オープン：14N14Nヒドラジン 29N
2を観ている
フィルド：アナモキソソーム オープン：Cell lysate ダイアモンド： 15NH 4+, 14NO2 - 四角：15N15Nヒドラジン 三角：15NH 4+, 14NO2 -, 15N15Nヒドラジン
アナモックス活性 (2/2)
30N
2を観ている
Results of the proteome analysis of
subcellular fractions and intact cells of K.
stuttgartiensis. Proteins with the highest
abundance in isolated anammoxosomes
were assigned to the anammoxosome.
Proteins that had the highest abundance
either in anammoxosomes surrounded by
the riboplasm (Riboplasm1) or both intact
cells and anammoxosomes surrounded
by the riboplasm (Riboplasm2), were
assigned to the riboplasm.
Ten hydroxylamine oxidoreductase (HAO)-related octaheme proteins in the Kuenenia stuttgartiensis genome.
The gene products are ordered according to their cluster position in the phylogenetic tree; highly homologous HAO-like proteins are
boxed by dashed lines. Lengths of the polypeptides are drawn to
scale (aa: amino acids) and homologous cytochrome c-rich parts are vertically aligned. Redox partners represent (potential) electron
transfer subunits found in the same gene cluster. Expression values are expressed as n–fold coverage of Solexa deep RNA sequencing of the K. stuttgartiensis transcriptome (Strous et al., 2006; Kartal et al., 2011b). Structural motifs: TMH: transmembrane-spanning helix, cleavage site: N-terminal cleavage site, multicopper oxidase:
multicopper oxidase domain, catalysis: catalytic heme, CXXCH: heme c-binding motif, CXXXXCH: unusual heme c-binding motif in kustc0694 and kustc1340, Tyrosine: tyrosine that covalently links the subunits, laminin: laminin sequence. a Classification according to
(Klotz et al., 2008). b This gene cluster also contains a gene encoding a cupredoxin-like blue copper protein (kustc0456).
Gene cluster organization of the hydrazine synthase
system in the Kuenenia stuttgartiensis genome (a)
and the proposed functional organization of its gene
products (b).
(a) Lengths of the gene products and the position of
structural motifs are drawn to scale (aa: amino acids).
(b) Putative heme b- and quinone (Q)-binding sites were
derived from sequence comparison with respect to the
highly homologous c subunit (FdnI, cyt b556) of
menaquinone-dependent formate dehydrogenase
(FDH-N, FdnGHI) from Escherichia coli (Jormakka et al., 2002).
Structural motifs are specified in the Figure. Numbers refer
to the kuste gene numbers. Cleavage site, N-terminal
Gene cluster organization of the three bc1 complexes in the Kuenenia stuttgartiensis genome (a) and the proposed
functional organization of their gene products (b).
(a) Lengths of the gene products and the position of structural motifs are drawn to scale (aa: amino acids). (b) Putative heme b- and
quinone (Q)-binding sites were derived from sequence comparison
with respect to the canonical bacterial bc1 complex (left-hand figure),
which is represented as its monomeric three-subunit complex for
simplicity (see also text). Numbers refer to the kust gene numbers as in (a). Structural motifs are specified in the Figure. 2Fe2S, Rieske
2Fe-2S iron-sulfur cluster; cleavage site, N-terminal cleavage site; tat signal, twin-arginine translocation signal; TMH,
Four ATPases in the Kuenenia stuttgartiensis genome.
Gene products are labeled according to the homologous
subunits in related
ATPases. Genes that have been annotated in the K.
stuttgartiensis genome as unknown proteins or that were
erroneously annotated are indicated with white circles on
top (Van Niftrik et al., 2010). Lengths of the polypeptides
are drawn to scale (aa: amino acids).
a. anammoxosome
membrane
1. cytoplasmic
membrane;
2. putative
peptidoglycan;
3.outermembrane;
4. S-layer.
Scale bars, 100nm
電子顕微鏡写真
Lysozyme-sensitive sacculi were obtained by boiling K. stuttgartiensis cells enriched by density centrifugation in SDS. (a) TEM of K.
stuttgartiensis sacculus using negative staining. (b) After lysozyme treatment, the K. stuttgartiensis sacculi were absent or had a fibrous
appearance, as observed by negative staining via TEM. Scale bars, 1 μm
(a) The negative control probe ELA-LA, which cannot be incorporated, shows only a faint background. (b) Septal incorporation of EDA-DA is present both in anammox and rod-shaped non-anammox species
present in the bioreactor (arrowhead). (c) SIM clearly shows that EDA-DA was incorporated specifically at the cell division site. Probe
incorporation was visualized with a complimentary fluorophore using
click chemistry (green) and cell surfaces were labelled by amine-reactive Pacific Blue N-hydroxysuccinimide ester (NHS) (blue). ELA-LA, ethynyl-L-alanyl-L-alanine. Scale bars,2 μm.
(a) UPLC analyses of K. stuttgartiensis
peptidoglycan. E. coli K12 PG profile is included as a reference. Peaks labelled 1–6 correspond to muropeptides of K. stuttgartiensis Histodenz-enriched cultures. RT, retention time; A204, absorbance 204 nm.
(b) Mass analysis of the PG peptidoglycan subunits isolated in panel A by MALDI-TOF. D44 was formulated based on its similar
retention time with E. coli D44 R.T; ND, not determined; %, relative abundance; theo, theoretical mass; det, determined mass. (c) Schematic representation of K.
stuttgartiensis detected PG species in their
reduced state.
M2,
N-acetylglucosamine-(GlcNAc)-N-acetylmuramic acid (MurNAc)-L-Ala-DGlu; M3, GlcNAc-MurNac-L-Ala-D-Glu-meso-DAP; M4, GlcNac-MurNAc-LAla-D-Glu-meso-DAP-D-Ala; D43, dimer muropeptide of a M4 D,D-crosslinked to a M3; M, MurNAc; G, GlcNAc.
