Journal of Environmental Biotechnology
Vol. 11, No. 1 · 2, 77–81, 2011

Original paper (regular paper)

Analyses of Three Dominant Membrane Proteinsfrom Anammox Planctomycete Candidatus ‘Brocadia sinica’
Fuyumi Tojo1, Yoshikane Itoh1, Satoshi Okabe2 and Masaaki Morikawa1*
Graduate School of Environmental Science, Hokkaido University, Kita-10, Nishi-5, Kita-ku, Sapporo,
Hokkaido 060–0810, Japan
2
Graduate School of Engineering, Hokkaido University, Kita-13, Nishi-8, Kita-ku, Sapporo, Hokkaido 060–8628, Japan1* TEL/FAX: +81–11–706–2253* E-mail: morikawa@ees.hokudai.ac.jp(Received; 27 October, 2011/Accepted; 15 November, 2011)
Anammox planctomycetes commonly possess a unique single membrane-bound organelle, the anammoxosome, that harbors enzymes central to anaerobic ammonia oxidation, anammox, metabolism. We found that three membrane proteins,
namely P35, P45, or P90, were dominantly produced in Candidatus ‘Brocadia sinica,’ an anammox planctomycete that has
been obtained from an eﬀective anammox reactor. N-terminal amino acid sequences suggested that all these three are components of hydrazine hydrolase which is a key enzyme in anammox metabolism. The genes encoding for P35, P45, and P90
formed a cluster preceded by sigma54 specific two-component regulator proteins. Heterologous gene expression of P35, P45
and P90 conferred high biofilm formation ability to the E. coli cells, suggesting that they were transported to cell membraneof E. coli and the membrane structure was modified to increase cell adhesion activity.
Key words: anammox planctomycete, Candidatus ‘Brocadia sinica’, anammoxosome, hydrazine hydrolase, biofilm

1. Introduction
The phylum planctomycetes of the domain Bacteria consists of unique budding, peptidoglycan-less organisms andpossess intracellular membrane compartmentation. planctomycetes consist of eleven genera, six of which are cultured,
Pirellula, Rhodopirellula, Blastopirellula, Planctomyces,
Gemmata, and Isosphaera, and fi ve so far uncultured,
Candidatus ‘Anammoxoglobus,’ Candidatus ‘Brocadia,’
Candidatus ‘Jettenia,’ Candidatus ‘Kuenenia,’ and Candidatus ‘Scalindua’ 4,5,6,8). The members in the latter fivegroups of ammonium-oxidizing chemoautotrophs perform
unique anammox, anaerobic ammonia oxidation, metabolism. Anammox metabolism produces a toxic hydrazine as
an intermediate that is further converted into dinitrogenby hydrazine oxidoreductase4). These uncultured anammox
panktomycetes commonly possess a single membranebound organelle, the anammoxosome, that harbors enzymes
central to the unique anammox metabolism 9,18,19). The
anammoxosome membrane contains ladderane lipids (alsounique to anammox planctomycetes), which are composedof concatenated cyclobutane rings and may posess both esterand ether links4,6). Molecular modeling indicated that theladderane lipids surrounding the anammoxosome yield an
exceptional rigid biomembrane, implicating that this dense
barrier may serve to separate the remainder of the cell fromthe intermediate hydrazine and to maintain concentration

gradients14). It has been recently shown that both hydrazinehydrolase (HH) and hydrazine oxidoreductase (HAO) arepresent inside anammoxosome6,7,10) in Candidatus ‘Kuenenia stuttgartiensis’.
Previously, we developed an up-flow, fi xed-bed anammox
biofi lm reactor with non-woven fabric sheets as biomass
carriers and attained a high nitrogen removal rate of
26.0 kgN/m3 day16,17) and the anammox planctomycetes has
recently been identified as Ca. ‘Brocadia sinica’ 2,11). The
half-saturation constants (Ks) for ammonium and nitrite
of Ca. ‘B. sinica’ were determined 28±4 and 86±4 μM,
respectively, higher than those of Ca. ‘B. anammoxidans’
and Ca. ‘K. stuttgartiensis’ 11). The ultrastructure of Ca.
‘B. sinica’ was also examined by transmission electron
microscopy to confirm the presence of the anammoxosome.
Here, we show that the three most abundantly produced
membrane proteins in Ca. ‘B. sinica’, P35, P45, and P90,
are components of HH. When these protein-encoding genes
were expressed in E. coli, the cells that produce either P35,
P45, or P90 formed more biofi lms than non-producing
control cells. This is the fi rst report that determines
membrane protein profi les in anammox planctomycete, Ca.
‘B. sinica’.

Tojo et al.

78

2. Materials and Methods
2.1. Bacterial strain and culture media
The microbial granules containing Ca. ‘B. sinica’ weregrown in an up-flow column reactor with mineral medium for anammox bacteria18). Briefly, it contained per liter,(NH4)2SO4, 660 mg; NaNO2, 345 mg; KHCO3, 500 mg;
KH 2PO4, 27.2 mg; MgSO4 · 7H 2O, 300 mg; CaCl2 · 2H 2O,
180 mg, and trace element solution. The previous study
determined the microbial composition in the granule sample
by fluorescence in-situ hybridization with probe AMX82012),
and found that anammox planctomycetes accounted forapproximately 90% of the total cells16,17).
2.2. Preparation and analyses of cytoplasmic andmembrane proteins from Ca. ‘B. sinica’
The anammox granules, 0.5 g, were disrupted by Multibeads shocker (Yasui Kikai Co.) using 0.1 mm glass beads
in 700 μl of 50 mM Tris-Cl (H7.5) containing 5 mM MgCl2.
After removing the glass beads by sedimentation, cell suspension was recovered. Cytoplasmic and membrane fractions
were separated by ultracentrifuge (4°C, 100,000 g, 120 min).
Each fraction sample was further prepared in SDS sample
buﬀer (50 mM Tris-HCl pH 6.8, 6% 2-mercaptoethanol,
10% glycerol, 2% SDS and 0.01% bromophenol blue) andboiled for 5 min. Clear supernatants after centrifuge wereanalyzed by SDS-PAGE using 12% acrylamide gel. Electrophoresis was carried out at 30 V for 16 hr. Protein bands
were visualized by Coomassie Brilliant Blue R-250 staining.
Major protein bands were blotted to PVDF membrane(Bio-Rad) in order to determine N-terminal amino acidsequence by Procise 492 (PERKIN ELMER) or Procise
491 cLC (Applied Biosystems). The protein sequence wasanalyzed by protein blast homology search program, http://
blast.ncbi.nlm.nih.gov/Blast.cgi.

PCR amplified DNA fragments were confi rmed no errors
by a genetic analyzer ABI 3130 (Applied Biosystems). EachDNA fragment was cloned into the gap of NdeI and EcoRI
sites of expression vector pET25b (+) to construct pET25bp35, pET25b-p45, and pET25b-p90 and the resultant
recombinant E. coli cells were examined as follows.
2.4. Biofilm formation and protein production byrecombinant E. coli
Biofi lm formation activity was measured for provisionally examining the change in cell adherence activity due tooverexpression of membrane proteins. Overnight shaking
cultures were diluted 1 : 100 by fresh LB medium, shaken
at 37°C for 2 hr, and then added with 1 mM IPTG (fi nal
concentration) for gene induction. The cultures were immediately dispensed to 96-well micro-titer plates and staticallygrown to form biofi lms at 26°C for 24 and 48 hr. This low
temperature condition was useful to overproduce hydrophobic membrane protein eﬀectively. Planktonic cell cultures
were discarded and the biofi lms formed inner-surface ofwells were washed with sterilized water. The biofi lms werethen stained by 0.1% crystal violet and bound crystal violet
was extracted by 33% acetic acid. Biofi lm formation activity
was determined by measuring absorbance of the solution
at 595 nm.
Protein production was analyzed by SDS-PAGE using
12% polyacrylamide gel as described elsewhere. The protein
sample was prepared from whole cells. The cells were similarlygrown as above in LB containing IPTG at 26°C except for
using glass test tubes in stead of micro-titer plates.

2.3. Expression of membrane protein genes from Ca.
‘B. sinica’ in E. coli
E. coli BL21(DE3) (Invitrogen) was used as the hoststrain to express membrane protein genes from Ca. ‘B.
sinica.’ Recombinant E. coli cells were cultured in LuliaBertani (LB) medium containing 100 μg/ml ampicillin.
The DNA regions encoding the P35, P45 and P90 were separately amplified by PCR using Ca. ‘B. sinica’ genome DNA
as a template, a set of following primers, and KOD plus DNA
polymerase (Toyobo). The primer sets were: 5’-CATATGAAGAGTAGTCTAAAGATTGGTTTA-3’ (NdeI site,
underlined) and 5’-GTGGAGTTCCTGAAGGCGCTATAAGAATTC-3’ (EcoRI site, underlined) for p35,
5’- CATATGA A AGAGAGAGGATGGATAGGAG-3’(NdeI site, underlined) and 5’-GATAAGGACGCCAGT
GGATTAATAGGAATTC-3’ (EcoRI site, underlined) for
p45, and 5’-CATATGAGTAAGAGGATAATAGGAG
GGG-3’ (NdeI site, underlined) and 5’-TACCCTGGC
TATCTGGCAAGATAAGAATTC-3’ (EcoRI site, underlined) for p90, respectively. The nucleotide sequences of theFig. 1. SDS-PAGE analysis of cytoplasmic and membrane proteinsfrom Ca. ‘B. sinica.’a, Size marker; b, cytoplasmic proteins; c, membrane proteins.

Analyses of Three Dominant Membrane Proteins from Anammox Planctomycete Candidatus ‘Brocadia sinica’

3. Results and Discussion
3.1. Identification of major membrane proteins in Ca.
‘B. sinica’
It was found that three proteins, namely P35, P45 andP90, were dominantly produced in the membrane fraction of
Ca. ‘B. sinica’ cells (Fig. 1). N-terminal amino acid sequencesof these proteins showed significant similarity with proteinsfrom anammox planctomycetes, including KSU-1 and Ca.
‘K. stuttgartiensis’ (Table 1). We had already determined
draft sequence of the whole genome of Ca. ‘B. sinica’(unpublished data). It was found that the genes encodingfor P35, P45, and P90 formed a cluster with a set of possible
sigma54 specific two-component transcriptional regulator
proteins (Sigma54-SK/RR) in the upstream region (Fig. 2a).
Sigma54 (RpoN) was identified as a sigma factor involved in
the transcription of genes involved in the cellular assimila-

Table 1. N-terminal amino acid sequence identities of P35, P45,
and P90 to other anammox planctomycete proteins.
Name

Amino acid sequence

P90
KSU-1
kuste2861
P45
kuste2859
P35
KSU-1
kuste2860

1 NQVMTGGSKQXKALXT 17
29 NQVMVGGSKQGKALWT 44
30 --VMTGGPVQGKALWT 43
1 GFIQGTHVKTDMPSPFFVT 19
35 GYIQGTHVKTDLPGPFHIT 53
1 GTPQVVATIQTGPEAW 16
28 GTPQVIATIQTGPE-W 42
40 GQPRVISTIQTGAT-W 54

Identity (%)
—
87
83
—
74
—
88
56

Note: kuste2859, kuste2860, and kuste2861 are hydrazinehydrolase components from Ca ‘Kuenenia stuttgartiensis’ 7).
KSU-1 denotes respective proteins from an anammox bacterium
KSU-1 13).

Fig. 2. a. Gene cluster structure of P35, P45, P90 and flanking regions.
Sigma54-SK, sigma54 specific sensor kinase; Sigma54-RR, sigma54 specific response regulator; hp, hypothetical protein;c554, cytochrome c554 homologue.
Homologous protein genes in Ca. ‘K. stuttgartiensis’ are also
shown in parentheses.
b. Signal peptide sequence and processing site (/).

79

tion of ammonia and glutamate under conditions of nitrogen
limitation1). It has been reported that Ca. ‘K. stuttgartiensis’ has similar gene organization of HH (kuste2859–2861)
and Sigma54-SK/RR (kuste2857/2858)15). However, it isinteresting that sigma54-SK/RR in the upstream of P35,
P45, P90 from Ca. ‘B. sinica’ is homologous to kuste1259/
kuste1258 at diﬀerent position rather than kuste2857/2858.
It may be also worth to note that the genes encoding
kuste1259/kuste1258 are orphan genes whose function is
yet unknown.
The amino acid sequence of P45 (376 a.a.) was 79% identical to kuste2859 (386 a.a.) from Ca. ‘K. stuttgartiensis,’while P35 (340 a.a.) was 91% identical to a protein from
KSU-1 (341 a.a.) and 86% identical to kuste2860 (353 a.a.).
P90 (825 a.a.) was 89% identical to protein of KSU-1 (810a.a.) and 82% identical to kuste2861 (809 a.a.), respectively.
It has recently been reported that kuste2859, 2860, and 2861
are anammoxosome proteins and HH components7). These
information strongly suggested that P35, P45, and P90 are
HH that locate in anammoxosome membrane rather thancell membrane of Ca. ‘B. sinica.’ High production levelsof these proteins may explain a quite eﬀective nitrogen removal rate of Ca. ‘B. sinica’ at 26.0 kgN/m3 day.
Anammoxosome membrane is composed of uniqueladderane lipids whose structure is quite diﬀerent from
popular bacterial cytoplasmic membrane lipids. We next
asked if the P35, P45, and P90 could be overproduced incytoplasmic membrane in E. coli.
3.2. Expression of p35, p45, and p90 in E. coli
Biofi lms are structures formed by surface attached microorganisms encased in matrices. Biofi lm formation is largely
aﬀected by cell surface property including membrane protein
profi les3). We hypothesized that the E. coli cells producing
P35, P45, or P90 in the cell membrane would have diﬀerent
biofi lm formation ability. It was found that the E. coli cells

Fig. 3. Biofilm formation activity of recombinant E. coli cells.
a, pET25b (+) vector; b, pET25b-p35; c, pET25b-p45; d,
pET25b-p90 harboring-strains.
Error bars indicate standard deviations in five independent
tests.

80

Tojo et al.

Acknowledgements
We thank to Dr. Kyoko Miwa and Dr. Mamoru Oshiki for
helpful discussion. This work has been supported by New
Energy and Industrial Technology Development Organization (NEDO).

References

Fig. 4. SDS-PAGE analysis of proteins from recombinant E. coli
cells.
a, Size marker; b, pET25b (+) vector; c, pET25b-p35; d,
pET25b-p45; e, pET25b-p90. Positions of the recombinant
P35 and P45 proteins are indicated by arrowheads.

that produced P35, P45, or P90 formed more biofi lms thanthe cells harboring pET25b vector only, although the difference is statistically not so much significant for P45 andP90. (Fig. 3). It may also worth to note that biofi lm formation of pET25b-p35 and pET25b-p45 harboring-strains
were slightly decreased at 48 hr while pET25b-p90 and
pET25b (+) harboring-strains increased or comparable at
48 hr.
Protein production was verified by SDS-PAGE (Fig. 4). It
was clear that P35 and P45 were abundantly produced in thecells. On the other hand, only a negligible amount of P90
was produced probably due to its unusual signal-peptide
processing site, 1-MSKRIIGGVMASALVVGALVCGDI
FAS/NQVMT-32 (Fig. 2b), with low transportation eﬃciency in E. coli cells. It has been previously shown that
HH is transported into anammoxosome in Ca ‘K. stuttgartiensis’ 6,7) and our result suggested that P35, P45, P90(HH) are localized in the membrane fraction of Ca ‘B.
sinica’. When we observed the cells by microscope, noneof these three recombinant cells formed inclusion body inthe cells. Although we do not have direct evidence that theproteins localized in the membrane fraction of the cells,
these results suggest that P35, P45, and P90 were produced
and transported to cell membrane of E. coli cells that has
no anammoxosome organelle. It also remains elucidated
whether conformation and topology of these proteins are
similar in anammoxosome membrane of Ca ‘B. sinica’ andin cell membrane of E. coli.

1) Buck, M., M-T. Gallegos, D. Studholme, Y. Guo, and J.D.
Gralla. 2000. The bacterial enhancer-dependent sigma54(sigmaN) transcription factor. J. Bacteriol. 182: 4129–4136.
2) Cho, S., Y. Takahashi, N. Fujii, Y. Yamada, H. Sato, and S.
Okabe. 2010. Nitrogen removal performance and microbialcommunity analysis of an anaerobic up-flow granular bed anammox reactor. Chemosphere 78: 1129–1135.
3) Danese, P.N., L.A. Pratt, S.L. Dove, and R. Kolter. 2000. The
outer membrane protein, antigen 43, mediates cell-to-cellinteractions within Escherichia coli biofilms. Mol. Microbiol.
37: 424–432.
4) Fuerst, J.A. 2005. Intracellular compartmentation in Planctomycetes. Annu. Rev. Microbiol. 59: 299–328.
5) Fuerst, J.A. 1995. The planctomycetes – emerging models for
microbial ecology, evolution and cell biology. Microbiology
141: 1493–1506.
6) Jetten, M.S.M., L. van Niftrik, M. Strous, B. Kartal, J.T.
Keltjens, and H.J.M.O. den Camp. 2009. Biochemistry andmolecular biology of anammox bacteria. Crit. Rev. Biochem.
Mol. Biol. 44: 65–84.
7) Karlsson, R., A. Karlsson, O. Backman, B.R. Johansson, and S.
Hulth. 2009. Identification of key proteins involved in the
anammox reaction. FEMS Microbiol. Lett. 297: 87–94.
8) Kartal, B., J. Rattray, L.A. van Niftrik, J. van de Vossenberg,
M.C. Schmid, R.I. Webb, S. Schouten, J.A. Fuerst, J.S. Damsté,
M.S. Jetten, and M. Strous. 2007. Candidatus ‘Anammoxoglobus propionicus’ a new propionate oxidizing species of anaerobic
ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30: 39–49.
9) Kowalchuk, G.A., and J.R. Stephen. 2001. Ammonia-oxidizing
bacteria: A model for molecular microbial ecology. Annu. Rev.
Microbiol. 55: 485–529.
10) Lindsay, M.R., R.I. Webb, M. Strous, M.S. Jetten, M.K. Butler,
R.J. Forde, and J.A. Fuerst. 2001. Cell compartmentalizationin planctomycetes: novel types of structural organisation forthe bacterial cell. Arch. Microbiol. 175: 413–429.
11) Oshiki, M., M. Shimokawa, N. Fujii, H. Satoh, and S. Okabe.
2011. Physiological characteristics of the anaerobic ammoniumoxidizing bacterium ‘Candidatus Brocadia sinica.’ Microbiology
157: 1706–1713.
12) Schmid, M., S. Schmitz-Esser, M. Jetten, and M. Wagner.
2001. 16S–23S rDNA intergenic spacer and 23S rDNA of anaerobic ammoniumoxidizing bacteria: implications for phylogeny and in situ detection. Environ. Microbiol. 3: 450–459.
13) Shimamura, M., T. Nishiyama, H. Shigetomo, T. Toyomoto, Y.
Kawahara, K. Furukawa, and T. Fujii. 2007. Isolation of amultiheme protein with features of a hydrazine-oxidizing enzyme
from an anaerobic ammonium-oxidizing enrichment culture.
Appl. Environ. Microbiol. 73: 1065–1072.
14) Sinninghe Damsté, J.S., W.I. Rijpstra, M. Strous, M.S. Jetten,
O.R. David, J.A. Geenevasen, and J.H. van Maarseveen. 2004.
A mixed ladderane/n-alkyl glycerol diether membrane lipid inan anaerobic ammonium-oxidizing bacterium. Chem. Commun. (Camb). 22: 2590–2591.
15) Strous, M., E. Pelletier, S. Mangenot, T. Rattei, A. Lehner, M.W.
Taylor, M. Horn, H. Daims, D. Bartol-Mavel, P. Wincker, V.
Barbe, N. Fonknechten, D. Vallenet, B. Segurens, C. SchenowitzTruong, C. Medigue, A. Collingro, B. Snel, B.E. Dutilh,
H.J.M.O. den Camp, C. van der Drift, I. Cirpus, K.T. van deAnalyses of Three Dominant Membrane Proteins from Anammox Planctomycete Candidatus ‘Brocadia sinica’
Pas-Schoonen, H.R. Harhang, L. van Niftrik, M. Schmid, J.
Keltjens, J. van de Vossenberg, B. Kartal, H. Meier, D.
Frishman, M.A. Huynen, H.W. Mewes, J. Weissenbach,
M.S.M. Jetten, M. Wagner, and D.L. Paslier. 2006. Deciphering the evolution and metabolism of an anammox bacterium
from a community genome. Nature 440: 790–794.
16) Tsushima, I., Y. Ogasawara, T. Kindaichi, H. Satoh, and S.
Okabe. 2007. Development of high-rate anaerobic ammoniumoxidizing (anammox) biofilm reactors. Water Res. 41: 1623–
1634.
17) Tsushima, I., Y. Ogasawara, M. Shimokawa, T. Kindaichi, and S.
Okabe. 2007. Development of a super high-rate anammox

81

reactor and in situ analysis of biofilm structure and function.
Water Sci. Technol. 55: 9–17.
18) van Niftrik, L., W.J. Geerts, E.G. van Donselaar, B.M.
Humbel, A. Yakushevska, A.J. Verkleij, M.S. Jetten, and M.
Strous. 2008. Combined structural and chemical analysis of
the anammoxosome: a membrane-bounded intracytoplasmic
compartment in anammox bacteria. J. Struct. Biol. 161: 401–
410.
19) van Niftrik, L.A., J.A. Fuerst, J.S. Sinninghe Damste, J.G.
Kuenen, M.S. Jetten, and M. Strous. 2004. The anammoxosome: an intracytoplasmic compartment in anammox bacteria.
FEMS Microbiol. Lett. 233: 7–13.