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Detection of Anaerobic Ammonium-Oxidizing Bacteria in Ago Bay Sediments

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Detection of Anaerobic Ammonium-Oxidizing Bacteria in Ago Bay Sediments 80117 (304) Biosci. Biotechnol. Biochem., 72, 80117-1–4, 2008 Note Detection of Anaerobic Ammonium-Oxidizing Bacteria in Ago Bay Sediments y Jun NAKAJIMA, Makiko SAKKA, Tetsuya KIMURA, and Kazuo SAKKA Graduate School of Bioresources, Mie University, Tsu 514-8507, Japan Received February 25, 2008; Accepted May 12, 2008; Online Publication, August 7, 2008 [doi:10.1271/bbb.80117] We identified 16S rRNA gene sequences in sediment samples from Ago Bay in Japan, forming a new branch of the anammox group or closely related to anaerobic ammonium oxidizing (anammox) bacterial sequences. Anammox activity in the sediment samples was detected by 15N tracer assays. These results, along with the results of fluorescence in situ hybridization FISH analy- sis, suggest the presence of anammox bacteria in the Ocean marineAdvance sediments. View Key words: anaerobic ammonium oxidizing (anam- mox); marine sediment; fluorescence in situ hybridization (FISH); Candidatus ‘‘Scalin- Fig. 1. Sampling Sites in Ago Bay in Japan. dua’’ In recent years, Ago Bay, a typical enclosed coastal from environmental DNA extracts was performed using sea in Mie Prefecture, Japan, is suffering eutrophica- anammox 16S rRNA oligonucleotide primer sets, tion1) including that due to nitrogen compounds, Brod541F-Brod1260R,3) Pla46-Amx820,4) and Pla46- 4) Proofs ammonium, nitrite, and nitrate ions. Aerobic nitrification BS820, and AmpliTaq Gold DNA polymerase (Ap- and subsequent anaerobic denitrification is part of the plied Biosystems, Foster City, CA). Cloning, sequenc- global nitrogen cycle. On the other hand, anammox is ing, and phylogenetic analysis were performed as the biological conversion of ammonium and nitrite to described by Rich et al.5) PCR of 16S rRNA genes dinitrogen gas. Since first discovery, the geographical using all the primer sets yielded the expected fragments distribution and importance of the anammox process from the DNA sample from site 20, but only primer set to the global nitrogen cycle have been of great interest Pla46-Amx820 gave amplified DNA fragments from and are currently being evaluated by several stra- site 4, site 6, site 10, and site 12 samples as template tegies, including detection of anammox bacterial 16S DNAs. Amplified fragments from the respective sedi- rRNA gene sequences and anammox reaction using ment samples were separately connected to a cloning 15N-labelled compounds.2) vector and introduced into E. coli to construct gene In this study, we investigated the distribution and libraries. We sequenced the inserts of more than 200 diversity of anammox-related Planctomycetales in sedi- recombinant plasmids. Phylogenetic analysis (Fig. 2A) ment samples from five sites in Ago Bay. All the indicated that DNA fragments amplified with Pla46- samples were taken from the upper 2–5 cm of the Amx820 clustered into 15 groups and clustered within sediments in April 2007. Five sites were site 4 (34 16.50 the anammox group to form a cohesive new branch. N, 136 47.40 E), site 6 (34 19.00 N, 136 48.00 E), Eight groups were highly similar to uncultured planc- site 10 (34 17.10 N, 136 51.30 E), site 12 (34 17.30 N, tomycete clone A6 (accession no. AY266449), which is 136 50.20 E), and site 20 (34 17.00 N, 136 45.00 E) recognized as an anammox-related bacterial 16S rRNA (Fig. 1). gene sequence.6) Sequencing of the inserts amplified DNA was isolated using the Ultraclean soil DNA kit with Pla46-BS820 detected a 16S rRNA gene highly (MoBio Laboratories, Solana Beach, CA). Amplifica- similar to that of anaerobic ammonium-oxidizing tion of 16S rRNA genes targeting the anammox group planctomycete JMK-2 (AB281489),7) which has recent- y To whom correspondence should be addressed. Tel: +81-59-231-9621; Fax: +81-59-231-9684; E-mail: [email protected] Abbreviations: anammox, anaerobic ammonium-oxidizing; PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization 80117-2 J. NAKAJIMA et al. A Advance View B Proofs Fig. 2. Phylogenetic Trees of 16S rRNA Genes Obtained with a Pla46F-Amx820 and Pla46F-BS820 Primer Set (A) and a Brod541F-Brod1260 Primer Set (B). The 16S rRNA gene sequences derived from the sediments in this study were compared with those of well-characterized anammox bacteria, Planctomycetes, and several uncultured clones. A clone obtained with Pla46F-Amx820 primer set from site 6, for example, was referred to as Am6-. The bar represents 2% estimated distance of sequence divergence. Sequence accession numbers are given in parentheses. ly been found in an enrichment culture derived from group (Fig. 2B). The latter observation indicates that Ago Bay sediment. Since JMK-2 is closely related to primer set Brod541F-Brod1260R provided efficient an authentic anammox bacterium, Candidatus ‘‘Scalin- amplification from marine sediments in Japan, although dua wagneri,’’ this bacterium might be important in the it is not commonly used in detection of anammox anammox reaction detected in this study. These find- bacteria. ings suggest the diversity of anammox bacteria in When sediments from five sites were incubated with marine systems. On the other hand, sequencing of three [15N] ammonium and [14N] nitrite under strictly anae- inserts amplified with Brod541F-Brod1260R showed robic condition, as described previously,7) concomitant that they clustered with the Candidatus ‘‘Scalindua’’ decreases in ammonium and nitrite ions were observed Anammox Bacteria in Ago Bay Sediments 80117-3 in site 6 and site 20 samples. Hence, the gas phases of site 6 and site 20 cultures were analyzed with a RMI-2 A mass spectrometer (Hitachi, Tokyo).7) Accumulation of 29 30 N2 and N2 was observed in the gas phase. The 29 relative anammox ratio ( N2 in total N2) was calculated at 0.81%, 6.1%, and 3.9% for control (the mineral medium was incubated without sediment), site 6, and site 20 cultures respectively. Although it is possible 29 30 that the production of N2 and N2 was caused by conventional denitrification, the contribution of anam- mox reaction to this phenomenon cannot be completely 30 denied. N2 gas might have been produced by the oxidation of [15N] ammonium under anoxic conditions B in the presence of manganese oxides.8) Since the reduction of nitrate to nitrite is the limiting step in the overall reduction of nitrate to N2, the ability of anammox bacteria to reduce nitrate coupled to the oxidation of organic acids must be a valuable survival strategy for them.9) FISH analysis was carried out as described previ- ously.9) In brief, Cy3-labeled anammox bacteria specific probes Amx820 and BS820 were used in the detection of anammoxAdvance bacteria, and Alexa-labeled probe Pla46 View was in the detection of Planctomycetes. A comparison of the C number of cells labeled with the anammox-specific probe with that of cells stained with DAPI (40,6- diamidino-2-phenylindole) indicated that anammox- related bacteria accounted for 1.1% and 0.5% of the DAPI stained bacteria at site 6 and site 20 (Fig. 3). The relative abundances of anammox bacteria at site 6 and site 20 are comparable to those in the Black Sea (0.75%),10) in the Benguela upwelling region 11) Proofs (0.5–1.3%), and in Baltimore inner harbor sediment (0.4%–0.6%).6) These observations clearly indicate the Fig. 3. FISH Image of Anammox Bacteria from Site 6 (A) and presence of anammox-related bacteria in Ago Bay Site 20 (B, C) Sediments. Cells were visualized after hybridization with probe Cy3-labeled sediments. Amx820 (A, B) or Cy3-labeled BS820 (C). White arrows indicate Although Amano et al. recently reported that representative Amx820, BS820 (Cy3) and Pla46 (Alexa) hybridized- anammox activity and anammox-related bacteria were cells that were also stained with DAPI. Scale bar indicates 10 mm. detected in coastal marine sediments in Japan,12) the anammox-related bacterial clones detected in this study (Fig. 2) were different from those reported in their References paper. Hence the results obtained for Ago Bay suggest 1) Nakanishi, K., Masuda, T., Hata, N., and Yamagata, Y., a great diversity of anammox-related Planctomycetales Present conditions and changes in recent years in organic and suggest the possibility of a global distribution pollution of bottom sediments in Ago Bay. Bull. Fish of anammox bacteria. It should be noted that a Res. Div. Mie, 10, 71–77 (2001). Candidatus ‘‘Scalindua wagneri’’-like clone was de- 2) Op den Camp, H. J., Kartal, B., Guven, D., van Niftrik, tected in a clone library constructed without thorough L. A., Haaijer, S. C., van der Sitear, W. R., van de enrichment cultivation, suggesting that this bacterium Pas-Schoonen, K. T., Cabezas, A., Ying, Z., Schmid, occurs in relatively large numbers in natural sea M. C., Kuypers, M. M., van de Vossenberg, J., Harhangi, sediments. H. R., Picioreanu, C., van Loosdrecht, M. C., Kuenen, J. G., Siterous, M., and Jetten, M. S., Global impact and Acknowledgments application of the anaerobic ammonium-oxidizing (anammox) bacteria. Biochem. Soc. Trans., 34, 174– 178 (2006). This study was supported financially by a grant under 3) Penton, C. R., Devol, A. H., and Tiedje, J. M., Molecular the Collaboration of Regional Entities for the Advance- evidence for the broad disiteribution of anaerobic ment of Technological Excellence Program from Japan ammonium-oxidizing bacteria in freshwater and marine Science and Technology Agency. We thank Dr. K. sediments. Appl. Environ. Microbiol., 72, 6829–6832 Sugimura of Mie University for FISH analysis. (2006). 80117-4 J. NAKAJIMA et al. 4) Schmid, M. C., Maas, B., Dapena, A., van de and Davies, I. M., Anoxic nitrification in marine sedi- Pas-Schoonen, K., van de Vossenberg, J., Kartal, B., ments. Mar. Ecol. Prog. Ser., 276, 37–51 (2004). van Niftrik, L., Schmidt, I., Cirpus, I., Kuenen, J. G., 9) Kartal, B., Kuypers, M.
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