Genotyping of Uncultured Archaea in a Polluted Site of Suez Gulf, Egypt, Based on 16S Rrna Gene Analyses

Egyptian Journal of Aquatic Research (2014) 40,27–33 National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com FULL LENGTH ARTICLE Genotyping of uncultured archaea in a polluted site of Suez Gulf, Egypt, based on 16S rRNA gene analyses Hosam Easa Elsaied * Aquagenome Resources and Biotechnology Research Group, National Institute of Oceanography, 101-El-Kasr El-Eini Street, Cairo, Egypt Received 3 February 2014; revised 11 March 2014; accepted 11 March 2014 Available online 18 April 2014 KEYWORDS Abstract Culture-independent 16S rRNA gene analysis approach was used to explore and evalu- Archaea; ate archaea in a polluted site, El-Zeitia, Suez Gulf, Egypt. Metagenomic DNA was extracted from a 16S rRNA gene diversity; sediment sample. Archaeal 16S rRNA gene was PCR amplified using universal archaeal primers, Sediment; followed by cloning and direct analyses by sequencing. Rarefaction analysis showed saturation, Suez Gulf recording 21 archaeal 16S rRNA gene phylotypes, which represented the total composition of archaea in the studied sample. Phylogenetic analysis showed that all recorded phylotypes belonged to two archaeal phyla. Sixteen phylotypes were located in the branch of methanogenic Eur- yarchaeota and more closely related to species of the genera Methanosaeta and Methanomassiliicoc- cus. Five phylotypes were affiliated to the new archaeal phylum Thaumarchaeota, which represented by species Candidatus nitrosopumilus. The recorded phylotypes had unique sequences, characteriz- ing them as new phylogenetic lineages. This work is the first investigation of uncultured archaea in the Suez Gulf, and implicated that the environmental characteristics shaped the diversity of archaeal 16S rRNA genes in the studied sample. ª 2014 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. Introduction Al-Mailem et al., 2010). Archaea have been considered as one of the main biological fractions in terms of bioremediation Archaea play an important ecological role for sustaining life in and environmental cleanup (Lovley, 2003). Hence, exploring aquatic heavy polluted environments (Le Borgne et al., 2008; and evaluation of archaea is the window, from which, we can look at what these microorganisms do to sustain life within * Tel./fax: +20 242185320. these harmful environments. E-mail address: [email protected] Archaea have been considered as a domain of single-celled Peer review under responsibility of National Institute of Oceanography prokaryotic microorganisms. In the past, archaea had been and Fisheries. classed with bacteria, the other known prokaryotes, as archae- bacteria (or Kingdom Monera), but this classification is re- garded as outdated (Woese and Fox, 1977; Pace, 2006). In Production and hosting by Elsevier fact, archaea have an independent evolutionary history and 1687-4285 ª 2014 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. http://dx.doi.org/10.1016/j.ejar.2014.03.002 28 H.E. Elsaied show many differences in their genome from other forms of exposed daily to high loads of petroleum wastes, coming from life, and so, they are now classified as a separate domain in the petroleum refining industry around the site, crude oil spills the three-domain system. In this system, the phylogenetically from the fixed oil pipes under the water and ships traveling distinct branches of evolutionary descent are the archaea, bac- through the Suez canal, as well as various other anthropogenic teria and eukaryote. wastes from the surrounding urban region (El-Agroudy et al., Archaea have been divided, based on rRNA gene analyses, 2006; Nemr et al., 2006). These harsh conditions have con- into five recognized phyla; Euryarchaeota, Crenarchaeota, structed an anoxic environment, which may favor the diversity Korarchaeota, Nanoarchaeota, and recently, Tha- of unique archaeal members. umarchaeota. It is surprising that most of the newly discovered Bacterial composition in the Suez Gulf site, El-Zeitia, has lineages seem to expand the two major phyla, Euryarchaeota been studied based on 16S rRNA gene (Elsaied et al., 2011). and Crenarchaeota, which have been defined early, based on This study focused on analyses of archaeal 16S rRNA gene only a few cultured archaeal species (Woese and Olsen, in order to complete the microbial profile, which helps in sus- 1986). However, more archaeal phyla may be established in taining life within this polluted environment. In addition, this the course of future research. study is looking forward to expand archaeal diversity by the Classification of archaea is still difficult, because the vast discovery of novel phylogenetic lineages within this domain. majorities have never been studied in the laboratory (Spang et al., 2013). Recent advances in environmental genomics have Materials and methods showed that we now have the necessary technical tools to char- acterize archaea, in the absence of laboratory cultivation and Sampling in the context of indigenous microbial communities (Treusch and Schleper, 2005). This approach involves direct cloning of Grab sample of surface sediment was collected at a water genomic DNA from the environment and storing this DNA depth of about 20 m from a site at El-Zeitia area, Suez Gulf, in clone libraries for sequencing and discovery of new phyloge- 29° 57.1600 N, 32° 31.7250 E(Fig. 1). The sample was put inside netic lineages. This molecular strategy has been applied mainly a clean sterile propylene tube and covered with Tris–EDTA based on 16S rRNA gene for exploring and evaluation of buffer, with a high concentration of EDTA (100 mM), to che- uncultured archaea in the aquatic ecosystems, especially in ex- late Mg++ and other nuclease co-enzymes. The sediment was treme marine environments, such as deep sea, hyper saline stored at À30 °C for DNA extraction. ponds and strictly anoxic polluted ecosystems (Auguet et al., 2009; Ceteciog˘lu et al., 2009; Zinger et al., 2012). Among the first discoveries of PCR-based molecular eco- Molecular analyses logical surveys was the detection of 16S rRNA genes of members of Crenarchaeota in the marine plankton (DeLong, 1992; Bulk microbial DNA was extracted from 50 g of sediment, Fuhrman et al., 1992). Hugenholtz (2002) dissected 18 differ- 10 g per each extraction, using PowerMax Soil DNA Isolation ent archaeal groups, 10 of which contained no cultivated rep- Kit (Catalog no. 12988-10, Mo Bio Laboratories, Carlsbad, resentatives, based on comparative analyses of 16S rRNA gene CA, USA) according to the manufacture’s protocol with mod- sequences. Meanwhile, almost 8718 uncultured archaeal 16S ifications. First, the sediment sample was fractionated for rRNA gene sequences from marine environments have been 10 min using glass beads. The fractionated sample was treated deposited in the public DNA databases. Many novel archaeal with a mixture of guanidine thiocyanate and 10% sodium groups seem (so far) to be confined to specific geographical dodecyl sulfate, SDS, at 75 °C for 40 min with strong shaking. locations or to ecosystems that have similar geochemistry, The sample homogenate was briefly centrifuged for 10 min at but other groups seem to be widely distributed. 5000 rpm and the supernatant lysate was removed in a clean Application of uncultured-dependent molecular techniques sterilized propylene tube. The DNA was purified from the for studying archaeal communities in Egyptian aquatic envi- crude lysate using a Sephadex column, provided in the Kit. ronments is still behind, and consequently, no available 16S The size of the extracted DNA was checked by electropho- rRNA gene database for exploring archaea within these envi- resis on a 0.9% agarose gel against a Lambda- HindIII digest ronments. However, few studies have been done to explore ar- marker (New England BioLabs, Hitchin, Hertfordshire, UK) chaea in extreme Egyptian habitats. Phylotypes, belonging to with ethidium bromide staining. Halobacteriaceae, Methanobacterium and Thermoplasma of PCR reaction mixture, 50 ll, contained 10· EX taq buffer II phylum Euryarchaeota, have been detected in the Solar Lake, (Mg2+ plus), 0.2 lM primer, 400 lM dNTP each, 2.25 U Taka- Sinai, Egypt (Cytryn et al., 2000). The 16S rRNA gene analy- ra EX-Taq Polymerase (Takara, Japan) and 5–30 ng DNA ses have showed dominance of Euryarchaeotes in hypersaline template. PCR amplification of the archaeal 16S rRNA genes, Lakes of Wadi An Natrun, Egypt (Mesbah et al., 2007). Six- from the purified genomic DNA, was carried out using the teen uncultured archaeal phylotypes, mostly belonging to primers Arch-21F (50-TTCCGGTTGATCCYGCCGGA-30) methanogenic Euryarchaeotes, have been recovered, based and Arch-958R (50-YCCGGCGTTGAMTCCAATT-30)(De- on 16S rRNA gene analyses, from sewage polluted sites at Long and Pace, 2001) with modification. PCR was performed the Manzala Lake, Egypt (Elsaied, 2008). with an initial denaturation step of 3 min at 95 °C. The touch- Suez Gulf has a wide variation of physico chemical charac- down PCR reaction continued with 30 cycles of 1 min at 95 °C, teristics, which favor high microbial diversities (Nemr et al., 40 s. at the desirable annealing temperatures, 56 °C with 2006; Elsaied et al., 2011). The Suez Gulf site, El-Zeitia, the decreasing 0.5 °C every 10 cycles, and 1 min extension at target of study, is located at the north terminus of the Suez 72 °C. The 30 thermal cycles were followed by a final extension Gulf and constitutes the south gate of the Suez Canal, Egypt. of 10 min at 72 °C to allow 30-A overhangs for the amplified El-Zeitia is the most contaminated site of the Suez Gulf. It is PCR product to facilitate TA-cloning. Cloning was done into Genotyping of uncultured archaea in Egypt 29 N Sampling S site Figure 1 A map showing the site of sampling. TOP10 Escherichia coli using a TOPO XL PCR-cloning kit, Table 1 Phylotypes and their accession numbers. according to the manufacturer’s instructions (Catalog no.

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