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, characterizing 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 30 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. Phylotype Accession number K4750-20, Invitrogen Life Technologies, Carlsbad, CA, 1SuezARCH AB899897 USA). Only cells containing XL-TOPO vector with the insert 2SuezARCH AB899898 were competent to grow with kanamycin. Plasmids were ex- 3SuezARCH AB899899 tracted from positive colonies and screened directly by sequenc- 4SuezARCH AB899900 ing using vector primers T7 and an ABI 3730xl 96-capillary 5SuezARCH AB899901 DNA analyzer (Applied Biosystems, Foster City, CA, USA). 6SuezARCH AB899902 7SuezARCH AB899903 Statistical analyses and construction of phylogenetic tree 8SuezARCH AB899904 9SuezARCH AB899905 10SuezARCH AB899906 Clone sequences were analyzed by FASTA screening to deter- 11SuezARCH AB899907 mine their similarity to known archaeal sequences in the DNA 12SuezARCH AB899908 database (http://ddbj.nig.ac.jp). The recovered sequences were 13SuezARCH AB899909 aligned using CLUSTAL W software (DDBJ, http://clu- 14SuezARCH AB899910 stalw.ddbj.nig.ac.jp). Grouping of sequences into phylotypes 15SuezARCH AB899911 was carried out, based on genetic distances, using the 16SuezARCH AB899912 MOTHUR software package V.1.7.2 (Schloss et al., 2009), 17SuezARCH AB899913 18SuezARCH AB899914 where the sequences that had >97% nucleotide identity, over 19SuezARCH AB899915 the region compared, were grouped into a single phylotype 20SuezARCH AB899916 (Godon et al., 1997). 21SuezARCH AB899917 The expected total composition of archaeal 16S rRNA genes, in the sediment sample, was determined by rarefaction analysis in the MOTHUR software package. mony and maximum likelihood, in the same MEGA software, Construction of the phylogenetic tree was done through data not shown. two bioinformatic processes. In the first processes, the nucleo- The current archaeal 16S rRNA gene sequences have been tide sequences of the recovered archaeal 16S rRNA gene phyl- registered in the DNA database under accession numbers otypes and their homologue sequences, from the DNA listed in Table 1. database, beside out-group sequences, were aligned using the online program ‘‘Clustal Omega’’, http://www.ebi.ac.uk/ Results and discussion Tools/msa/clustalo/. In the second processes, the aligned sequences, including the sequence gaps, were submitted to the The recovered 16S rRNA gene phylotypes represented the total MEGA software, V. 6.06, http://www.megasoftware.net/, for archaeal composition within the sediment sample drawing the phylogenetic tree. Bootstrap method, provided as a phylogeny test, in the MEGA software, was performed Archaeal 16S rRNA gene phylotypes had sizes from 912 bp, as using a number of 500 Bootstrap replications. Phylogenetic in phylotype 1SuezARCH, to 940 bp, phylotype 19Suez- tree was constructed by applying the neighbor joining algo- ARCH. These amplification results indicated the possibility rithm. The branching patterns of the constructed tree were in of bias in PCR amplification was minimized. This is because agreement with other compared algorithms, maximum parsi- the earliest cycles of used touchdown polymerase chain reac- 30 H.E. Elsaied

25 methanogenic Euryarchaeota (Fig. 3). Cluster 1 was represented by 5 phylotypes, which were rooted with the genera 20 Methanosaeta, Methanosalsum, Methanolobus and Metha- nococcoides (Ma et al., 2006). Cluster 3 formed of 7 phylotypes showed afﬁliation to the Methanomassiliicoccus luminyensis 15 (Dridi et al., 2012). Both clusters 2 and 4 contained 4 phylotypes, which represented unique phylogenetic lineages within 10 the branch of methanogens, characterizing the sampling site. Dominance of methanogens was in parallel with that of sulfate-reducing deltaproteobacteria, recorded previously, from 5 No. Phylotypes

Number of archaeal phylotypes of archaeal Number the same sample (Elsaied et al., 2011). This observation represented a kind of social evolution, where sulfate-reducing bacte- 0 ria benefit from methanogens in utilizing of methane in the 0 5 10 15 20 25 30 35 Number of sequences sampled reduction of sulfate (Susanti and Mukhopadhyay, 2012). Hence, both of the two phylogenetic groups, methanogens Figure 2 Rarefaction curve for the expected number of archaeal and sulfate reducers, have co-occurred in the most studied 16S rRNA gene phylotypes. marine samples (Orphan et al., 2001). Methanogenic archaea are widely distributed in polluted tion had a high annealing temperature, 56 C, which is least- aquatic area (Clementino et al., 2007; Vieira et al., 2007; Haller permissive of nonspecific binding that it is able to tolerate. et al., 2011; etc.). Methanosaeta 16S rDNA-like phylotypes Thus, the first sequence amplified is the one between the re- have been recorded, as a dominant archaeal fraction in two gions of greatest primer specificity; it is most likely that this sites, Genka and Bashtir, of the Manzala Lake, Egypt (Elsaied, is the sequence of interest. These fragments were further ampli- 2008). Both the current site and those of Manzala are charac- fied during subsequent rounds at lower temperatures, from terized by heavy hydrocarbon pollution, where the oxygen le- 55.5 to 55 C, and consequently, out compete the nonspecific vel is zero, favoring the abundance of methanogens (El- sequences to which the primers may bind, if a lower annealing Agroudy et al., 2006; Elsaied, 2008). Uncultured phylotypes temperature was applied in the first cycles of PCR. Thus, the closely related to Methanobacterium and Methanococcus have used touchdown PCR increased the specificity of the reaction been recorded in anoxic hypersaline solar Lake, Sinai, Egypt at higher temperatures and increased the efficiency towards (Cytryn et al., 2000). In addition, two phylotypes closely re- the end by lowering the annealing temperature, avoiding non lated to the genus Methanomethylovorans have been detected specific amplifications, which resulted in standard PCR in ear- in the Wadi An Natrun, Egypt (Mesbah et al., 2007). Diversity lier works (DeLong and Pace, 2001). Moreover, PCR was of methanogens in a wide range of Egyptian aquatic environ- tested using a range from 26 to 30 cycles, and then, the ampli- ments may be controlled by two ecological factors, the anoxic cons were combined for cloning (Suzuki and Giovannoni, condition, which enhances the abundance of methanogens; 1996). Hence, the PCR could screen as much as possible of and type of utilized substrate for the production of methane. the actual composition of archaeal 16S rRNA genes in the sed- On the other hand, existence of uncultured 16S rRNA gene iment sample. phylotypes, represented by clusters 2 and 4, in the phylogenetic Twenty-nine clones were analyzed by sequences. The rarefac- branch of methanogens, expanded the diversity of methano- tion curve showed saturation after analyses of 25 clones, record- gens to explore new genera and species. ing 21 phylotypes, which represented the total composition of The cluster 5 included 5 phylotypes, which formed mono- archaeal 16S rRNA genes in the studied sample (Fig. 2). In com- phyletic and paraphyletic clades with anoxic ammonia-oxidiz- parison with bacterial 16S rRNA genes, which have been re- ing archaeon, Candidatus nitrosopumilus, belonging to corded previously from the same sample (Elsaied et al., 2011), Thaumarchaeota (Matsutani et al., 2011). Thaumarchaeotes the current archaeal 16S rRNA genes showed lower diversity, have constituted a new discovered phylum of the archaea, pro- 21 phylotypes, than those of bacteria, 32 phylotypes. Generally, posed in 2008 (Brochier-Armanet et al., 2008). About 997 archaea in the studied site of the Suez Gulf showed lowest 16S uncultured 16S rRNA gene phylotypes, affiliated to Tha- rRNA gene diversity compared with those recorded in Solar umarchaeota, from different marine resources, have been re- Lakes, Sinai, 165 phylotypes, and Wadi An Natrun, Egypt, corded in the DNA data base (Belmar et al., 2011; Lund 198 phylotypes (Cytryn et al., 2000; Mesbah et al., 2007). On et al., 2012). The current phylotypes within the cluster 5 repre- the other hand, the current archaeal phylotypes were more di- sented new phylogenetic lineages of Thaumarchaeotes, increas- verse than those recovered from sewage polluted sites of the ing the diversity within this new discovered phylum. However, Manzala Lake, Egypt, which recorded 16 phylotypes in the the current phylotypes, cluster 5, represented the first discovery Bashtir sediment and 9 phylotypes in the Genka sediment (Elsa- of phylotypes belonging to Thaumarchaeota in the Suez Gulf. ied, 2008). These diversities may be related with the environmental characteristics, of each sampling site, which may shape the diversity of archaeal domain members. Genotyping of uncultured archaea in studied Suez Gulf sediment

Dominance of both of methanogens-, Euryarchaeota, and The phylotypes 2-, 6-, 9- and 21SuezARCH, constituting the Thaumarchaeota-like phylotypes clusters 2 and 4, respectively, showed the lowest homologies with other phylotypes in the branch of methanogens (Figs. 3 The sediment sample contained 16 phylotypes, of total 21 and 4a). The phylotypes 2- and 6SuezARCH had nucleotide phylotypes, which were located in 4 clusters, belonging to identities average of 80% with other methanogen-like Genotyping of uncultured archaea in Egypt 31

98 12SuezARCH 17SuezARC H 97 16SuezARCH 98 20SuezARC 100 H Cluster 1 15SuezARCH 99 Methanosaeta harundinacea (AY970347)

100 Methanosalsum sp. (HM053965) 100 Methanolobus bombayensis (FR733684) 71 Methanococcoides sp. NM1 (HE862408) 98

2SuezARCH Cluster 2 100 6SuezARCH 97 10SuezARCH Euryarchaeota, Methanomassiliicoccus luminyensis (HQ896499) Methanogens 1SuezARCH 100

51 4SuezARCH 75 3SuezARC H 100 Cluster 3 88 63 18SuezARCH

56 7SuezARCH

53 97 13SuezARCH

76 85 9SuezARCH Cluster 4 21SuezARCH

55 Methanobacterium oryzae (AF028690) Methanococcus voltaei (AF306670)

100 5SuezARCH

74 Candidatus nitrosopumilus (AB546961) 81 8SuezARCH Cluster 5 Thaumarchaeota 74 100 11SuezARCH 19SuezARCH 72 14SuezARCH Korarchaeota 99 Uncultured korarchaeote (AB175572) Nanoarchaeum equitans (AJ318041) Nanoarchaeota Out-group 100 Ignisphaera aggregans (DQ060321) Crenarchaeota Bacteria Escherichia coli (J01859)

0.1

Figure 3 A phylogenetic tree based on archaeal 16S rRNA gene partial sequences, corresponding to nucleotide positions from 8 to 974 of Escherichia coli (accession no. J01859). The tree shows the phylogenetic relationship between the recovered phylotypes, shaded, homologues from database and out-group, which included representatives of 16S rRNA gene sequences from the archaeal phyla, Korarchaeota, Nanoarchaeota and Crenarchaeota, beside the bacterial species, Escherichia coli. The tree was constructed by neighbor- joining analysis and conﬁrmed by maximum-parsimony and maximum-likelihood algorithms. Average Bootstrap values, of compared algorithms, are indicated at the branch roots. The bar represents 0.1 changes per nucleotide. Accession numbers of database extracted sequences are in brackets.

phylotypes. The phylotypes 9- and 21SuezARCH were more On the other hand, the phylotypes 8-, 11-, 14- and 19Suez- divergent, displaying nucleotide identities average of 75% with ARCH were characterized by the occurrence of 8 nucleo- other methanogen-like phylotypes. tides, located between nucleotide positions 217 and 218 of The sequence region from 81 to 109, belonging to 16S E. coli, which were missed in the phylotype 5SuezARCH rRNA gene of E. coli, was considered as a ﬁnger print se- (Fig. 4b). In addition, the phylotype 8SuezARCH contained quence, which could differentiate the current methanogen-like 4 nucleotides, CGTT, which were missed in other phylo- phylotypes (Fig. 4a). Both phylotypes of clusters 3 and 4 types. These ﬁnger print nucleotides may constitute the showed the deletion of 7 nucleotides, from position 81 to 88, key sequences, which marked new phylogenetic lineages, rep- while those nucleotides characterized the cluster 1, phylotypes resented by phylotypes 8-, 11-, 14- and 19SuezARCH, with- 12-, 15-,16-,17- and 20SuezARCH; and cluster 2, which in- in the phylum Thaumarchaeota. cluded phylotypes 2-, and 6SuezARCH (Fig. 4a). However, this study was the base line for exploring and The phylotype 5SuezARCH showed the highest homol- evaluation of uncultured Thaumarchaeota in the Suez Gulf. ogy, 98.25%, with the known Thaumarchaeota species C. More metagenomic survey should be done, based on func- nitrosopumilus. Other phylotypes in the cluster 5 showed tional genes, in order to understand the ecological role of Tha- homologies from 85% to 90% with the C. nitrosopumilus. umarchaeota within this polluted environment. 32 H.E. Elsaied

(a)

(b)

Figure 4 Multiple alignments for sequences from current archaeal 16S rRNA gene phylotypes and those from other known methanogenic Euryarchaeotes (a) and Thaumarchaeotes (b) in database. The nucleotide alignments are only in the sites of speciﬁc sequences, ﬁnger prints, marked by shading. Species used in the alignments had accession numbers listed in Fig. 3. The numbers between brackets, at right sites, mentioned to clusters at the phylogenetic tree, Fig. 3. Identical nucleotides are represented by dots. The missed nucleotides, gaps, are showed by dashes.

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