Genetic Diversity among thermophilic bacteria ...

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Genetic Diversity among thermophilic bacteria Isolated from geothermal sites by Using Two PCR Typing Methods Samy Selim

a b

c

b

, Mervat El Sherif , Saher El-Alfy & Nashwa Hagagy

b d

a

Department of Medical Laboratory Sciences, College of Applied Medical Science , Al Jouf University , Sakaka , P.O. 2014 , Saudi Arabia b

Microbiology section, Botany Department, Faculty of Science , Suez Canal University , Ismailia , P.O. 41522 , Egypt c

Department of Biological and Geological Sciences, Faculty of Education , Alexandria University , Egypt d

Department of Human and Animal Biology , Università degli Studi di Torino , Torino , Italy Accepted author version posted online: 22 Jul 2013.

To cite this article: Geomicrobiology Journal (2013): Genetic Diversity among thermophilic bacteria Isolated from geothermal sites by Using Two PCR Typing Methods, Geomicrobiology Journal, DOI: 10.1080/01490451.2013.822614 To link to this article: http://dx.doi.org/10.1080/01490451.2013.822614

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ACCEPTED MANUSCRIPT Genetic Diversity among thermophilic bacteria Isolated from geothermal sites by Using Two PCR Typing Methods

Samy Selim1,2*, Mervat El Sherif3, Saher El-Alfy2, Nashwa Hagagy2,4 1

Department of Medical Laboratory Sciences, College of Applied Medical Science, Al Jouf

University, Sakaka, P.O. 2014, Saudi Arabia

Downloaded by [46.251.128.129] at 06:08 23 July 2013

2

Microbiology section, Botany Department, Faculty of Science, Suez Canal University,

Ismailia,P.O. 41522, Egypt 3

Department of Biological and Geological Sciences, Faculty of Education, Alexandria

University, Egypt 4

Department of Human and Animal Biology, Università degli Studi di Torino, Torino, Italy

*Corresponding Author: E-mail address: [email protected]

Abstract Microbial communities thriving at two hot springs, Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs), in Egypt was studied by cultural and molecular methods. Thirteen morphologically distinct strains of facultative anaerobic thermophilic bacterial isolates have been characterized and identified using phenotypic and genotypic characters including RAPD-PCR, ERIC-PCR typing, plasmid analysis and 16S rRNA sequencing. All isolates produced plasmid DNA with various sizes ranging from 0.7 kb to a larger plasmid 7.2 kb. The bacterial strains could tolerate a temperature range between 45 to 85°C and a pH between 4–11. Also, sulphate-reducing bacteria (SRB) in the thermal springs were investigated with combined

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ACCEPTED MANUSCRIPT biochemical and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 30 to 50 oC and could use pyruvate, lactate and ethanol as electron donors. The dissimilatory sulphite reductase (DSR) gene sequences of eleven representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio


vulgaris. 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Bacillus (one species), Anoxybacillus (four species) and Geobacillus (eight species). In this study phenotypic and genotypic diversity were applied for the first time to differentiate thermophilic bacteria of such geothermal sites in Sinai, Egypt. Keywords: Thermophilic bacteria, Sulphate-reducing bacteria, RAPD-PCR, ERIC-PCR typing, Hot spring, Egypt. Introduction Hot springs are prevalent all over the earth, on every continent and even under the oceans and seas. Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs) are two hot springs in Egypt which have been not yet explored in details from the microbiological point of view. Hammam Pharaon and Oyoun Mossa, approximately 45 km south of Ras Sudr in Sinai. These places are most important sites for therapeutic tourism with the highest rate of sulfuric water known to the world. Hammam Pharaon is located 250 kms from Cairo consisting of a group of natural hot sulfuric springs whose temperature is 90o C. The water flows directly from the mountain into a 100 m long natural lake directly beside the sea shore. Oyoun Mossa is

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ACCEPTED MANUSCRIPT located in South Sinai's official capital, Al-Tor. Oyoun Mossa area comprises twelve springs, situated in the extreme north of the eastern side of the Suez Gulf. A large number of aerobic or facultatively anaerobic, rod shaped, endospore-forming bacteria are widely distributed in the environment. Many of which belong to the genus Bacillus, They are classified as acidophilic, alkalophilic, thermophilic or others according to their nature. Thermophilic Bacillus species with an optimal growth temperature between 45 and 70 °C have


been isolated from a wide range of environments (Studholme et al. 1999), and crucial features associated with heat-tolerance have been widely documented. Sulphate-reducing bacteria (SRB) are a unique group of prokaryotes which have the capability of using sulphate and other oxidized sulphur compounds as final electron acceptors during anaerobic respiration (Keith et al. 1982). The SRB are widely distributed on earth and play a significant role in nature by virtue of their potential for numerous interactions such as geochemical transformations, fuel production, food spoilage, biocorrosion, bioremediation, nutrient cycling, associations with animal diseases, organic matter turnover, biodegradation of chlorinated aromatic pollutants in anaerobic soils and sediments, and mercury methylation (Fauque 1995; Barton and Tomei 1995). These bacteria possess very similar characteristics though they are found in many diverse habitats across the world. Recently, molecular phylogenetic approaches studying the small-subunit rRNA gene (16S rDNA) have been used to examine the microbial diversity of various hot springs such as the Yellowstone National Park, USA (Huber et al. 1998; Reysenbach et al. 2000; Skirnisdottir et al. 2000; Schaffer et al. 2004), the Bakreshwar hot spring, India (Ghosh et al. 2003), the Garhwal Himalaya hot spring, India (Sharma et al. 2009), the Grensdalur hot spring, Iceland, the Garga

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ACCEPTED MANUSCRIPT hot spring, Russia (Nazina et al. 2004), the Nalychevskie, Oksinskie, Apapelskie, and Dachnye hot springs in Kamchatka Peninsula, Russia (Belkova et al. 2007), the El Biban hot spring in Northeast of Algeria (Kecha et al. 2007), and the Rotoura hot spring, New Zealand (Niederberger et al. 2008). Kamchatka Peninsula (Wemheuer et al. 2013). In view of understanding the biodiversity or applications, these studies have been conducted using cultureindependent or culture-dependent methods, respectively. Investigations of microorganisms in


isolated terrestrial hot springs would enhance our understanding of both thermophilic microbial diversity in nature and their evolutionary mechanisms (Reysenbach et al. 2000). Though substantial information is available on geological, geophysical, hydrological and geothermal properties, studies on biological properties of the thermal springs of Egypt such as the presence of SRB are sparse. Therefore these springs provide particularly interesting environments for studies, as they may yield some novel thermophilic microorganisms. In this study microbial communities living at two hot springs in Egypt, Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs), were evaluated in water and sediment. cultural methods applied with particular consideration to thermophilic aerobic bacteria and sulphate reducing bacteria (SRB). The phenotypic characteristics of the isolates were evaluated, as well the genotypic diversity of aerobic thermophilic bacteria thriving at these springs was studied by RAPD-PCR, ERIC-PCR typing methods. MATERIALS AND METHODS Sampling sites and sample collection Water and soil samples were collected in sterile containers from the Egyptian hot springs, Hammam Pharaon (two sites, I and II) and Oyoun Mossa in Sinai desert at the red sea beach

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ACCEPTED MANUSCRIPT (Figure 1). The temperature of water ranged between 40 to 90° C according to the site of collection and the water pH was around 7.5. Each sample containing an estimated ratio of 1:1 (water, solid) (v/w) hot spring water and surface sediment was collected using a sterile spoon, transferred to a 20 ml sterile bottle, and divided into aliquots (5 ml of each) for parallel analyses. ). The details of the study sites and chemical constituents have already been reported (Selim et al.


2009). All samples were kept on ice until reaching the laboratory.

Culture Assays

Bacterial strains and growth conditions

Cultivation techniques targeted different metabolism types. The search for aerobic and/or anaerobic heterotrophic thermophilic bacteria, sulphate reducing bacteria (SRB) was carried out on non-selective as well as selective media using the serial dilution technique (Johnson and Curl, 1972). Tryptone yeast extract glucose agar and starch casein agar were used for the isolation of bacteria and actinomycetes, respectively. Petri dishes were incubated at 30, 50, 65 and 80° C in the dark. For incubation at 50° C and above, the medium was gelled with 2.0% agar (instead of 1.6%, w/v). Observations were recorded after one week of incubation. For isolating obligate thermophilic organisms, a mixture of 1 g soil and 9 ml tryptone yeast extract broth in culture tubes were incubated at 75 or 80° C. The tubes were observed for turbidity 36 h after incubation. All experiments were conducted in triplicate. Thirteen morphologically distinct facultative

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ACCEPTED MANUSCRIPT anaerobic strains of thermophilic bacteria have been characterized and identified using phenotypic and genotypic characters. Enumeration of sulphate reducing bacteria (SRB) SRB in water were enumerated by the most probable number (MPN) technique using tenfold dilutions in three parallels of anoxic medium tubes with Starkey’s medium (Saharani et al. 2008). The lactate-sulphate medium contained 4 ml of 60% (w/v) sodium lactate, 0.20 g


magnesium sulphate, 0.01 g dipotassium phosphate, 0.01 g ferrous ammonium sulphate, 10.0 g sodium chloride, 1.0 g yeast extract and 0.10 g ascorbic acid in one liter. Enumeration was also done simultaneously by the total plate count (TPC) method using Starkey’s medium. The tubes and plates were incubated in anaerobic conditions at 40° C for 15–20 days. The presence of SRB was indicated by the formation of black colour of the medium and presence of gas bubbles while black coloured colonies indicated its presence on plates. Eleven distinct SRB isolates were selected for further studies. Phenotypic studies Phenotypic studies including morphological, physiological and biochemical tests on thermophilic isolates were performed as suggested by Harley and Prescott (2002). Isolates were first Gram stained and examined under the light microscopy. Sporulation was examined under compound microscope. The thirteen thermophilic isolates were then subjected to some physiological tests on nutrient agar plates for 1–3 days: growth at 37, 45, 65 and 70 o C; in the presence of 0.5, 1, 3 and 6% (w/v) NaCl at 55o C; at pH 8, 9 and 10 at 55o C. The fermentation of various sugars was examined by changes in the colour of Andrade peptone water containing 0.1% Andrade indicator. The sensitivity to antibiotics was tested at 65° C for 24 h in TY agar

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ACCEPTED MANUSCRIPT media. For the determination of oxygen requirements the strains were grown in TY broth for 24 h. Characterization of the sulphate reducing bacterial isolates included the determination of growth temperature and the utilization of a range of substrates as sole sources of carbon was evaluated according to Widdel and Pfennig (1984). The ability of isolates to grow at different temperatures was determined using sulphate-reducing bacteria medium containing lactate. Broth


cultures of test strains were inoculated into the medium and incubated at 25, 35, 45 and 55o C in a thermostat regulated incubator. The presence of desulfoviridin was determined as described by Widdel and Pfennig (1984). Molecular Assays Plasmid DNA extraction and analysis Bacteria were streaked from frozen glycerol stocks onto nutrient agar plates and were then incubated overnight at 55o C. The cells were scraped and suspended in 1.5 ml sterile water. The method of Sullivan and Klaenhammer (1993) was followed for the extraction of small plasmid DNA. After digesting with HaeIII restriction enzyme, restriction products were resolved in a 1 % agarose gel and the size of the plasmid DNA molecules were calculated in a gel documentation system (Vilber-Lourmat, Torcy, France). DNA extraction The cultures were grown in broth to saturation and centrifuged at 10,000 g for 10 min prior to sediment the bacteria. The pellet was washed in 1 ml phosphate buffered saline (PBS) and centrifuged again. DNA was extracted from SRB and thermophilic bacterial isolates using a

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ACCEPTED MANUSCRIPT DNeasy kit (Qiagen, USA) and following manufacturer’s instructions. The purity and concentration of DNA preparation was determined spectrophotometrically at 260 nm. RAPD-PCR analysis DNA amplification reactions were conducted in a Model No. 9700, Perkin–Elmer thermocycler. After an overnight culture on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.), a 10-ml volume of cells was scraped from the agar plate with a calibrated


inoculation loop and washed in 300 ml of sterile saline. Cells were resuspended in 300 ml of distilled water and heated at 100o C for 10 min. The boiled suspensions were kept at 4 o C for later use as target DNA templates for RAPD analysis. Each 25-ml reaction mixture contained 0.025 U of Taq polymerase (AmpliTaq; Roche Diagnostics, Branchburg, N.J.) per ml; dATP, dGTP, dTTP, and dCTP at 200 mM each (Boehringer Mannheim); and 1 mM primer A (5`CTGGCGGCTG-3`) or B (5`-CAGGCGGCGT-3`) (DNA Synthesis Facility, Biotechnology Service Centre, Ontario, Canada) in PCR buffer (100 mM Tris-HCl [pH 8.5], 50 mM KCl, 4 mM MgCl2). After an initial 2 min of denaturation at 94o C, reaction mixtures were run through 35 cycles of denaturation for 30 s at 94o C, annealing for 30 s at 35o C, and extension for 1 min at 72oC. A final extension was carried out for 4 min at 72o C. Samples were electrophoresed for 3 h at 90 V on 1.5% agarose containing 0.5 mg of ethidium bromide per ml in 1X TAE buffer (40 M Tris-acetate, 1 mM EDTA [pH 8.0]). Reproducibility of results was examined by repeating the AP-PCR procedure twice for each isolate. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR typing The ERIC-PCR method was used for the study the genotyping of bacterial isolates (Versalovic et al. 1991). A single colony was inoculated into Mueller–Hinton broth and

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ACCEPTED MANUSCRIPT incubated for 20 h at 37o C. After centrifugation at 10 000 g for 10 min, each pellet was washed three times in 750 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and then resuspended in 500 µL TE buffer. The solution was boiled for 20 min and centrifuged at 10 000 g for 10 min, and the supernatant was then used as a crude DNA extract in PCRs. Amplification reactions were performed in a final volume of 25 µL, with 15.5 lL H2O, 2.5 lL 25 mM MgCl2, 2.0 lL each 2.5 mM

dATP,

dTTP,

dGTP

and

dCTP,

0.3

lL

1

mM

ERIC2

primer

(5`


AAGTAAGTGACTGGGGTGAGCG-3`) (Trilink Biotechnologies, San Diego, CA, USA), 2.6 lL 10· PCR buffer (Sigma-Aldrich, Munich, Germany), 0.13 lL Taq polymerase (Sigma-Aldrich) and 2 lL DNA extract. A negative control with H2O instead of DNA extract was used in each run. PCRs comprised one cycle for 3 min at 94o C, two cycles of 45 s at 94o C, 1 min at 30o C and 1 min at 72o C, and 44 cycles of 30 s at 94o C, 30 s at 55o C and 1 min at 72o C, with a final extension for 4 min at 72o C. The PCR products were analysed by electrophoresis with DNA Molecular Weight Marker XIV (Roche Diagnostics) for 2 h at 110 V in an agarose 1.5% w⁄ v gel and staining with ethidium bromide 0.1% w⁄v. The amplicon patterns were evaluated with a Gel Documentation System (Vilber-Lourmat, Torcy, France). Differences of two or more DNA bands were considered to represent different strains, while isolates differing by only one band were considered to be subtypes. PCR amplification of DSR genes For sulphate reducing bacteria (SRB), primers specific for DSR gene DSR 1F 5'ACACACTGGAAGCACG-3' and DSR 4R 5'-GTGTAGCAGTTACCGCA-3' were used. PCR was carried out in a 30 µL reaction mixture consisting of a 10 µl PCR buffer [100 mM Tris–Cl pH 8.0, 500 mM KCl, 15 mM MgCl2 and 0.1% gelatin, 10 picomoles each of forward and

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ACCEPTED MANUSCRIPT reverse primers, 200 µM of each of 4 dNTPs, 1.5 U of Taq polymerase and 500 ng of genomic DNA. Amplification of DSR gene was performed at 94o C for 1 min, 53o C for 1 min and 72o C for 3 min. All PCR reactions were performed for 35 cycles. An initial denaturation at 94o C for 5 min and a final extension of 72o C for 10 min were included in all reactions. The PCR products were resolved on a 1.6% agarose gel, stained with ethidium bromide and photographed using a gel documentation system.


Analysis of 16S rRNA gene sequences For the sequence analysis, two primers annealing at the 5` and 3` end of the 16S rDNA were 5`-GAGTTTGATCCTGGCTCAG-3` (positions 9–27 [Escherichia coli 16S rDNA numbering]) and 5` -AGAAA GGAGG TGATC CAGCC-3` (positions 1542–1525 [E. coli 16S rDNA numbering]), respectively (Lane 1991). PCR amplification was performed in a final reaction volume of 100 µl, and the reaction mixture contained each primer at a concentration of 0.5 µM, each deoxynucleoside triphosphate at a concentration of 200 µM, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl , 0.01% (w/v) gelatin, and 2.5 U of Taq DNA polymerase. The PCR reaction was conducted for 35 cycles in a DNA thermal cycler (Model No. 9700, Perkin–Elmer Co. Wellesley, USA). The following thermal profile was used for the PCR: denaturation at 94° C for 1 min, primer annealing at 60° C for 1 min and extension at 72° C for 2 min. The final cycle included extension for 10 min at 72° C to ensure full extension of the products. The amplified PCR products were then analyzed in a 1.0 % (w/v) agarose gel, excised from the gel, and purified. The amplified 16S-rDNA was cloned into pGEM®-T vector (Promega, Madison, USA), and transformed into Escherichia coli. Plasmid DNA was isolated from the positive clones using Qiaprep® Spin Miniprep kit (Qiagen, Hilden, Germany)

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ACCEPTED MANUSCRIPT according to the manufacturer’s instructions. Plasmid specific primers USP and RSP and the prokaryote specific forward primers, 16F530, 16F926 and reverse primers 16R519 and 16R907 (Lane 1991), were used as infra-red dye labelled dideoxyoligonucleotides. 16S-rDNA was sequenced using the Cycle ReaderTM Auto DNA sequencing kit (MBI Fermentas, St. Leon, Germany) and LI-COR® automated DNA sequencing machine (MWG-Biotech) according to manufacturer’s instructions. The 16S-rDNA gene sequence of the isolates was aligned with


reference 16S-rRNA sequences of the European Microbiological Laboratory (EMBL), GenBank (gb in Germany) and the data base of Japan (dbj) using the BLAST algorithm available in NCBI (National Centre for Biotechnology information) in internet (Altuschul et al. 1997). Phylogenetic analysis Using CLUSTALX version 1.8312, the sequences of the 16S rDNA of the bacterial isolates were aligned and clustered against those available from the GenBank. Blocks of sequence data contain 255 characters with a data matrix of 173 taxa. Phylogenetic trees were constructed in MEGA 4.0 version (www.megasoftware.net) using neighbour joining algorithms (NJ).

RESULTS

While the microbes could be isolated from the water and soil samples on various media across a broad temperature range (30–80o C), the optimum temperature for growth of microbial populations was 50o C. Moderate numbers of microorganisms were isolated on diluted tryptone yeast extract glucose agar assisting as well to enumerate a few colonies being morphologically

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ACCEPTED MANUSCRIPT different from those which developed on full strength medium. Some filamentous organisms were isolated from soil samples of both sites, using broth culture at 75 or 80o C. A total of 121 morphologically distinct representative colonies were obtained as pure cultures. On the basis of microscopic examination, three types of microbial populations were observed (bacteria, yeast and filamentous organisms from Hammam Pharaon, and bacteria and filamentous organisms from Oyoun Mossa). Most bacilli were observed with an endospore. Yeast colonies from


Hammam Pharaon samples revealed the presence of branched mycelium with spores (spore size 3–7 mm). The isolates have been grouped on the basis of their temperature, pH and salt requirements (Table 1). The enumeration of SRB was done by most probable number (MPN) and total plate count (TPC) methods. The number of SRB by MPN method ranged from 5 to 246 cells per 100 ml of water samples and from 34 to 332 cells per g of soil. The number of SRB using the total plate count method ranged from 2.0 x 101 to 213.0 x 101 CFU/g soil. The results obtained indicated that suitable conditions existed for the occurrence of SRB. The colony morphology, microscopic features and growth characters of 13 thermophilic strains and 11 strains of sulphate reducing bacteria (SRB) are presented in Table 2 and 3 respectively. Plasmid DNA samples were digested with HaeIII restriction enzyme (Figure 2). All isolates were found to have plasmid DNA, with various sizes ranging from 0.7 kb to a larger plasmid of 7.2 kb. Seven types of plasmid (P1, P2, P3, P4, P5, P6 and P7) molecules were identified (Figure 3). Thus it was clear that almost all of the isolates from sediments contained a plasmid DNA. RAPD analysis of the 18 isolates led to a minimum of 4 and a maximum of 7 discrete visible bands ranging in size from 0.1 to 3.5 kb (Figure 3). RAPD-PCR with primer A

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ACCEPTED MANUSCRIPT was most informative since the highest numbers of polymorphisms were observed among the isolates. Primers A and B were uninformative because no polymorphism could be detected among the isolates (data not shown). The genetic variation within thirteen thermo-tolerant bacterial isolates was also assessed using ERIC-PCR and was shown as a useful method for differentiating closely related bacteria. ERIC-PCR analysis led to a minimum of 9 and a maximum of 13 characteristic bands ranging in size from 0.2 to 5.1 kb as shown in Figure 4.


Isolate 6 shared 82% similarity with isolates 7, 8, 9, 11 and 18. Isolate number 1 shared 86% similarity with bacterial isolates 2, 3, 4 and 5. The electrophoretic (RAPD and ERIC-PCR) patterns of bacterial isolates showed that the bacterial isolates 11 and 18 were predominant and represented 38.5 and 30.7%, respectively, of the 13 thermophilic bacterial isolates. For molecular characterization of SRB isolates obtained in this study, amplification of DSR genes were performed. All the 11 isolates yielded a PCR product of 1.9 kb for DSR gene with DSR gene specific primers (Figure 5). The morphological and physiological characterization of the 11 isolates proved that the isolated sulphate-reducing bacteria belong to the species D. vulgaris. The 16S rRNA sequence analysis showed that there was a similarity (≥98%) between test strains and representative strains of the gene bank of Bacillus spp., Anoxybacillus and Geobacillus (Figure 6, Table 4), indicating that 16S rRNA gene sequence data are helpful for identification of thermophilic bacteria at genus, but not at species level. The phylogenetic and BLAST analysis of the sequencing results revealed the following 16S rRNA gene’s partial sequence features of the representative isolates. Out of 13 strains, one isolate was identified as Bacillus spp., four as A. flavithermus, two as G. thermodenitrificans, two as Geobacillus spp.

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ACCEPTED MANUSCRIPT The rest of isolates were identified as G stearothermophilus, G. thermoleovorans, G. subterraneus, G. kaustophilus. The phylogenetic affiliations of 13 strains under study showed nearest phylogenetic neighbours in the gene bank database with the homology values between 98 to 100%. All sequences have been accessioned by NCBI (Table 4).

DISCUSSION


The microbial biodiversity in extreme environments is generally considered to be rare compared to most other environments (Kvist et al. 2005). The isolations conducted at mesophilic and thermophilic temperatures indicated a suitability of such environments for growth and multiplication of thermophiles. The colonization of endospore forming bacilli under harsh environments is well known (Sneath 1986). The occurrence of pink filamentous communities in Octopus spring and black filamentous communities associated with thermal springs at Calcite spring in Yellowstone/USA has been recorded. Thermal areas protected in the Yellowstone have received attention concerning microbial ecological research (Reysenbach et al. 1994 and 2000; Reysenbach and Cady 2001). Isolates belonging to the mesophilic range are maintained by routine subculturing or storing in 10% glycerol in a deep freezer (-20o C). Isolates of a thermophilic range could be maintained by prolonged incubation at the optimum temperature on agar plates (agar concentration 2%), till the agar gets dried (usually after 3 months). The cultures could be easily revived by inoculating small piece of dried agar on fresh medium and an incubation at the optimum temperature. The extremophiles, in particular thermophiles and alkalophiles have been a subject of extensive investigations (Stetter 2013). These are particularly interested for the production of industrially useful enzymes like protease, cellulase, cellulase free

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ACCEPTED MANUSCRIPT xylanase and amylase for their industrial applications in the detergent, leather, pulp and paper, and other industries (Pennisi 1997; Srinivasan and Ingale 1999; Budhiraja et al. 2002). On the other hand, SRB constitute a chemoorganotrophic group that exhibits diverse morphological and physiological diversity. SRB reduce sulphur and generate H2S which can react with toxic metals to produce insoluble metal sulphides. Hence, SRB can potentially be used as bioremediators for removal of metal contaminants in water and soil (McIntyre et al. 1990;


White and Gadd 1996). SRB are present in most soils and water, but are outnumbered by other types of bacteria. Hence, enrichment of the target population is usually necessary before isolation (Postgate 1984). Lactate was used as carbon and energy source in accordance with the traditional isolation procedure, where sulphate-reducing bacteria are grown on a narrow range of substrates like lactate, pyruvate and ethanol, organic acids and alcohols (Widdel and Pfennig 1984). The key enzyme of dissimilatory sulphate reduction DSR catalyzes the reduction of sulphite to sulphide, an essential step in anaerobic respiration. The gene coding for this essential enzyme has been found in all sulphate-reducing bacteria tested so far (Klein et al.

2001;

Pavissich et al. 2010; Wan et al. 2010). In the present study for characterization and identification of 13 isolates of thermophilic bacteria both phenotypic and genotypic characters are given emphasis. These are confirm with the criteria of thermophilic bacteria, which grew at temperatures above 50° C (Perry and Staley 1997). Based on the growth and biochemical characters, such as tolerance to high temperature, low nutrient environments and production of lipase, all detected 13 strains appeared to be closer to the genera Bacillus spp., Anoxybacillus and Geobacillus. 16S rRNA sequence analysis showed that there was a strong similarity (≥98%) between the test strains and representative strains from

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ACCEPTED MANUSCRIPT the gene bank of Anoxybacillus, Geobacillus, and Bacillus spp., which may indicate that 16S rRNA gene sequence data is helpful for identification of thermophilic bacteria at genus, but not at species level. Our findings confirmed the previously reported data of Sung et al. (2002), Belduz et al. (2003), Dulger et al. (2004), Zeigler (2005) and Meintanis et al. (2008). Thus, sequence data needs to be supported by another genomic fingerprint method, like rep-PCR, is expected to be more powerful and reliable approach to discriminate thermophilic bacteria even at


strain levels. In the literature, it is also known that the analysis of 16S rRNA sequences may be insufficient to distinguish between some closely related species (Vandamme et al. 1996). Besides, rRNA sequences used alone can be misleading because of the existence of multiple 16S rRNA operons and the occurrence of a recombination within strains (Meintanis et al. 2008). This study is an attempt toward a synthetic taxonomy possible through the development on new mathematical and information strategies (Vandamme et al. 1996). While these results are important for further taxonomic work, positive results on lipase and amylase activities are indicative of potential applications of these bacterial cultures. Thermophiles as a group of microorganisms have been recognized for novel applications in biotechnology. Amongst thermophiles, the genus Geobacillus has received considerable attention mainly due to its taxonomic features and potential applications, such as production of industrially important enzymes. The strains used in the present study were isolated from the samples showing the absence of important nutrients, such as, carbon and nitrogen (Selim et al. 2009). Environmental conditions and the nutritional status available in a natural habitat may indicate toward the development of a particular group of microbial population. Low nutrient status and high temperature under a temperate Sinai location allowed the development of a

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ACCEPTED MANUSCRIPT unique population dominated by a large number of bacilli including Anoxybacillus and Geobacillus. While more attempts are being made toward understanding of unculturable microorganisms in modern biotechnology, new emphasis is also given to obtain the maximum number of isolates using modified methods such as a range of media, modified media or low nutrient media. The importance of culturable microorganisms has also been emphasized in recent literature (Zinder 2002).


High temperature, alkaline nature and poor nutrient status of the soil at both sites seem to provide a unique environment for the development of specialized microbial communities. Identification of areas with extreme environments including the isolation of extremophiles and investigations on the associated microbial wealth for biotechnological applications are of great importance, both for basic and applied research. To the best of our knowledge this is the first report of microbial diversity associated with hot spring sites in the Sinai Peninsula. The isolates obtained from the two sites are maintained in the laboratory for further investigations. In this study, using RAPD-PCR and ERIC-PCR typing methods provided the first insight into the true diversity of these environments, but only DNA sequencing of the amplified targets allowed that new microorganisms, most of them non-cultivable, could be identified and classified. More recently, the development of next-generation sequencing technologies allows metagenomic libraries to be rapidly constructed and sequenced. Therefore the sequences of the 16S rRNA genes or other relevant genes can easily be analyzed, providing a more comprehensive view of microbial diversity (López et al. 2013). In the future study, using this new molecular methodology, metagenome, gives support to the study of the whole biological diversity and might help to characterize all the microorganisms living in an environment.

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ACCEPTED MANUSCRIPT References Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipmana DJ. 1997. Gapped Blast and PSI-Blast: a new generation of protein database search programs. Nucleic acid program Res 25:3389-3402. Barton LL, Tomei FA. 1995. Characteristics and activities of sulphate-reducing bacteria. In: Barton LL (ed) Sulphate-reducing bacteria, Plenum Press, New York, 1–32.


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Table 1. Grouping of total aerobic isolates based on different physiological parameters Parameters Thermotolerant

Rangea 30–55oC

Thermophile

45–65 oC

Number of strains 95 41

o

Hyperthermophile

55–85 C

22

Halophile

0.5–2.0% 2.0–5.0% 10%

79 37 5

Alkaliphiles

8–9 10 11

121 20 3

Acdiophiles

4 5 6

0 11 121

a

Based on Seackbach (2000).

24

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Table 2. Phenotypic studies of thermophilic bacterial isolates

Gram Reaction Cell morphology* Motility Spore Shape Relation to O2 Oxidase Catalase Citrate utilization Growth at or in: 37oC 45oC 65 oC 80 oC pH 9 pH 12 1% Nacl 2% Nacl 3% Nacl Hydrolysis of: Aesculine Gelatin Production of acid: Arabinose Cellobiose Glucose Glycerol Lactose Maltose Mannitol Sorbitol Sucrose Trehlose Xylose Methyl red test Nitrate reduction Substrate utility: Casein Tween 80 Antibiotic sensitivity Amikacin (30µg) Ampicillin (30µg) Gentimicin (10µg) Kanamycin (30µg) Urease production Indole Production Voges–Proskauer test

HP1 + R + S F + + -

HP2 + R + S F + + -

HP3 + R + S F + + -

HP4 + R + S F + + -

HP5 + R + S F + + -

HP6 + R + S F + + +

HP7 + R + S F + + +

HP8 + R + S F + + -

HP9 + R + S F + + -

HP10 HP11 HP15 HP18 + + + + R R R R + + + S S S S F F F F + + + + + + + + -

+ + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

-

-

-

-

+

+

-

-

-

-

-

-

-

+ + + + + + -

+ + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + + +

+ + + + + + + + +

+ + + + + -

+ + + + + -

+ + + + + +

+ + + + + + +

+ + + + + + -

+ + + +

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + + -

+ + + -

+ + + + -

+ + + + -

+

 Cellular morphology was studied using bright field microscopy of a Gram-stained preparation. When observed under the microscope, Gram positive cells were purple or blue in colour while Gram negative cells were pink or red in colour. R: Rod shaped, S: Swollen, F: Facultative anaerobes, +: Positive result and -: Negative result.

25

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ACCEPTED MANUSCRIPT Table 3. Selected characteristics of taxonomic significance and of differential value amongst 11 strains of sulphate-reducing bacteria isolated from hot springs in Sinai Peninsula,

SR2

SR3

SR4

SR5

SR6

SR7

SR8

SR9

SR10

SR11

Gram Reaction Flagella Possession of desulfoviridin Electron donors Lactate Pyruvate Formate Malate Fumarate Ethanol Electron acceptor (with lactate) Sulfate Drug resistance kanamycin (1 mg) vancomycin (5 µg) colistin (10 µg) Biochemical reaction Catalase Indole Nitrate Growth on bile salt Urea

SR1


Egypt

P +

P +

P +

P +

P +

P +

P +

P +

P +

P +

P +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+

+

+

+

+

+

+

+

+

+

+

S R R

S R R

S R R

S R R

S R R

S R R

S R R

S R R

S R R

S R R

S R R

+ + -

+ + -

+ + -

+ + -

+ + -

+ + -

+ + -

+ + -

+ + -

+ + -

+ + -

P: Single polar flagellum. S: Sensitive. R: Resistant.

Table 4. Closest sequences and % similarity of the thermophilic bacterial strains

26

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ACCEPTED MANUSCRIPT Strain no. HP 1 DQ055416

Geobacillus sp-G6

% Similarity 100

HP 2

EF392653

Geobacillus sp-174

100

HP HM161632

HP 3

FJ808712

Geobacillus stearothermophilus- Ah22

99

GU984043

HP 4

AY248717

Geobacillus thermoleovorans-G1

100

HP HM038230

HP 5

FN428689

Geobacillus subterraneus-R-35641

100

HP HM038231

HP 6

EU381189

Geobacillus kaustophilus-GBPI-4

100

HP HM038232

HP 7

AJ785764

Geobacillus thermodenitrificans-HRO10

100

GU903484

HP 8

GQ184213

Anoxybacillus flavithermus- SK3-4

99

HP8 HM217196

HP 9

EU816689

Anoxybacillus flavithermus- LK4

99

HP9 HM217197

HP 10 FJ950739

Anoxybacillus flavithermus- WL 16S

99

HP10 HM217198

HP 11 AY461746

Bacillus sp-H-05

98

HP11 HM217199

99

GU984042

99

HP18 HM217200

HP 15 AJ586360

Closest sequence

Anoxybacillus flavithermus-R-18857

HP 18 AY608963 Geobacillus thermodenitrificans- BGSC W9A26

Accession no.* HP HM161631

* Accession nos. as assigned by NCBI.

Sinai Suez Canal

EGYPT

* Oyoun Mossa * Hammam Pharaon

Red Sea

27

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ACCEPTED MANUSCRIPT FIG. 1. Geographical location of Hammam Pharaon and Oyon Mossa, 123 km north of Sharm El-Sheikh east of the coast of Gulf of Suez, Sinai, Egypt.

M

1

2

3

4

5

6

7

Kb


2.6

0.8 0.7 0.6 0.5 0.4 0.2 0.1

Fig. 2. Plasmid profiles with HaeIII restriction enzyme. Lanes 1 to 7, P1-P7 plasmids; M, DNA ladder.

M

1

2

3

4

5

28

6

7

8

9

10

11 15 18

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Fig. 3. RAPD-PCR products from thirteen thermophilic bacterial isolates generated by random primers A., Lane M, Molecular Marker (10 Kb).


M

1

2

3

4

5

6

7

8

9

10 11

15

18

Fig. 4. ERIC-PCR profile generated with the ERIC 2 primer products from thirteen thermophilic bacterial isolates; M, Molecular Marker (10 kb).

M 1 2

3 4 5 6 7

8

9 10 11

1.9 Kbp

29

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ACCEPTED MANUSCRIPT Fig. 5. PCR amplification of DSR genes of representative isolates of D. vulgaris strains isolated in this study. Lane M: Molecular weight marker (GeneRuler 100 bp DNA Ladder Plus);


Lanes 1 to 11: D. vulgaris strains.

Fig. 6. Dendogram estimated phylogenetic relationships on the basis of 16S rRNA gene sequence data of the thermophilic bacteria isolated from hot springs in Sinai, Egypt, using the neighbour-joining method. The accession numbers are given in parentheses. Only bootstrap values N50% are shown at nodes (based on 1000 bootstrap resamplings). The scale bar represents 1% divergence.

30

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