The cyanobacterial community of the Zerka Ma'in hot springs, Jordan: morphological and molecular diversity and nitrogen fixation

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The cyanobacterial community of the Zerka Ma'in hot springs, Jordan: morphological and molecular diversity and nitrogen fixation
  The cyanobacterial community of the Zerka Ma’in hot springs, Jordan: morphological and molecular diversity and nitrogen fixation D ANNY  I ONESCU1,2 , A HARON  O REN1,* , O RLY  L EVITAN3 , M UNA  H INDIYEH4 , H ANAN  M ALKAWI5   and  I LANA  B ERMAN -F RANK3   1  The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 2  The School for Marine Sciences, The Ruppin Academic Center, Emek-Hefer, Israel 3  Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel 4  Department of Water and Environmental Engineering, German Jordanian University, Amman, Jordan 5  Yarmouk University, Irbid, JordanWith 5 figures and 2 tables Abstract: The area of the Dead Sea, bordering Jordan and Israel, contains many springs with different physical and chemical properties. Hot springs are found on the eastern shore of the lake: the springs of Zara (the ancient Callirrhoe) with tem-peratures up to 59 °C and those of Zerka Ma’in, 5 km inland. The latter springs, mentioned by J OSEPHUS  (1 st  century C.E.), yield fresh water with a low sulfide content, near-neutral pH and temperatures up to 63 °C. Although the growth of green microorganisms was already noted in 1807 by the German explorer U LRICH  J ASPER  S EETZEN , the microflora of the springs has remained unexplored. In 2005 we began a series of surveys characterizing the microbial diversity of the springs. The sources of the Zerka Ma’in springs and their outflow channels are covered by green to orange mats, containing a diverse community of unicellular and filamen-tous cyanobacteria. Conspicuous types include Thermosynechococcus , unicellular Gloeocapsa  types, and Spirulina -like filaments. Large colonies of Scytonema  were also found. Several representative types of Thermosynechococcus , Gloeocapsa , and Mastigocladus / Fischerella  were isolated and cultured. Amplification of cyano-bacterial 16S rRNA genes from DNA extracted from the mats showed a high di-versity of Thermosynechococcus  in the springs. Low concentrations of ammonium and nitrate in the spring water and the presence of heterocystous cyanobacteria indicated that biological nitrogen fixation may be important in the spring ecosys- * corresponding author Algological Studies 130  109–124 Stuttgart, October 2009 DOI: 10.1127/1864-1318/2009/0130-0109 1864-1318/09/0130-109 $ 4.00 © 2009 E. Schweizerbart’sche Verlagsbuchhandlung, D-70176 Stuttgart  110   D. I ONESCU   et al.   tem. nifH   genes related to those of Fischerella ,  Phormidium sp., and Lyngbya sp. were amplified from the community DNA. Laboratory experiments with hetero-cystous isolates showed the occurrence of nitrogen fixation (acetylene reduction) at 52 °C but not at 63 °C. However, the possibility that nitrogen is fixed in situ  at 63 °C cannot be excluded as reverse transcription PCR with mRNA isolated from the site yielded an amplified gene with 100 % homology to the nifH   gene found in a filamentous cyanobacterial culture obtained from the site, and low but significant rates of acetylene reduction were found during in situ  incubations.Key words: cyanobacteria, 16S rRNA gene sequences, hot springs, nitrogenase, thermophilic Introduction The Dead Sea Rift, within the Syrian-African Rift Valley, is rich in thermal springs. Some are freshwater springs, others are saline or hypersaline. Ex-amples are Hamat Gader (up to 52 °C) and the hot springs of Tiberias (up to 60 °C). The area of the Dead Sea, on the border between Jordan and Israel, contains many springs which differ in their physical and chemical properties. The eastern bank of the Dead Sea (Jordan) is especially rich in thermal springs. These include the springs of Zara on the Dead Sea shore (the ancient Callirrhoe; D ONNER  1963) with temperatures up to 59 °C, and the hot springs of Zerka Ma’in, 5 km inland, up to 63 °C ( A BU  A JAMIEH   Fig. 1. Map showing the location of the Zerka Ma’in and the Zara hot springs east of the Dead Sea and sampling sites A1, A2, B1, and B2.  The cyanobacterial community of hot springs 111 1980, 1989, S WARIEH  2000) (Fig. 1, Table 1).Very little is known about the phototrophic communities inhabiting the hot springs of the Dead Sea Rift. A morphological description of the cyanobacteria of the Tiberias hot springs has been given ( D OR  1967), and taxonomic identifications of the species present in the Zara springs (limited to areas in the temperature range 35–40 °C) were published long ago ( F RÉMY  & R AYSS  1938). The German explorer U LRICH  J ASPER  S EETZEN  (1767–1811) wrote after his visit to the hot springs of Zerka Ma’in in 1807: “In dem Wasser wuchs eine grüne schleimige Conferve” [In the water grew a green slimy microscopic alga] ( S EETZEN  1854). B LANCKENHORN  (1912) provided a somewhat more detailed macroscopic description of the green material in the springs, yet no further studies are available on their biota.We have recently begun a microbiological survey of the Jordanian hot springs ( I ONESCU  et al. 2007). Here we present information on the morphological diversity of the cyanobacteria inhabiting the Zerka Ma’in springs and 16S rRNA gene sequence-based diversity, as found both in environmental samples and in cyanobacteria cultured from these samples. In addition, we evaluated the potential for nitrogen fixation by the cyanobacterial community, by examining the presence of nifH   genes, their expression, and nitrogen fixation activity. Table 1. Chemical and physical characteristics of water samples collected at the dif-ferent sampling sites of the Zerka Ma’in springs, as analysed at the Department of Earth and Environmental Sciences, Yarmouk University.Sampling site A1 A2 B1 B2 TemperaturepHH 2 SConductivityTotal dissolved saltsAlkalinityHardness[Cl – ][NO 3– ][SO 42– ][HCO 3– ][Mg 2+ ][Ca 2+ ][K + ][Na + ][Sr 2+ ](°C)(mM)(µS cm –1 ) (ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)(ppm)636.690.321401306120560860.10197239 1 901853386.33.3 1 626.620.341801267110520810.41.02196ND911863585.6ND586.380.0324601428130540900.90.72210ND931933778.5ND586.81NA24701445130580790.60211ND921953878.2ND 1 As determined by I. G AVRIELI , The Geological Survey of Israel. ND = not determined.  112   D. I ONESCU   et al.   Materials and methods Samples were collected from the sampling sites indicated in Fig. 1 between December 2005 and June 2007 into sterile plastic 50 mL tubes and stored in a refrigerated coolbox until processed within 40 h of sampling. Photomicro-graphs were made in a Zeiss Axiovert model 135 TV microscope equipped with phase-contrast optics. Cultures were prepared in 250 mL Erlenmeyer flasks containing 100 mL BG-11 growth medium ( S TANIER  et al. 1971), and incubated in a New Brunswick Innova 44 illuminated incubator at 45 °C and an incident light intensity of 175 µ mol quanta m –2  s –1 . For DNA ex-traction, cultures and environmental samples were incubated for 30 min at 100 ºC in lysis buffer (100 mM Tris-HCl, 50 mM EDTA, 10 mM NaCl, 1 % SDS, pH 8), followed by extraction with phenol-chloroform-isoamyl alco-hol (25:24:1). The extracts were washed with chloroform-isoamyl alcohol (24:1) until no interphase could be observed. DNA was then precipitated with cold ethanol, washed with ice-cold 70 % ethanol, and resuspended in water. Fragments of the 16S rRNA gene were then amplified by PCR, using cyanobacteria-specific primers as specified in Table 2. The primer sets 29F – 809R and 740F – 1494R were used for cultures, while 106f and 781R were used for environmental samples. Amplicons srcinating from environmen-tal sequences were cloned using the InsTAclone kit (K1214, Fermentas, Lithuania) and verified using colony PCR before being sequenced.Samples for RNA analysis were collected into 1 mL of TriReagent (TR 118, Molecular Research Center Inc., OH, USA) and immediately placed in liquid nitrogen to be later processed according to the manufacturer’s in-structions. To evaluate the existence of the nifH   mRNA in total RNA ex-tracts, cDNA was produced using a first strand cDNA synthesis kit (K1612, Fermentas, Lithuania) together with the nifHR primer (Table 2). The Table 2. PCR primers used in this study. Primer Primer target Sequence 27FGeneral 16S rRNA 1 AGA GTT TGA TTT ACG CGA CA809RCyanobacterial 16S rRNA 1 GCT TCG GCA CGG CTC GGG TCG ATA740FCyanobacterial 16S rRNA 2 GGC YRW AWC TGA CAC TSA GGG A1494General 16S rRNAGGY TAC CTT GTT ACG ACT106FCyanobacterial 16S rRNA 3  CGG ACG GGT GAG TAA CGC GTG A781RCyanobacterial 16S rRNA 3 GAC TAC WGG GGT ATC TAA TCC CWT TnifHFCyanobacterial nifH 4 CGT AGG TTG CGA CCC TAA GGC TGAnifHRCyanobacterial nifH 4 GCA TAC ATC GCC ATC ATT TCA CC 1   P OMATI  et al. 2004. 2 F. G OH , University of New South Wales, personal communi-cation. 3   N ÜBEL  et al. 1997. 4 M AZARD  et al. 2004.  The cyanobacterial community of hot springs 113 cDNA was further amplified using the nifHF and the nifHR primers. The same primer was used to screen our cultures for the nifH   gene.Nitrogen fixation activity was assessed using the acetylene reduction as-say ( S TEWART  et al. 1967). Subcultures of 32 mL or 11 mL in case of in situ experiments were placed in rubber-stopped glass bottles, 72 mL and 25 mL respectively. Acetylene (8 mL and 2 mL respectively) was injected into the head space. Control experiments included systems without acetylene and sterile medium with acetylene. To preserve the ethylene/acetylene ratio ob-tained immediately after the in situ  incubation was terminated, 2.5 mL of gas from the head-space were transferred to 4 mL vacuum tubes (Vacuette, Greiner Bio-One) containing saturated NaCl solution. Acetylene reduction was assessed by injecting 1 mL of gas taken from the head space into a gas chromatograph equipped with a flame ionization detector (FID-GC SRI 310). To evaluate various factors which affect the N 2  fixation abilities of our isolates, we tested activity in: 1) cultures which were initially grown in N-free BG-11 medium (BG-11 0 ), transferred into fresh BG-11 0  medium and incubated at 31, 45, 53 and 63 ºC under constant light for 24 h; 2) cultures initially grown in BG-11 medium, transferred to BG-11 0  two days prior to acetylene reduction assay, and incubated at 45 ºC under constant light and under a 12 h dark/light regime for 48 h; 3) cultures initially grown in BG-11 medium, washed with and transferred to BG-11 0  immediately prior to acetylene reduction assay initiation and incubated at 48 ºC under a 12 h dark/light regime for 100 h. Cyanobacterial mats were collected from site A1 and from two nearby streams running at a temperature of 51 ºC and 39 ºC. Duplicate samples were incubated in situ  at their respective sampling sites under various conditions: 1) in pond/stream water under constant light; 2) dark incubation in bottles covered with aluminum foil; 3) in BG-11 0  medium under constant light. Incubation time ranged between 1:45–2:45 hours.The Bunsen gas solubility coefficient for ethylene was calculated for each of the various incubation temperatures according to B REITHBART  et al. (2004). N 2  fixation rates were calculated according to C APONE  (1993); a ratio of 4:1 was used to convert reduced acetylene to nitrogen fixed accord-ing to S TAAL  et al. (2001) and J ENSEN  & C OX  (1983). Chlorophyll was ex-tracted either by 90 % methanol at 80 ºC (experiments 1 and 2) or by 90 % acetone (experiment 3). Results The microbial mats at sites A1 and A2 (Fig. 1) were dark-green in colour. At sites B1 and B2 and along the channel connecting them, the dominant colour was orange, with prominent patches of green material being present as well. Microscopic examination shows a highly diverse community of cy-
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