Phylogeny and Ecophysiological Features of Prokaryotes Isolated from Temporary Saline Tidal Pools

Ann Microbiol (2014) 64:599–609 DOI 10.1007/s13213-013-0693-y

ORIGINAL ARTICLE

Phylogeny and ecophysiological features of prokaryotes isolated from temporary saline tidal pools

Spyridon Ntougias

Received: 19 March 2013 /Accepted: 8 July 2013 /Published online: 31 July 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract Although hypersaline environments have been Introduction extensively examined, only a limited number of microbial community studies have been performed in saline tide pools. Halophilic and halotolerant microorganisms are distributed We have studied a temporary salt-saturated tide pool and in several terrestrial and aquatic habitats, such as the Dead isolated prokaryotes from the water. Chlorinity measure- Sea, the hypersaline lakes of Asia and Africa, the benthic ments revealed that the tide pool brine could be characterized zone, salt marshes and marine sediments (Takai and as one of the most hypersaline ecosystems on earth. Enu- Horikoshi 1999; Mutlu et al. 2008; Tsiamis et al. 2008; meration of microorganisms at different salinities showed Bodaker et al. 2010). From a biotechnological point of view, that the tide pool was dominated by moderate halophiles. halophiles can be used in environmental applications, such Based on 16S rRNA gene sequence analysis, the prokaryotic as the bioremediation of highly polluted marine and saline strains isolated were related to the bacterial genera Rhodo- terrestrial ecosystems, and in the development of new bio- vibrio, Halovibrio, Aquisalimonas, Bacillus and Staphylo- technological products, such as the use of their metabolites coccus and to the haloarchaeal species Haloferax alexan- (e.g. ectoine and β-carotene) in the cosmetic and food in- drinus. Four bacterial isolates were distantly related to their dustry (Oren 2010). closest validly described species Aquisalimonas asiatica Microbial communities in hypersaline environments have (96.5 % similarity), representing a novel phylogenetic link- been extensively studied during the last decade (Burns et al. age. Ecophysiological analysis also revealed distinct pheno- 2004; Sørensen et al. 2005; Caton et al. 2009), resulting in typic profiles for the prokaryotic strains analyzed. The her- the identification of a plethora of novel halophilic bacteria bicide 2,4-dichlorophenoxyacetate could be effectively uti- and archaea. However, the identification of halophilic diver- lized by selected strains as the sole carbon source, but phe- sity in the tidal and intertidal zone is limited. To our knowl- nolic compounds could not be utilized by any of the halo- edge, only Bolhuis and Stal (2011) have indirectly examined philic isolates examined. None of the halophilic strains were the halophilic community in coastal microbial mats located able to grow without the presence of sea salt or seawater. along a tidal gradient, reporting a massive increase in Based on these results, we conclude that moderate halophilic halobacterial sequences during the summer. Thus, saline bacteria rather than extremely halophilic archaea dominate in tidal pools are ideal environments for exploring novel halo- such a hypersaline environment. philic microbiota as to date no microbial community studies have been performed in such naturally occurring saline ecosystems. The only related work reported thus far has been an Keywords Ecophysiology . Extremophiles . Halophilic examination of fecal contamination levels in tidal pools by bacteria . Haloarchaea . Marine ecosystems . Tidal zone Genthe et al. (1995), although these authors provided no information on tide pool microbiota. Tidal or tide pools are formed after seawater entrapment in pools of the coastal zone. Several physical and biogeo- chemical parameters affect microbial life in such tidal pools * S. Ntougias ( ) (Huggett and Griffiths 1986; Kooistra et al. 1989; Netto et al. Department of Environmental Engineering, Democritus University of Thrace, Vas. Sofias 12, 67100 Xanthi, Greece 2003). The pools can be shallow or deep, large or small, with e-mail: [email protected] rocky or muddy bottoms. Large and shallow pools are more 600 Ann Microbiol (2014) 64:599–609 exposed to the surface than small and deep ones. These dilution, aliquots of 0.1 mL were placed in five different features impact nutrient availability and exposure of the isolation media. The procedure was repeated in triplicate. microorganisms to certain environmental factors. Air tem- The isolation media used were prepared as follows: (1) 0.10 perature and sunlight influence the temperature of the tide volume of seawater per volume of isolation medium pool and consequently the photosynthetic and respiration (≅ 0.35 % salt concentration), (2) 0.85 volumes of seawater rates, thereby affecting the levels of carbon dioxide and per volume of isolation medium (0.85×seawater salt oxygen in the water of the pools. The size, shape and volume concentration≅3.0 % salt concentration), (3) 0.85 volume of the tide pool also affect the concentration of dissolved of seawater per volume of isolation medium and 90 g L−1 oxygen, while increasing levels of dissolved carbon dioxide NaCl (≅12.0 % salt concentration), (4) 0.85 volume of turn the water acidic (Schulte 2007). The water evaporation seawater per volume of isolation medium and 220 g L−1 rate (as influenced by the water surface area, air temperature NaCl (≅25.0 % salt concentration) and (5) 0.85 volume of and speed) and rainfall affect the salinity of the tidal pool. seawater per volume of isolation medium, 200 g L−1 NaCl −1 The aim of our study was to identify the diversity of and 50 g L MgCl2.6H2O(≅25.0 % salt concentration— cultured aerobic heterotrophic prokaryotes isolated from addition of high amount of Mg). Peptone and yeast extract temporary rocky tide pools of high salinity and to investigate were in all media tested at concentrations of 5 and 1 g l−1 the ecophysiology of both the bacteria and archaea isolated, respectively. Media were solidified with 17 g L−1 bacterio- with special reference to their halophilicity and ability to logical agar. For both isolation and enumeration, plates were utilize certain organic substrates. incubated at 37 °C for 2 months, with the exception of cultures obtained from the isolation medium containing a high concentration of Mg, which were incubated at 50 °C. Materials and methods All other experimental tests were carried out at 37 °C, unless otherwise specified. After colony enumeration, the cultured Sample and physicochemical analysis prokaryotic population was expressed as colony forming units (CFUs) per milliliter of tide pool saline water. Saturated brine was obtained from a tide pool with a rocky bottom located at the seaside of Kardamili, South Greece. Kardamili tidal pools are formed seasonally on the top of Morphology and salt tolerance huge rocks located at the edge of the shore (latitude 36.885589, longitude 22.23187). The tidal pool under study Cell morphology and presence of flagella were examined was approximately 50×20 cm, with a depth of 10–15 cm. using a Zeiss Axiostar Plus microscope (Carl Zeis AG, Electrical conductivity (EC) and pH were determined using Oberkochen, Germany) after flagella staining (Flagella Stain CRISON CM-35 (CRISON Instruments, Alella, Spain) and Droppers; Becton Dickinson & Co., Franklin Lakes, NJ). Metrohm 632 (Metrohm, Herisau, Switzerland) conductivity Salt tolerance was investigated in media consisting of the and pH meters respectively. Dissolved oxygen in the tide nutritional base described above (i.e. 5 g L−1 peptone and pool water was measured using a CRISON oxi 45 oxymeter. 1gL−1 yeast extract) and the following saline solutions: 0.10 Ions in the brine were determined using a ICS-3000 Dual Ion v/v seawater (≅ 0.35 % salt concentration), 0.85 v/v seawater Chromatography System (Dionex Corp. Sunnyvale, CA). (≅ 3 % salt concentration), 0.85 v/v seawater and 3 % NaCl The anion/cation analysis was carried out using Dionex (≅ 6 % salt concentration), 0.85 v/v seawater and 6 % NaCl AG22/CG12A guard (4×50 mm) and analytical columns (≅ 9 % salt concentration), 0.85 v/v seawater and 9 % NaCl (4×250), 4-mm ASRS-300 (31 mA)/CSRS-300 (59 mm) (≅ 12 % salt concentration), 0.85 v/v seawater and 12 % suppressors and CR-ATC/CR-CTC trap columns. Sodium NaCl (≅ 15 % salt concentration), 0.85 v/v seawater and carbonate (4.5 mM)/sodium hydrogen carbonate (1.4 mM) 15 % NaCl (≅ 18 % salt concentration), 0.85 v/v seawater and methanesulfonic acid (20 mM) were used as eluents for and 18 % NaCl (≅ 21 % salt concentration), 0.85 v/v seawa- anions and cations at flow rate of 1.2 and 1 mL min−1, ter and 22 % NaCl (≅ 25 % salt concentration) and 0.85 v/v respectively. The sample loop size was 50 μL and a conduc- seawater and 27 % NaCl (≅ 30 % salt concentration). Tem- tivity detector was used for the determination of ions. perature and pH range for growth were tested in media consisting of the nutritional base and the appropriate saline Isolation and enumeration of prokaryotes solution. Temperatures of 5, 10, 15, 20, 25, 30, 37, 40, 45, 50, 55 and 60 °C were examined. pH within 4 and 10 (in In order to isolate and enumerate the cultured prokaryotic steps of 1 pH) was tested, and a pH check was performed population, 10 mL of saline water collected from the tide before the inoculation and after the end of the experiment. pool was mixed with 90 mL of sterile tide pool saline water, Before and after sterilization, the pH was adjusted by using a and a series of tenfold dilutions was performed. From each sterile 0.5 M HCl or 0.5 M NaOH solution. Ann Microbiol (2014) 64:599–609 601

Utilization of organic compounds as the sole carbon source Table 1 Physicochemical characteristics of the tide pool brine Brine characteristics Value To investigate substrate utilization, we prepared media con- − taining a specific organic substrate, 0.02 g L 1 yeast extract pH (1/50 diluted) 7.07 and saline solution (at the optimum salt concentration of each Electrical conductivity (EC) (1/50 diluted) (mS cm−1) 11.9 −1 isolate examined). Saline solutions of 0.85 v/v seawater (for Dissolved O2 (mg L ) 1.98 strain K12-25), 0.85 v/v seawater and 6 % w/v NaCl (for Temperature (°C) 38.3 strains K12-9A and K12-17A) and 0.85 v/v seawater and Na+ (g L−1) 109.21±0.49 + −1 12 % w/v NaCl (for strain K25M-2B) was prepared. No NH4 -N (mg L ) 12.6±0.29 growth was detected in medium containing the above yeast K+ (g L−1) 7.76±0.20 extract concentration as the sole carbon source. The concen- Mg2+ (g L−1) 24.69±0.14 tration of each substrate examined was 0.05 M, unless the Ca2+ (g L−1) 1.46±0.21 water solubility of the substrate was lower than the target F− (g L−1) 0.01 concentration in which case a concentration up to saturation Cl− (g L−1) 244.94±4.25 point was used. The D-amygdalin hydrate, cetyltrimethyla- Br− (g L−1) 0.29±0.10 mmonium bromide and phenol concentrations tested were 5, 5 − −1 NO3 -N (g L ) 0.18±0.05 −1 and0.4gL , respectively, while the concentration for ethyl 2− −1 SO4 (g L ) 23.54±0.05 acetoacetate or glycerol tributyrate was 0.5 % v/v. Acid production from sugars was examined in media μ consisting of the nutritional base and the appropriate saline 2mMMgCl2,200 M each dATP, dTTP, dCTP and dGTP, the μ solution in the presence of 10 g L−1 of the substrate tested, appropriate primer pair, 0.5 M each primer and 2.5 U DNA 0.02 g L−1 yeast extract and 18 mg L−1 phenol red. Phenylal- polymerase (GeneON, Ludwigshafen, Germany) was prepared. anine deaminase and DNAse activity were tested at the opti- PCR amplification was performedinagradientPCR(TaKaRa, mum salt concentration using phenylalanine agar (23 g L−1) Otsu, Japan) using a denaturation step of 2 min at 94 °C, and DNase test agar with toluidine blue (42 g L−1), respec- followed by 35 cycles of denaturation at 94 °C for 30 s, primer tively. Urea hydrolysis was examined using urea broth under annealing at 52 °C for 30 s and DNA chain extension at 72 °C optimum salinity. Gelatin, starch, milk, tributyrin and Tween for 1 min, with a final DNA chain extension at 72 °C for 30 80 hydrolysis were examined in media consisting of the min. nutritional base using 120 g L−1 gelatin, 10 g L−1 starch, 50 g L−1 skimmedmilk,1%v/vtributyrinand1%v/vTween Cloning and sequencing of the PCR products 80, respectively, at optimum salt concentration. Antibiotic susceptibility was investigated in media consisting The PCR products were purified, ligated in the pGEM T- of the nutritional base and the appropriate saline solution Easy vector and transformed into E. coli JM-109 cells fol- (optimum salt concentration per each isolate examined) re- lowing the manufacturer’s instructions (Promega). Blue and ported above, in the presence of antibiotic tested. Anaerobic white colonies were selected, and recombinant plasmids growth was examined using Anaerocult A (Merck, Whitehouse were extracted using the GF-1 Plasmid DNA Extraction kit Station, NJ). (Vivantis, Selangor, Malaysia). The insert size in plasmids was confirmed by PCR amplification using primers SP6 (5′- PCR amplification of prokaryotic isolates Table 2 Enumeration of cultivated prokaryotic population in the tide pool brine DNA from cultures grown at the appropriate salinity was extracted using the Promega DNA Extraction kit (Promega, Salinity of the media Incubation Enumeration −1 Madison, WI). The nearly full-length 16S rRNA gene was examined (%) temperature (°C) (CFU mL ) amplified for bacterial isolates using the primer sets pA (5′- 0370a AGA GTT TGATCC TGG CTC AG-3′;sense;positions8–27 3 37 7.2±1.6 (×105)b by Escherichia coli numbering) and pH (5′-AAG GAG GTG 12 37 11.7±0.2 (×105)c ATC CAG CCG CA-3′;antisense;positions1542–1522), while 25 37 14.3±0.8 (×103)a PCR amplification for archaeal isolates was achieved using 25b 50 12.3±3.3 (×102)a primers w003 (5′-ATT CYG GTT GAT CCY GSC-3′;sense; positions 1-18 by Haloferax volcanii numbering) and w002 Values followed by a different lowercase letter are statistically signifi- (5′-GNT ACC TTG TTA CGA CTT-3′; antisense; positions cantly different (Duncan’s test a<0.05) 1447-1430) (Tsiamis et al. 2008). A reaction mixture (50 μL) a Presence of only two colonies in one of the three replicates − containing 1 μL (50 ng μL 1) genomic DNA, 10× PCR buffer, b Medium containing Mg 602 Ann Microbiol (2014) 64:599–609

ATT TAG GTG ACA CTA TAG-3′) and T7 (5′-AAT ACG relatives obtained were included in a subsequent phylogenetic ACT CAC TAT AG-3′). Two individual clones per strain analysis, and alignment of the sequences was performed using were sequenced and the nucleotide sequence data was sub- ClustalW (http://www.genome.jp/tools/clustalw/). TREECON mitted to GenBank under accession numbers KC309441– for Windows was used to construct the phylogenetic trees KC309470. Sequencing reactions were carried out by (Van de Peer and De Wachter 1994). Evolutionary distances Macrogen (Seoul, South Korea). were calculated using the method of Jukes and Cantor (1969), and the topology was inferred using the “neighbor-joining” Phylogenetic and cluster analyses approach (Saitou and Nei 1987) based on bootstrap analysis of 1,000 trees. The 16S rRNA gene sequences were assembled and similarity A dendrogram of the physiological characteristics of the searches against the NCBI data base and the Ribosomal Data- isolates analyzed was constructed using NTSYSpc ver. 2.2 base Project were carried out using the basic local alignment (Exeter Software, Setauket, NY; Rohlf 2007). A binary 0/1 search tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) matrix was made based on the absence or presence of a and the SEQUENCE_MATCH program (http://rdp.cme.msu. physiological feature, respectively, and the ‘average taxo- edu/seqmatch/seqmatch_intro.jsp), respectively. The closest nomic distance (DIST) coefficient was calculated using the

Table 3 Salinity range of prokaryotes isolated from the tide pool brine

Isolate Salinity Optimum salinity (%)

0% 3% 6% 9% 12% 15% 18% 21% 25% 30%

K0-1B + + + + + + +/−− − − 0–3 K0-2B + + + + + + +/−− − − 0–3 K3-2Ba + + + + −−−− −− 0 K3-6B − +++++++/−−− 6–9 K3-7B − +++++++/−−− 6–9 K3-11B − +++++++ +/−− 3–6 K3-12B − +++++++/−−− 6–9 K3-15B − +++++++/−−− 6–9 K3-16B − +++++++/−−− 6–9 K3-25B − +++++++/−−− 6–9 K12-1B + + + + + + +/−− − − 0–3 K12-3A − +++++++ +− 6–9 K12-7B − +++++++ +− 3–6 K12-9A − +++++++ +− 6–9 K12-11A − +++++++ +/−− 6–9 K12-12B − +++++++/− +/−− 6–9 K12-14Α − +++++++ +/−− 6–9 K12-16A − +++++++ +− 6–9 K12-17A −−++++++/−−− 6–9 K12-22B −−++++++/−−− 6–9 K12-23B − +/− ++++++/−−− 6–9 K12-25A − +++++++ +− 3–6 K12-26A −−++++++/−−− 6–9 K25-29A −−++++++ +/−− 6–9 K12-34B −−++++++/−−− 6–9 K25-2C − +/− ++++++ +/−− 6–9 K25-7C − +++++++ +− 6–9 K25-9C − +++++++ +− 3–6 K25M-1B −−+/− +++++ ++15–18 K25M-2B −−+/− +++++ ++15–18

+, Growth; +/−, marginal growth; −, no growth Ann Microbiol (2014) 64:599–609 603

Simint option of the package to obtain dissimilarities. For The non-halophilic population in the tide pool was re- clustering, the neighbor-joining algorithm (Njoin option) of stricted since microbial growth was almost absent in medium the program was employed using the unweighted pairgroup with no salinity (Table 2). The tide pool was dominated by method with arithmetric average (UPGMA). halophilic microbiota which were favored on media with 12 % salinity, while the microbial population of the brine was drastically reduced by at least twofold in hypersaline Results media (Table 2). The halophilic population was present in higher numbers under mesophilic conditions comparted to The saline brine obtained from the tide pool was saturated as thermophilic conditions. Strains K0-1B and K0-2B were the indicated by the extremely high ionic concentrations and the EC only isolates obtained from medium with 0 % salinity, and values determined (Table 1). The dominant ion species were the number of halotolerant isolates grown on media with 3 + − 2+ −2 Na ,Cl,Mg and SO4 (Table 1). The pH in the tide pool and 12 % salinity was also limited (isolates K3-2Ba and brine was neutral owing to seawater entrapment. The tempera- K12-1B) (Table 3). The majority of isolates were halophilic ture was 38 °C and the dissolved oxygen in the brine was and extremely halophilic microorganisms which were able to estimated to be at near-hypoxic concentrations (1.98 mg L−1). grow in a wide range of salinity (Table 3). The lower and

Fig. 1 Phylogenetic allocation of prokaryotes identified in the tide pool Percentage bootstrap values (>50 %) based on 1,000 replicates. Pro- brine. Evolutionary distances were calculated by the Jukes and Cantor karyotic strains from the present study are depicted in bold. Scale bar: (1969) method and the topology was inferred using the neighbor- 0.02 substitutions per site joining algorithm (Saitou and Nei 1987). Numbers on the nodes: 604 Ann Microbiol (2014) 64:599–609 upper salinity limit for growth of the halophilic isolates (96.5 % similarity). The sole isolate of OTU#4 was closely varied between 3–6and21–30 %, respectively, depending related to Bacillus cereus (99.9 % similarity), while OTU#5 on the halophilicity of the strain examined (Table 3). The isolates were affiliated to Staphylococcus hominis (99.7 % optimum salinity for growth was 9 % for the majority of the similarity). Isolates of all OTUs were halophilic microorgan- isolates obtained, although isolates were also identified with isms, with the exception of members of OTUs#4 and 5. a salinity optimum of 3 or 15 %. Ecophysiological features of members of halophilic clus- Using universal 16S rRNA gene-targeted PCR primers, we ters were further investigated by determining the morphology sequenced the almost full-length 16S rRNA gene from all 30 and physiological profile of one representative isolate per each isolates. The phylogenetic allocation of the prokaryotic strains halophilic OTU. Cells of the halophilic strains from the tide isolated from the tide pool brine is presented in Fig. 1.Pro- pool brine examined were Gram-negative, nonmotile and karyotic isolates were classified into six distinct operational cocco-bacilloid, growing as small round colonies (Table 4). taxonomic units (OTUs). The majority of OTUs represented Color of the cell biomass varied among the representative phylogenetic linkages among bacteria, while a single OTU isolates, with strains K12-9A, K12-17A, K12-25A and (OTU#6) consisted of archaeal isolates closely related to the K25M-2B forming yellow, white, subyellow and orange col- halobacterial species Haloferax alexandrinus (99.7 % simi- onies, respectively (Table 4). Endospores were not observed. larity in the 16S rRNA gene). The largest OTU (OTU#3) All strains examined were strictly aerobic, oxidase and cata- comprised 13 isolates which were affiliated with the species lase positive, mesophilic and neutrophilic microorganisms Rhodovibrio sodomensis (94.7 % similarity) and R. salinarum (Table 4). The optimum temperature and pH for growth were (93.8 % similarity), although their closest phylogenetic rela- 35–37 °C and 7–8, respectively (Table 4), while the four tive was Rhodovibrio sp. 2 Mb1 (99.0 % similarity). The representative halophilic strains differed in their optimum sa- representatives of OTU#1 (7 isolates) were associated with linity for growth (Table 3). The halophilic isolates examined the species Halovibrio denitrificans (97.6 % similarity) and were slow growers, growing in a narrow pH range (near pH of Pseudomonas halophila (97.5 % similarity), while their clos- seawater). Only isolate K12-25A could grow at 5 °C, while the est match was clone SN144 (99.3 % similarity). The four other isolates could not grow below 15 °C. The upper temper- isolates representing OTU#2 were related to clone LL45B ature limit for growth was lower than 50 °C, with the exception (99.3 % similarity), while they were distantly related to their of the archaeal strain K12-25A which could grow at 50 °C. No closest validly described species Aquisalimonas asiatica growth was observed at 55 °C for any isolate tested. None of

Table 4 Morphology and re- sponse to environmental condi- Morphological characteristics/ Halophilic prokaryotes tions of halophilic prokaryotes environmental conditions (one representative strain per K12-9A K12-17A K12-25A K25M-2B OTU) isolated from the tide pool Colony shape Small round Small round Small round Small round Colony color Yellow White Sub-yellow Orange to red Appearance of colonies on 2-days after 3-days after 2-days after 3-days after solid medium Cell shape Small cocco- Small cocco- Small cocco- Small cocco- bacilloid bacilloid bacilloid bacilloid Motility Non-motile Non-motile Non-motile Non-motile Oxygen requirement Strict aerobe Strict aerobe Strict aerobe Strict aerobe Oxidase Positive Positive Positive Positive Catalase Positive Positivea Positive Positivea Gram stain Negative Negative Negative Negative pH range 6–96–96–96b-8 pH optimum 7–87–87–87 Temperature range 15–45 15–40 5–40 20–50 Temperature optimum 37 35–37 35–37 35–37 Optimum salinity 9 % 9 % 3 % 15 % Growth on NaCl-containing −−−− OTU, Operational taxonomic medium (of optimum salinity) unit Growth on sea salt-containing +++− +, Growth; −, no growth medium (of optimum salinity) Growth on seawater-containing ++++ a After a long application period medium (of optimum salinity) b Marginal growth Ann Microbiol (2014) 64:599–609 605

Table 5 Physiology of halophilic prokaryotes (one representative Table 5 (continued) strain per OTU) isolated from the tide pool brine Physiological Halophilic prokaryotes Physiological Halophilic prokaryotes characteristics characteristics K12- K12- K12- K25M- K12- K12- K12- K25M- 9A 17A 25A 2B 9A 17A 25A 2B −−−− D-Saccharate a Biochemical tests Succinate −−+ − −−−− Casein hydrolysis Sugar alcohols DNase activity −−−− − − − − D(−)-Mannitol +/ +/ +/ +/ −−−− Gelatin liquefaction myo-Inositol +/− +/− +/− +/− Indole test − +/−−+ −−−− D-Sorbitol +/ +/ −−−− Nitrate reduction Ethyl ester −−−− Methyl red reaction Ethyl acetoacetate +/−−+ − −−−− Phenylalanine Cetrimide (antiseptic agent)b deamination Cetyltrimethylammonium −−−− Starch hydrolysis −−−− bromide −−−− Tributyrin hydrolysis Herbicide − −− Tween 80 hydrolysis + 2,4-Dichlorophenoxy- −−+ − Urea hydrolysis −−−− acetateb b Voges-Proskauer reaction −−−−Phenolics −−−− Sugars and related compoundsb Catechol − − − − Cinnamate −−−− L(+)-Arabinose +/ +/ +/ +/ − − Ferulate −−−− D(−)-Amygdalin hydrate +/ ++ − − − p-Nitrophenol −−−− D(+)-Cellobiose +/ +/ +/ + − − Phenol −−−− D(+)-Galactose +/ +++/ − − − Protocatechuate −−−− D(+)-Glucose ++/+/ +/ −−−− Lactose +/− +/− +/− +/− Syringaldehyde −−− − Syringate −−−− D(+)-Maltose +/ +/ +/ − −−− Vanillate −−−− D(+)-Mannose +/ +/ −−− − o-Vanillin −−−− D(+)-Melezitose +/ +/ α-D-Melibiose −−−+/− a Biochemical test: +, positive; +/−, weak positive, −, negative − − − − D(+)-Raffinose +/ +/ +/ +/ b Organic compound utilization: −, no utilization (OD600 nm <0.04); +/−, Sucrose +/−−+/− +/− weak utilization (OD600 nm 0.04–0.1); +, utilization (OD600 nm >0.1) − − − − D(+)-Trehalose +/ +/ +/ −−− − D-Xylose +/ +/ +/ Amino acidsb the representative halophilic strains could grow in the absence −−−of sea salt or seawater. The archaeal representative required L-Arginine ++/ − − − seawater and not sea salt for growth (Table 4). L-Asparagine + +/ +/ −−−− The utilization of organic compounds by the selected L-Cysteine − − isolates is given in Table 5. All of the isolates examined L-GLutamine +/ +/ ++ could not hydrolyze complex organic macromolecules (e.g. Glycine −−−− proteins, starch, lipids, DNA), with the exception of strain ++/− +/− +/− L-Histidine K12-17A which could hydrolyze Tween 80. None of the −−++/− L-Isoleucine halophilic isolates were capable of 2,3 butanediol fermenta- +/− +/− +/− +/− L-Lysine tion following mixed-acid fermentation. Tryptophan deami- +/− +/− +/− +/− L-Proline nation was carried out by strains K12-17A (weak reaction) +/− +/− +/− +/− L-Serine and K25M-2B (strong reaction), while none of the isolates Organic acidsb tested carried out phenylalanine deamination. Halophilic Acetate + − ++/− isolates were negative for urea hydrolysis and nitrate reduc- − − − Citrate +/ +/ + tion. All of the halophilic isolates examined showed weak −−−− Maleate +/ +/ utilization of sugars and sugar alcohols, with the exception of Oxalate −−−− an effective utilization of D(+)-glucose by strain K12-9A, D(−)- Propionate +/−−++/− amygdalin and D(+)-galactose by strains K12-17A and K12-25A 606 Ann Microbiol (2014) 64:599–609

Table 6 Antibiotic susceptibility of halophilic prokaryotes (one repre- erythromycin and rifampicin and resistant to bacitracin, sentative strain per OTU) isolated from the tide pool brine cyclohexidine, tetracycline and trimethroprim. The archaeal Antibiotics Halophilic prokaryotes isolate was resistant to all antibiotics tested, with the exception of trimethroprim (Table 6). K12- K12- K12- K25M- 9A 17A 25A 2B

Ampicillin (30 μgmL−1)+− +/− + Discussion Bacitracin (10 μgmL−1)++ + + Cefotaxine (30 μgmL−1)+− ++ A limited number of studies have been performed in tide Chloramphenicol −− − + pools, and these have focused mainly on the mineralogy and (30 μgmL−1) the structure of the algal (Zhuang 2006; Tujula et al. 2010) − Cyclohexidine (10 μgmL1)+ + + + and ichthyic (Godinho and Lotufo 2010) communities. Our − Erythromycin (30 μgmL1) −− − + study is the first attempt to investigate halophilic microbiota − Kanamycin (30 μgmL1) − ++/− + in the tide pool by identifying the cultured halophilic pro- − Neomycin (30 μgmL1) − ++/− + karyotic diversity using both physiological and molecular Penicillin (50 u) + − +/− + approaches. Polymixin b (100 u) + − ++ Based on the chlorinity determined, the tide pool brine RIfampicin (5 μgmL−1) −− − + examined was an extremely hypersaline environment, Streptomycin (30 μgmL−1) − ++/− + possessing salinity levels similar to those reported for the most Tetracycline (10 μgmL−1)+ + + + saline environments on earth, such as Lake Assal (Wood and Trimethroprim (25 μgmL−1)+ + + − Talling 1988). Magnesium was the third most prevalent ion in tide pool brine, as expected (due to seawater entrapment), − − +, Resistant; +/ , resistant (weak growth); , susceptible although the Mg2+ to Na+ ratio was almost double than that reported for seawater, likely attributable to extensive evapora- and D(+)-cellobiose by strain K25M-2B. Strains K12-9A, K12- tion, which could lead to the precipitation of halite (NaCl), 25A and K25M-2B were able to effectively utilize the amino creating a magnesium-rich brine (Baati et al. 2011). acids L-arginine, L-asparagine and L-histidine; L-glutamine The prokaryotic microbiota isolated were moderate halo- and L-isoleucine; L-glutamine, respectively. A wide range of philes since they grew optimally within a salinity range of 3– organic acids were utilized by strain K12-25A. Only acetate 9 %, thereby differing from the archaeal isolates which were was effectively utilized by strain K12-9A. The herbicide 2,4- considered to be extreme halophiles owing to their optimum dichlorophenoxyacetate (2,4-D) could be effectively utilized growth at a salinity of 15–18 % (Andrei et al. 2012). H. by strain K12-25A as the sole carbon source, while phenolic alexandrinus isolates had an absolute requirement for seawa- compounds could not be utilized by the halophilic isolates ter. In addition, the halophilic bacteria isolated from the satu- examined. rated brine of the tide pool required seawater or sea salt, and The bacterial isolates tested showed distinct antibiotic patterns no growth was observed in the presence of NaCl only. These (Table 6). However, they were susceptible to chloramphenicol, findings reveal the marine origin of the isolates dominating

Fig. 2 Dendrogram of 36 morphological and physiological character- was created based on the absence or presence of a characteristic, istics tested for the halophilic representative isolates obtained from the respectively, while missing data were denoted. Dissimilarities were tide pool brine and the type strains of Aquisaliminas asiatica and calculated with the Simint option of NTSYSpc ver. 2.2 computer Haloferax alexandrinus. The restricted number of phenotypic charac- program by employing the average taxonomic distance-DIST coeffi- teristics reported for the type strains of the species Rhodovibrio cient. The Njoin option of the package was employed using the sodomensis, Pseudomonas halophila and Halovibrio denitrificans did unweighted pair group method with arithmetric average for clustering not permit their inclusion in the numerical analysis. A binary 0/1 matrix Ann Microbiol (2014) 64:599–609 607

Table 7 Prokaryotes identified in the tide pool brine: ecological niches of their closest phylogenetic relatives

OTU No. of isolates Closest phylogenetic relative Ecological niche Similarity (%) References

17 Rhodovibrio sp. 2 Mb1 Maras salterns 99.0 Maturrano et al. 2006 Rhodovibrio sodomensis DSM 9895T Dead Sea 94.7 Mack et al. 1993 Rhodovibrio salinarum NCIMB 2243T Portuguese saltern 93.7 Nissen and Dundas 1984 2 4 clone LL45B evaporitic crust, Lindsey Lake 99.3 Sahl et al. 2008 Aquisalimonas asiatica CG12T alkaline saline lake, Mongolia 96.5 Márquez et al. 2007 3 13 clone SN 144 pristine soil, Jidong Oilfield 99.2 Liu et al. 2009 strain GSP65 Great Salt Plains of Oklahoma 99.1 AY553126 Pseudomonas halophila DSM 3050T Great Salt Lake, U.S.A. 98.9 Sorokin et al. 2006 Halovibrio denitrificans HGD 3T hypersaline lake, Mongolia 98.7 Sorokin et al. 2006 Halospina denitrificans HGD 1-3T hypersaline lake, Siberia 97.6 Sorokin et al. 2006 41 Bacillus cereus HVR22 Not reported 99.9 JQ739719 Bacillus cereus GUFBSS253-84 Northern Arabian Sea 99.9 JN315893 53 Staphylococcus hominis DM 122T Human pathogen 99.7 Kloos and Schleifer 1975 62 Haloferax sp. FB247_14 Messolonghi solar saltern 99.7 Tsiamis et al. 2008 Haloferax alexandrinus JCM 10717T Alexandria solar saltern 99.7 Asker and Ohta 2002

the tide pool brine examined since seawater components were were distinct from Rhodovibrio spp. in terms of cell shape, essential for growth. In halophiles, the absolute requirement colony color and oxygen requirement for growth (Mack et al. for seawater has been rarely reported, with only Yanase et al. 1993). However, the small number of phenotypic character- (1992) reported the isolation of a seawater-depended bacteri- istics reported for the type species of the above genera did not um from a submarine brine. allow the comparison of their physiological profiles with those Hyperhalophilic archaea are usually favored under of members of OTUs#1 and 3. On the other hand, the detailed hypersaline conditions (Oren 2002). However, in our satu- descriptions of Aquisalimonas asiatica and Haloferax alexan- rated brine from tide pools, moderate halophilic bacteria drinus permitted the statistical evaluation of the morphological rather than haloarchaea dominated the hypersaline environ- and physiological characteristics of members of OTUs#2 and 6, ment (Fig. 1), possibly due to the fluctuating salinity of the respectively. Numerical analysis of 36 morphological and phys- tide pool, as moderate salt concentrations can do not favor iological characteristics commonly reported for both OTUs#2 archaeal microbiota. The dominance of extremely halophilic and 6 isolates and their type species revealed the distinct phe- bacteria that can compete haloarchaea at conditions of salt notypes of the four representative halophiles examined (Fig. 2). saturation has rarely been reported (Ventosa et al. 1998; In brief, members of OTU#2 differed from A. asiatica in pH Sorokin et al. 2006). range, oxygen requirement and lower temperature limit for Isolates K25M-1A and K25M-2B were the only growth, motility, nitrate reduction, Tween 80 hydrolysis and haloarchaeal strains obtained from the tide pool brine which utilization of L-asparagine, D(+)-galactose, L-histidine, lactose, required the presence of seawater for growth in the media D(−)-manitol, myo-inositol, D(+)-raffinose, L-serine, D-sorbitol used. Their closest relatives were Haloferax alexandrinus and D(+)-trehalose (Márquez et al. 2007). OTU#6 isolates dif- strains isolated from Messolonghi (West Greece) and Alexan- fered from H. alexandrinus in gelatin liquefaction, Tween 80 dria (North Egypt) solar salterns (Tsiamis et al. 2008; Asker hydrolysis and utilization of D(+)-galactose, lactose, D(+)-man- and Ohta 2002). Interestingly, the H. alexandrinus strains nose and succinate (Asker and Ohta 2002). from these two previous studies are both inhabitants of the All of the halophilic prokaryotes isolated from the tide pool Eastern Mediterranean basin. Moreover, the haloarchaeal brine showed a late appearance on solid media under optimum strains which were isolated from the tide pool showed a growth conditions. Moreover, the majority of organic com- similar antibiotic pattern to H. alexandrinus and its closely pounds tested were marginally utilized by the halophilic iso- related species (Asker and Ohta 2002), indicating that this lates. Taken together, these findings suggest a slow-growing antibiotic profile is common within the members of this halophilic population which was established owing to the taxon. limitation of nutrients since the tidal pool was covered by the OTU#1 isolates differed from Halovibrio denitrificans in salt crust. A plethora of microorganisms can cope with persis- their salinity range for growth (Sorokin et al. 2006), nitrate tent starvation in saline environments, providing evidence for reduction and utilization of sugars, while OTU#3 isolates mechanisms supporting long-term survival of halophiles 608 Ann Microbiol (2014) 64:599–609

(Kunte et al. 2002). Interestingly, Rhodovibrio spp. from Baati H, Jarboui R, Gharsallah N, Sghir A, Ammar E (2011) Molecular hypersaline environments have been reported to grow slowly community analysis of magnesium-rich bittern brine recovered from a Tunisian solar saltern. Can J Microbiol 57:975–981 under optimum culture conditions (Mack et al. 1993). Bodaker I, Sharon I, Suzuki MT, Feingersch R, Shmoish M, Andreishcheva Regarding the ecological niches, the closest relatives of the E, Sogin ML, Rosenberg M, Maguire ME, Belkin S, Oren A (2010) halophilic isolates from the tide pool brine were microorgan- Comparative community genomics in the Dead Sea: an increasingly – isms obtained from hypersaline environments (Table 7). Our extreme environment. 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