.
UNIVERSITAT DE LES ILLES BALEARS
PROGRAMA DE DOCTORAT EN MICROBIOLOGIA AMBIENTAL I BIOTECNOLOGIA DEPARTAMENTO DE BIOLOGIA
Insights into the biogeography, survival, and dispersal mechanisms of the extremely halophilic bacterium Salinibacter ruber
JOCELYN BRITO ECHEVERRIA
TESI DOCTORAL
Directores de tesis
Ramon Rosselló Móra Aránzazu López López
Institut Mediterrani d’Estudis Avançats (CSIC-‐UIB) Departament de Ecologia i Recursos Marins
Grupo de Microbiología Marina
Palma de Mallorca Julio 2011
Dissertation presented by Jocelyn Brito Echeverría for the Ph.D. degree in the Programme of Environmental Microbiology and Biotechnology organized by Universitat de les Illes Balears (UIB)
Ramon Rosselló-‐Móra Aránzazu López López Director de tesi Directora de Tesi
Jocelyn Brito Echeverría Doctorando
Tesis doctoral presentada por Jocelyn Brito Echeverría para obtener el título de Doctor en el Programa de Microbiología Ambiental y Biotecnología de la Universidad de las Islas Baleares (UIB)
Acknowledgements
This work was supported by the projects CLG2006-‐12714-‐C02-‐01/ 02, CLG2009-‐ 12651-‐C02-‐01/ 02; and CE-‐CSD2007-‐0005 of the Spanish Ministry of Science and Innovation, which were also co-‐financed with FEDER support from the European Union. Jocelyn Brito-‐Echeverría was financed by the Government of the Balearic Islands through the Ministry of Economy and Finance.
Also I want to thank some people who made this work possible. First, to my thesis supervisors Dr.
Ramon Rosselló-‐Móra and Dr. Arantxa López-‐López for the opportunity provided and the intellectual, personal and technical help received during the consecution of this work.
Second, to the members of the Marine Microbiology Group of IMEDEA for all the great experiences we lived.
Third, to Prof. Philippe Schmitt-‐Kopplin and Dr. Marianna Lucio from Helmholtz Zentrum Müenchen for their technical and intellectual support and for all the hours spent on my training.
Finally, to Prof. Josefa Antón and Dr. Arantxa Peña of the Universidad de Alicante for their precious advices and technical support.
And lastly, but not least, to my lovely husband Dr Echeveste for his infinite patience, wisdom and love.
A mis abuelos A mis niños Florencia y Leonardo
TABLE OF CONTENTS
ABSTRACT………..1
I. INTRODUCTION...7
1. Hypersaline environment………..…….9
1.1. Hypersaline environments: diversity and dispersal of microorganisms...10
2. Solar salterns, a thalassohaline environment……….………....….12
2.1. Microbiota of solar salterns...13
2.2. Extremely halophilic bacteria inhabiting solar salterns………..………....14
3. The extremely halophilic bacterium Salinibacter ruber ……….………15
3.1. Salinibacter characteristics of the genus………….………..16
3.2. Phylogeny………..18
3.3. Abundance and distribution..………...19
3.4. The genome of Salinibacter ruber DSM 13855T...………..21
3.5. Genomic comparisons among Salinibacter strains………..……….……….23
4. Biogeography………..……….24
4.1. Biogeographical patterns of S. ruber……….25
5. Genomics and metabolomics of cultured organisms……….………26
5.1. Genotypic characterization of prokaryotes: Multilocus Sequence Analysis (MLSA)...27
5.2. Phenotypic characterization of prokaryotes: Metabolomics ………29
II. AIMS AND SCOPES OF THESIS PROJECT………33
III. MATERIALS AND METHODS……….……….37
1. Samples, strains and cultures………...………...39
1.1. S. ruber strain collection and culture conditions………..………...39
1.2. Isolation of prokaryotes from nostrils of Calonectris diomedea ………..……….40
1.3. Reference strains………..………...40
1.4. Growth curves……….40
1.5. Stress conditions………41
1.6. Culturability of cells……….………41
2. Nucleic acids extractions and quantifications……….………..41
2.1. DNA extraction of Halococcus sp. strains isolated from nostrils and reference strains………….……….41
2.2. DNA extraction of S. ruber strains for Polymerase Chain Reaction (PCR)………..…..43
2.3. Spectrophotometric determination of DNA……….………43
2.4. Agarose Gel Electrophoresis for DNA quantification……….………...43
3. DNA amplifications by PCR……….………43
3.1. 16S rRNA genes of Archaea and Bacteria domain……….………..43
3.2. Ribosomal internal transcribed spacer (ITS)……….44
3.3. Housekeeping or protein-‐coding genes………..44
3.4. Fingerprinting techniques……….………..44
3.4.1. Random Amplification of Polymorphic DNA (RAPD)……….……….44
3.4.2. Denaturing Gradient Gel Electrophoresis (DGGE) amplifications………...………..……..45
3.5. Purification of PCR products………..46
4. Whole-‐DNA analyses………51
4.1. Determination of G+C mole percentage……….51
4.2. DNA-‐DNA hybridizations (DDH)……….………..51
4.2.1. Labelling and separation of DNA………..……….………..……….51
4.2.2. Detection of eluted DNA………..………53
4.2.3. Treatment of hybridization data………..………...53
5. Sequencing of genes and phylogenetic reconstructions………..………..54
5.1. Sequencing………..……….54
5.2. Sequence analyses………..………...54
5.3. Phylogenetic reconstructions of 16S rRNA and concatenated genes………..………54
6. Proteins extraction and quantification………...………55
6.1. Outer membrane protein extraction………...……….55
6.2. Inner membrane protein extraction ………...………..56
6.3. Proteins quantification………...………56
6.4. Matrix Assisted Laser Desorption/Ionization time of flight Mass Spectrometry (MALDI-‐ TOF MS).…57 6.4.1. Mass spectra analyzes………..………...58
7. Lipopolysaccharide (LPS) analysis………..58
7.1. LPS extraction……….……….58
7.2. Detection of LPS by silver staining………..………..59
8. Electrophoresis……….………..60
8.1. Agarose gel electrophoresis ……….……….60
8.2. Polyacrylamide gel electrophoresis (SDS-‐PAGE)……...60
8.3. Denaturing Gradient Gel Electrophoresis (DGGE)…..……….………...61
9. Physiological and biochemical tests……….………...62
10. Metabolomics……….……….62
10.1. Metabolite extracts preparation………….……….………62
10.2. Solid-‐phase extraction……….63
10.3. ICR-‐FT/MS procedure……….……….63
10.4. Statistical analysis...64
10.5. Metabolites identification...65
11. Microscopy techniques...65
11.1. Sample fixation...……….65
11.2. -‐4'-‐6-‐diamidino-‐2-‐phenylindole stain (DAPI)………...66
11.3. Fluorescence in situ hybridization (FISH)……….……….……….66
IV. RESULTS AND DISCUSSION………..67
CHAPTER 1: Intraspecific diversity and biogeography of S.ruber strains 1.1. Background……….……….69
1.2. Multilocus sequence analysis (MLSA)………..……….69
1.2.1. Amplification of protein-‐coding genes………...69
1.2.2. Sequencing of protein-‐coding genes…….……….……….70
1.2.3. Phylogenetic reconstruccions ……….……….72
1.3. Metabolomic comparisions of strains………..……….74
1.3.1. Metabolome composition analysis………74
1.3.2. Statistical analysis and proposed models………..………..74
1.3.3. Discriminative analysis of Mediterranean strains……….………..79
1.4. Conclusions………..………81
CHAPTER 2: Survival and response to adverse conditions in S. ruber M8 and M31 strains 2.1. Background……….……….85
2.2. Growth curves………85
2.2.1. Growth kinetics……….……….85
2.2.2. Variations of cells numbers and FISH-‐detectable cells along the growth……….………...86
2.2.3. Culturability along the growth ……….89
2.3. Stress dynamics……….………89
2.3.1. Cells abundances and FISH-‐detectable cells numbers under stress conditions………...90
2.3.2. Culturability changes in stress conditions……….…...90
2.3.3. Cells numbers FISH counts and culturability changes during prolonged temperature
conditions...91
2.4. Metabolome comparisions………..……….93
2.4.1. Common metabolome composition in both strains along the different phases of the growth curve.……….……93
2.4.1.1.Statistical analysis and models……….………..93
2.4.1.2. Analysis of discriminative masses……….95
2.4.1.3. Identification of masses…….……….………97
2.4.2. Metabolome composition in M8 and M31 strains during stress conditions.………102
2.4.2.1. Statistical analysis and models of each strain……….…102
2.4.2.2.Common response discriminative analysis in the assayed stresses……….………...105
2.4.2.3. Common long term response discriminative analysis in temperature stress……….107
2.4.2.4. Identification of stress response masses………109
2.4.3. Identification of masses and metabolic implications………..………121
2.5. Membrane proteins analysis of strains during stress conditions……….122
2.5.1. SDS-‐PAGE of outer membrane proteins……….……….………..122
2.5.2. SDS-‐PAGE of inner membrane proteins………...124
2.6. Lipopolysaccharide (LPS) analysis of strains during stress conditions……….……….126
2.7. Matrix assisted laser desorption/ionization-‐time-‐of-‐flight mass spectrometry analysis of strains during stress ………..…....128
2.7.1. Statistical analysis………..………128
2.7.2. Clustering analysis……….130
2.8. Conclusions…….………..131
CHAPTER 3: Dispersal mechanisms of extremely halophilic microorganisms
3.1. Background………137
3.2. Initial molecular screening of samples………138
3.3. Isolation of prokaryotes from nostril of Calonectris diomedea individuals………..138
3.3.1. Sequencing of 16S rRNA genes and phylogenetic analysis...………....139
3.4. Taxomonic study of the isolates……….141
3.4.1. G+C content ………...141
3.4.2. DNA-‐DNA hybridization ……….141
3.4.3. Physiological and biochemical tests………142
3.5. Intraspecific diversity of new Halococcus strains………145
3.5.1. Random Amplification of Polymorphic DNA (RAPD)………145
3.5.2. Multilocus sequence analysis (MLSA) ………..147
3.5.3. Phylogenetics reconstructions………..150
3.6. Conclusions……….………..152
V. CONCLUSIONS……….157
REFERENCES………159
SUPLEMENTARY MATERIAL……….187
-‐1-‐
ABSTRACT
Salinibacter ruber is the first extremely halophilic member of the Bacteria domain with demonstrated ecological relevance in hypersaline environments. To date, members of S. ruber have been isolated from a large number of widely separated salterns, but all of them have shown an enormous degree of taxonomic resemblance. The extreme conditions and geographical isolation of its environments are optimal circumstances for observing allopatric speciation and to investigate possible mechanisms of dispersal of halophilic organisms. The screening of nostril salt-‐excreting glands from migratory seabirds revealed the occurrence of Halococcus strains which may constitute one the most rapid and effective mechanisms to blurry allopatric speciation of the members of this archaea and other halophilic species.
On the other hand, a metabolomic approach by using ultrahigh resolution mass spectrometry revealed that isolates from five different sites in the world, could be distinguished by differences in the membrane composition, and these differences could be correlated to their geographical isolation site distances. In addition, this approach allowed the study of the environmental changes response of the two closest relative strains M8 and M31. This response could be attributed to modifications in the cell membrane, specifically in the length or saturation ratio of fatty acids involved in the glycerolipid, and glycerophospholipid metabolisms. Thus, the pathways related to the synthesis and metabolism of cell envelope compounds in the extremely halophilic bacteria Salinibacter ruber, could be the first line of defense guarding the organism from adverse environmental conditions.
-‐3-‐
RESUMEN
Salinibacter ruber es el primer miembro reconocido del dominio Bacteria con relevancia ecológica en ambientes hipersalinos. Hasta la fecha, S. ruber ha sido aislado en salinas geográficamente muy distantes y, sin embargo, desde un punto de vista taxonómico constituyen un grupo altamente homogéneo. Tanto las condiciones extremas, como el aislamiento geográfico de los ambientes en los que se han aislado, son óptimas circunstancias para observar especiación alopátrica e investigar posibles mecanismos de dispersión en organismos halófilos extremos. La detección cepas de Halococcus en las glándulas secretoras de sal, en narinas de aves marinas migratorias, reveló que éstas podrían constituir uno de los mecanismos más rápidos y eficientes para la posible especiación alopátrica de arqueas y otros procariotas halófilos extremos. Por otro parte, la aproximación metabolómica, mediante el uso de espectrometría de masas ultra alta resolución, reveló que aislados provenientes de cinco lugares geográficamente distantes, pueden ser distinguidos por diferencias de sus componentes de membrana, las cuales se correlacionan con la distancia geográfica de los aislados. Además, esta aproximación permitió estudiar la respuesta de dos de las cepas más cercanas, M8 y M31, frente a cambios en las condiciones ambientales. De esta forma, la respuesta pudo atribuirse a modificaciones de la membrana celular, concretamente en la longitud o grado de saturación de los ácidos grasos que participan en el metabolismo de glicerolípidos y glicerofosfolípidos. Por lo tanto, las vías relacionadas con la síntesis y metabolismo de compuestos que constituyen la envoltura celular de la bacteria halófila extrema Salinibacter ruber, parecen ser la primera línea de defensa que protege al organismo frente a las condiciones adversas de su medio circundante.
-‐5-‐
RESUM
Salinibacter ruber és el primer membre reconegut del domini Bacteri amb rellevància ecològica en ambients hipersalins. Fins ara, S. ruber ha estat aïllat en salines geogràficament molt distants però, les quals des d'un punt de vista taxonòmic constitueixen un grup homogeni. Tant les condicions extremes, com la distància geogràfica dels seus aïllats, són òptimes circumstàncies per observar especiació alopàtrica i investigar possibles mecanismes de dispersió en organismes halòfils extrems. La detecció de soques de Halococcus en les glàndules secretores de sal en narius d'aus marines migratòries, va revelar que aquestes podrien constituir un dels mecanismes més ràpids i eficients per a la especiació alopàtrica d'arqueges i altres halòfils extrems. D'altra banda, l'aproximació metabolòmica, mitjançant l'ús d'espectrometria de masses d’ultra alta resolució, va revelar que aïllats provinents de cinc llocs geogràficament diferents, poden ser distingits per diferències en els seus components de membrana, les quals es correlacionen amb la distància geogràfica dels aïllats. A més, aquesta aproximació va permetre estudiar la resposta de dues de les soques més properes, M8 i M31, davant els canvis del medi.
D'aquesta manera, la resposta es va poder atribuir a modificacions de la membrana cel·∙lular, concretament en la longitud o grau de saturació dels àcids grassos que participen en el metabolisme de glicerolípids i glicerofosfolípids. Per tant, les vies relacionades amb la síntesi i metabolisme de compostos que constitueixen l'embolcall cel·∙lular del bacteri halòfil extrem Salinibacter ruber, podrien ser la primera línia de defensa que protegeix l'organisme enfront de les condicions adverses del medi que l’envolta.
I. INTRODUCTION
Introduction
-‐9-‐
1. Hypersaline environments
The oceans are the largest bodies of saline water on the Earth, with average salinities ranging from 32 to 35 g L-‐1(Kerkar, 2004). Hypersaline environments, with higher salinities than those found in seawater, are generally originated as a result of evaporation of seawater (Kerkar, 2004). In this kind of environments, also known as thalassohaline, the salt composition is similar to seawater, where the Na+ and Cl-‐ are the dominating ions, followed by SO42-‐, and the pH is near neutral to slightly alkaline (Oren, 2002c). During the seawater evaporation, some changes occur in the ionic composition due to the precipitation of minerals as CaCO3 (as calcite or aragonite) and gypsum (CaSO4*2H2O), and other minerals that exceed their solubility. Salt lakes of marine origin, as Great Salt Lake in Utah (41º O’N-‐112º O’E) and the multipond solar salterns located mainly in tropical and subtropical areas worldwide, are examples of this kind of environments.
Moreover, there are also hypersaline environments whose composition differs from seawater, which are called athalossohaline environments. The dominating ions in these ones are commonly K+ and Mg2+, while Na+ and SO42-‐ remains at low concentrations (Oren, 2002a). A well known example is the Dead Sea, a slightly acidic lake (pH around 6.0), where the concentration of divalent cations, such as Mg2+ and Ca2+, exceeds that of monovalent cations, such as K+ and Na+ (Oren, 2002c). Chloride and bromide are the dominant anions, with sulphate concentrations being very low (Oren, 2002c). There are other hypersaline athalassohaline lakes worldwide in which the ionic composition widely varies (Javor, 1989;
Rodríguez-‐Valera, 1988).
Depending on the geographic area, the chemical composition of hypersaline environments may be affected by several factors, as the decrease of oxygen when increasing salinity or high and low temperatures (Rodríguez-‐Valera, 1988; Ventosa, 2006). In addition, factors such as the atmospheric pressure, nutrient availability, solar radiation and/or the presence of trace metals and other toxic compounds, affect, not only the chemical composition, but also the biological processes that occur in these environments (Rodríguez-‐Valera, 1988). Microorganisms living in hypersaline environments can be classified through their salt dependence and salt tolerance, which are the terms generally used in microbial taxonomy to describe the phenotypic characteristics of microorganisms inhabiting this kind of environments (Oren, 2008). The most widely used definitions were formulated thirty years ago by Kushner (1978), who distinguished different categories: extreme halophiles, whose best growth occurs in media containing 2.5-‐5.2 M salt; borderline extreme halophiles, whose best growth occurs in media containing 1.5-‐4.0 M salt; moderate halophiles, whose best growth occurs in media containing 0.5-‐2.5 M salt; and halotolerant microorganisms, which do not show an absolute requirement of salt for growth
Introduction
-‐10-‐
but they often grow well up to very high salt concentrations (considered extremely halotolerant if the growth range extends above 2.5 M salt) (Kushner, 1978). Currently, microorganisms that grow optimally at salt concentrations of 50 g L-‐1 (0.8 M) or higher, and tolerate 100 g L-‐1 (1.7 M) at least, are considered halophilic microorganisms (Oren, 2008).
1.1.Hypersaline environments: diversity and dispersal of microorganims
Life exists over the whole range of salt concentrations encountered in natural habitats: from freshwater environments to hypersaline lakes such as the Dead Sea (31º20’N 35º30’E, Israel, Jordan and Palestine), saltern crystallizer ponds, and other habitats saturated in sodium chloride. The microbial community diversity adapted to these prevailing conditions depends on the properties of the saline and hypersaline habitats on Earth. These extreme environments periodically support dense microbial blooms (Oren, 1988), as observed in saltern ponds that often display a bright red coloration due to the large number of pigmented microorganisms they harbour (Oren & Rodriguez-‐Valera, 2001).
Based on the small subunit rRNA sequences, halophiles are found in all the three domains of life:
Archaea, Bacteria and Eucarya (Figure 1). Within Archaea, specifically within the phylum Euryarchaeota, the most salt-‐requiring microorganisms are found in the order Halobacteriales, which contains a single family, the Halobacteriaceae. The most representative genera in the family are Halobacterium (Larsen, 1989), Haloarcula (Torreblanca et al., 1986) and Haloquadratum (Walsby, 1980), and require over 100-‐
150 g L-‐1 of salt to grow and stabilize. Besides, within the order Methanococci we also find halophilic or highly halotolerant species as Methanohalophilus, and Methanohalobium. Moreover, and to date, within the Crenarchaeota kingdom no halophiles have been yet identified (Oren, 2002a).In the bacterial domain, the halophily is widespread within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes (Oren, 2008). However, not many bacterial species had been considered as ecologically relevant in such environments. The most important cases of ecological relevance are the members of the genus Salinibacter (Antón et al., 2000) and Salicola (Maturrano et al., 2006b).
Finally, within Eucarya the most important representative member of the halophilic organisms is the unicellular green algae Dunaliella, which seems to be the main or sole primary producer in many hypersaline environments (Oren, 2005a). Other representative members of this domain are the brine shrimp Artemia salina (Linnaeus, 1758), the meristematic fungus Trimmatostroma salinum (Zalar P, 1999), the black yeast Hortaea werneckii (Gunde-‐Cimerman et al., 2000), and flagellates as Pleurostomum flabellatum (Cho, 2005) and Halocafetaria seosinensis (Park, 2006).
Introduction
-‐11-‐
Figure 1: Distribution of halophilic microorganisms within the tree of life based on the small subunit rRNA gene sequences. Figure indicates the groups of microorganisms containing halophilic representatives found in all three domains: Archaea, Bacteria, and Eukarya.
Most of the extreme halophiles exhibit a relatively high-‐salt requirement for their growth, both in cultured species (Oren, 1994b) or natural communities (Antón et al., 2000), being growth absent below 10–15% salt. However, other haloarchaea may survive as viable cells in low-‐salinity environments close to seawater salt concentrations (Purdy et al., 2004). Despite the distantly, scattered distribution of hypersaline environments, in many cases the microbial composition is highly similar (Pedrós-‐Alió, 2005), suggesting possible mechanisms of dispersal of extremely halophilic microorganisms.
The first dispersal mechanism was proposed thirty years ago by Rodríguez-‐Valera and colleagues (1979), who argued that the halophilic organisms throughout the Earth may be a consequence of the circulation of the seawater streams around the world’s oceans. Through this mechanism, extreme halophiles may be transported in a dormant stage, which may be applicable for coastal high-‐salt environments (Rodríguez-‐Valera et al., 1979), but may not explain the occurrence of extreme halophiles in salterns at high elevations. For example, widely spread extreme halophiles have been detected in salterns at more than 3000 m above the sea level in the Andean cordillera (Maturrano et al., 2006a), probably through organisms that may have survived trapped in ancient evaporitic formations (Stan-‐Lotter et al., 2002).
Introduction
-‐12-‐
However, there are other mechanisms, such as aerial transport that may act as plausible dispersal systems too (Kellogg & Griffin, 2006). For example, natural and artificial hypersaline environments are frequented by migratory birds that feed from Artemia spp. thriving in their brines. Microorganisms may be trapped in their feathers or extremities and be dispersed through similar environments, as it has been hypothesized for cladocerans and bryozoans (Figuerola et al., 2005), constituting one of the most rapid and effective mechanism to allopatric speciation for the halophilic organisms.
2.Solar salterns: a thalassohaline environment
Coastal solar saltern evaporation ponds are thalassohaline, semi-‐artificial hypersaline environments, in which the seawater is evaporated for the production of salt (Oren, 2009). These systems are found worldwide in dry tropical and subtropical climates (Oren, 2009), being habitually subjected to high solar radiation, wide temperature fluctuations and low oxygen concentration due to the saturation of salts (Figure 2). Seawater, pumped to the first set of ponds, is evaporated until certain salinity is reached. The water is then transferred to the next series of ponds, in where the salinity increases in each stage. The process results in a discontinuous salinity gradient, in which the salt concentration in each pond is kept approximately constant over the time. In an early stage, when the salinity reaches twice or three times that of seawater, CaCO3 precipitates in the form of aragonite and/or calcite. When the salt concentration has reached four times the seawater concentration, the solubility limit of CaSO4 is reached, deriving in a massive precipitation of gypsum (CaSO4*2 H2O) (Javor, 2002). Gypsum deposits are characteristically found on the bottom of the saltern ponds (Caumette P., 1994). NaCl crystals (halite) are formed when the total salt concentration reaches values above 300 g L-‐1. After most of the NaCl has precipitated to the bottom of the crystallizer ponds, the remaining concentrated brines (“the bitterns”) mainly contain Mg2+, K+, Cl-‐, and SO42-‐.
In this sense, Mg2+ is the third most abundant element dissolved in seawater and is ubiquitous in the Earth's crust, where it exists in association with a variety of anions (Hallsworth et al., 2007). Magnesium chloride is exceptionally soluble in water, achieving high concentrations (> 5M) in brines (Hallsworth et al., 2007). Finally, after collecting the sodium chloride, the brines are generally returned to the sea or further processed to harvest potash (KCl) and other salts (Oren, 2002a).
Many halophilic and halotolerant microorganisms in cultures have been isolated from these environments from both, water and sediments underlying the brines (Oren, 2005a).
Introduction
-‐13-‐
Each pond enables the growth of those microbial communities adapted to the specific salinity of their brines, providing an excellent model system for the study of the microbial biodiversity, community dynamics, and physiological adaptations at high salt concentrations (Oren, 2005a).
Figure 2: Solar salterns consist of a series of shallow ponds connected in a sequence of increasingly saline brines.
(A) The extreme halophiles that inhabit these environments grow best at the highest salinities (3.4–5 mol L-‐1 NaCl), forming dense blooms, and resulting in the red color of many salterns (Guixa-‐Boixareu, 1996). (B) Panoramic view of a Mediterranean solar saltern (Majorca, Spain) where the seawater pumped to the first set of ponds (arrow), evaporates until certain salinity is reached. The water is then transferred to the next series of ponds, where the salinity increases in each stage, resulting in a discontinuous salinity gradient, in which the salt concentration in each pond is kept approximately constant over the time. Crystallizers are the last ponds and have salinity above 30%.
2.1.Microbiota of solar salterns
The microbiota of saltern ponds has been deeply studied and reviewed by conventional and molecular techniques (Antón et al., 1999; Antón et al., 2000; Benlloch et al., 2001; Benlloch et al., 2002; Casamayor et al., 2002; Estrada et al., 2004; Litchfield & Gillevet, 2002; Maturrano et al., 2006a; Oren, 1994b; Oren
& Rodriguez-‐Valera, 2001; Oren, 2002a; Oren, 2002b; Rodríguez-‐Valera et al., 1999).
Studies along a gradient of salinities in the solar salterns of Santa Pola (Alicante, Spain), showed a consistent decreasing trend in the number of microbial species with increasing salinity, and indicated a selective effect of extremely high salinities (Pedrós-‐Alió et al., 2000). Although the diversity of this microbiota is very low (Oren, 2002b), there is a considerable microdiversity, since the coexistence of several closely related clones of microorganisms has been detected in such environments (Benlloch et al., 2002).
Introduction
-‐14-‐
According to the salt concentration increases, the brines take on a red color due to the presence of a prokaryotic community dominated by dense population (70-‐95% of cell counts) of the halophilic square Archaea Haloquadratum walsbyi, with a lower proportion (from 5 to 30% of cell counts) of extremely halophilic members of the Bacteria such as Salinibacter ruber (Antón et al., 2000; Antón et al., 2008b). In the Eukaryotic domain, the unicellular green alga Dunaniella (rich in β-‐carotene) acts as the primary producer (Oren, 2005b). In addition, these environments show one of the highest number of virus–like particles (VLP) reported for aquatic systems (Guixa-‐Boixareu, 1996; Santos et al., 2007). The total prokaryotic cell counts detected in crystallizer ponds from different solar salterns ranges betwee 107-‐108 cells mL-‐1 (Oren, 2002c). However, as is commonly observed in most ecosystems, the number of colony forming units recovered from the ponds is generally several orders of magnitude lower than the number of microscopically recognizable cells. Therefore, those organisms isolated with the highest frequencies are not necessary those that are present in the highest numbers (Oren, 2002a).
Analyses of genomic libraries of these environments have shown that usually there is a co-‐existence of related clones of Bacteria (S. ruber) and Archaea (H. walsbyi), being fewer the cases of co-‐existence of other species of Halobacteriaceae, Proteobacteria, and other members of Bacteroidetes (Baati et al., 2008; Benlloch, 1995; Estrada et al., 2004). Nevertheless, in some cases the contribution of these groups may vary. For example, in the Adriatic and Mediterranean salterns a square-‐shaped Haloquadratum relative dominates the crystallizer community (Antón et al., 2000; Pasic et al., 2005), whereas the bacterial community of the Andean Maras salterns is dominated by Salicola marasensis, where S. ruber relatives remains undetectable (Maturrano et al., 2006a).
2.2. Extremely halophilic bacteria inhabiting solar salterns
For many years, it was assumed that hypersaline environments with NaCl concentrations close to saturation were dominated by halophilic members of the Archaea domain, while Bacteria were not relevant in this kind of environments (Guixa-‐Boixareu, 1996).
Thus, the prokaryotic diversity of the crystallizers of solar salterns was considered low and described as
“almost monospecific cultures of halophilic Archaea” (Guixa-‐Boixareu, 1996). Molecular diversity studies performed in Santa Pola salterns (Alicante) revealed that the bacterial population in crystallizers was different to that found in the low salinity ponds (Martínez-‐Murcia AJ., 1995), suggesting that crystallizers may be highly specialized niches for bacteria, which constituted only a small proportion of the total biomass of these ponds (Martínez-‐Murcia AJ., 1995).
Introduction
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The construction of 16S rRNA genes libraries showed that Bacteria were largely minoritary, with only a cluster of closely related sequences, all belonging to Alphaproteobacteria but distantly related to any known species (Benlloch, 1995).
In addition, the authors considered that due to the high concentration of salts (30.8%), bacteria found were probably metabolically inactive (Martínez-‐Murcia A.J., 1995). Thus, the idea that this kind of environment could only be inhabited by Archaea became a tenet, and cristallyzers were considered as monocultures of members of that domain (Antón et al., 1999). However, when fluorescence in situ hybridization (FISH) was used to quantify the diversity of crystallizers ponds of Santa Pola salterns (around 37% salinity), putatively active members of the domain Bacteria were found in high numbers (3x106 cells mL-‐1)(Antón et al., 1999). These cells, which accounted for 18% of the total cells counts, displayed high ribosome content, as indicated by the intensity and uniform FISH signals obtained with 16S rRNA-‐targeted oligonucleotide probe EUB 338 (Amann et al., 1990), a specific probe for members of the Bacteria Domain (Antón et al., 1999).
Later, the characterization of the bacterial community by rRNA approach showed a high abundance and growth of a new group of hitherto-‐uncultured bacteria from multipond solar salterns (Antón et al., 2000). These bacteria constituted 5-‐25% of the total prokaryotic community, and were represented by two main phylotypes, named as EHB-‐1 and EHB-‐2 (from Extremely Halophilic Bacterium). The sequence similarity among the two phylotypes was 97.6% and their study showed that EHB-‐1 was five times more abundant than EHB-‐2, and a new bacterial group was provisionally proposed, Candidatus “Salinibacter gen. nov.” (Antón et al., 2000).
3. The extremely halophilic bacterium Salinibacter ruber
An in-‐deep study of five extremely halophilic bacteria belonging to the EHB-‐1 phylotype that were isolated from different crystallizer ponds in Alicante (P13 and P18 isolates) and Majorca (M1, M8 and M31 isolates), Spain, suggested that they were sufficiently similar to each other, and sufficiently different from their closest relative (Rhodothermus marinus) to be considered a single species (Antón et al., 2002). The isolate M31T from Majorca salterns, chosen as type strain (DSM 15388T), was classified as Salinibacter ruber (Antón et al., 2002). The new species affiliated with the super phylum Bacteroidetes-‐
Chlorobi, with Rhodotermus marinus, a species comprising slightly halophilic-‐thermophilic bacteria, being the closest cultivated relative (Alfredsson, 1988; Sako et al., 1996).
Introduction
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Thus, S. ruber has been classified in the phylum BXX, Bacteroidetes, class II Sphingobacteria, Order I Sphingobacteriales, Family V Crenotrichaceae, as new genera (Genus IV) and species (Garrity, 2003).
3.1. Salinibacter characteristics of the genus
Microorganisms belonging to this genus are Gram-‐negative, aerobic, heterotrophic bacteria that grow optimally at total salt concentrations of 150-‐300 g L-‐1 and require at least 150 g salt L-‐1 for growth (Antón et al., 2002). Their optimal pH range for growing is 6.5-‐8.0, and no growth is observed below pH 6.0 or above pH 8.5. Their optimum temperature ranges from 37 to 47ºC, showing slow growth below 27ºC and no growth above 52ºC (Antón et al., 2002). The G+C content ranges from 66.3 to 67.7 mol % (HPLC) (Antón et al., 2002). Other major phenotypic characteristics of the genus have been summarized in table 1 and figure 3. In addition, colonies and liquid cultures of S. ruber have a bright red color due to acyl-‐
glycoside C40-‐carotenoid pigment (salinixantin), which has an absorption maximum at 478nm (Lutnaes, 2002) and shoulder at 506-‐510nm (Figure 3) (Antón et al., 2002).
Table 1: Description of phenotypic characteristics of S. ruber (adaptation from Antón et al, 2002).
Phenotypic characteristic Salinibacter ruber
Cells Motile, straight or slighty curved rods (2-‐6 x 0.4 µm)
Agar colonies Red, about 1 mm diameter, circular and convex with an entire margin.
Oxidase Positive
Catalase Positive
Nitrate Negative reduction
Production of acids from sugars Negative Starch and gelatine hydrolysis Positive
Tween hydrolysis Tween 80 is negative; tween 20 is weakly or not at all.
Indol production form L-‐
tryptophan Negative
Antibiotics sensitivity Penicillin G, ampicilin, chloranphenicol, streptomycin, novobiocin, rifampicin and ciprofloxacin
Antibiotics insensitivity Kanamicin, bacitracin, tetracycline, colistin, anisomysin and aphadicolin
Introduction
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Due to the environmental conditions, Salinibacter enzymes are adapted to function in the presence of high salt concentrations (Oren & Mana, 2002). The amino acid composition of its bulk protein shows a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine (Oren & Mana, 2002).
Moreover, S. ruber contains extremely high concentrations of K+ ions in its cytoplasm, about 11.4 ± 1.1 µmol K+ (mg protein)-‐1 (Oren et al., 2002). These values are in the same range as those detected in halophilic Archaea, which use KCl osmotically to balance the high NaCl concentration in their surrounding medium and to adapt the entire intracellular enzymatic machinery to the presence of high salt concentrations (Balashov et al., 2005; Christian & Waltho, 1962). Table 2 shows some phenotypic characteristics that S. ruber shares with halophilic Archaea of Halobacteriaceae families.
Figure 3: Phenotypic characteristics of Salinibacter genus. (a) Epifluorescence micrographs from a brine sample incubated for 24 h with the radioactive amino acid mixture, where square and pleomorphic-‐shaped cells correspond to the archaeal population (blue stain) and rod-‐shaped cells correspond to Salinibacter ssp. populations (green stain with probe EUB338 fluosprime labeled) (b) Scanning Electronic Microscopy (SEM) shows the straight or slightly curved rods cells of Salinibacter ruber M31T (c) S ruber cells grown in liquid culture (d) Isolation plate which shows the red pigmentation of the colonies (a and b pictures adapted from Antón et al., 2002 and Rosselló-‐Móra et al., 2003).
Introduction
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Table 2: Comparison of the most important characteristics of Salinibacter spp. and Halobacteriaceae (adaptation from Oren, 2008)
Salinibacter Halobacteriaceae
Salt requirement >150 g l-‐1 >150 g l-‐1 (in most) Salt optimum 150-‐300 g l-‐1 200-‐250 g l-‐1 (in most)
%G+C 66.2 59-‐71 (46.9 en H. walsbyi)
Compatible solute KCl KCl
Enzymes Salt dependent and tolerant The most are salt dependent
Lipids bacterial archaeal
Carotenoid pigment C40-‐ salinixantin C50-‐bacterioruberin
Retinal pigment XR, HR, SR BR, HR, SR(not all)
*XR: xanthorhodopsin; HR: halorhodopsin; SR: sensory rhodopsin; BR: bacteriorhodopsin.
3.2. Phylogeny
Members of S. ruber have been isolated from a large number of widely remote salterns, but all of them have shown an enormous degree of taxonomic resemblance (Ludwig & Klenk, 2001). Phylogenetic reconstructions based on the 16S rRNA gene, and on the ITS (Internal Transcribed Spacer) between the 16S and 23S rRNA genes, affiliated S. ruber with the phylum Bacteroidetes, being its closest related cultured organism R. marinus (Antón et al., 2002). The clade comprising R. marinus and S. ruber appeared as a deep branch within the phylum, and placed close to the node of bifurcation of the superphylum that comprises Bacteroidetes and Chlorobi (Figure 4)(Ludwig & Klenk, 2001). Subsequently, the phylogenetic study based on protein alignments of 22 genes selected from the genome of M31 type strain, showed that reconstructions based on concatenating large numbers of protein-‐coding genes seem to produce topologies with similar resolution to that of the single 16S rRNA genes trees (Soria-‐
Carrasco et al., 2007).
Introduction
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Figure 4: Phylogenetic reconstruction of based on 16S rDNA sequences from M31T and M8 Salinibacter ruber strains. Reconstruction shows the S. ruber strains within of superphylum Bacteroidetes-‐Chlorobi. Numbers above the branches represent bootstrap support from 100 replicates. The scale bar represents 0.1% estimated sequence divergence
3.3. Abundance and distribution
Salinibacter representatives have been detected or isolated in environments all over the world using different techniques with different levels of sensitivity (Figure 5; Antón et al., 2008). In Europe, Salinibacter representatives have been found in crystallizer ponds salterns in mainland Spain (Alicante and Tarragona), Balearic (Majorca and Ibiza), and Canary Islands (Antón et al., 2008b). In Asia, close relatives have been found in hypersaline Tuz Lake in central Anatolia, Turkey (Mutlu et al., 2008) and in water samples from Eilat salterns, Israel (Elevi-‐Bardavid et al., 2007). In Africa, sequences related to S.
ruber were also retrieved from water and sediments of three different soda lakes in the Wadi An Natrum depression in Egypt, while in water samples from Shabka in Suez were only detected by FISH (Mesbah et al., 2007). Moreover, most of the sequences generated by clone libraries constructed from three ponds with different salt concentrations of Sfax salterns (Tunisian) were grouped within the class Bacteroidetes, specifically with Salinibacter (Baati et al., 2008). S. ruber related sequences have also been reported for other locations in America, such as the Great Salt Lake in Utah, in the athalassohaline Andean Lake Tebenquiche in Northern Chile, and salterns in Baja California, Mexico (Antón et al., 2008b).
Introduction
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Besides, in the Andean Maras salterns (Peru), S. ruber has been isolated from brine samples taken in different years, despite it was not detected by FISH or clone libraries analyses (Maturrano et al., 2006a).
As illustrated by all these examples, S. ruber and relatives have been detected in a wide variety of environments, being therefore Salinibacter spp. one of the most abundant and most likely ecologically relevant organisms in hypersaline environments (Pedrós-‐Alió, 2006). However, preliminary data with crystallizer ponds from solar salterns indicated that although the bacterium may experience changes in abundance along the year; these changes may not be very dramatic, at least under “normal”
environmental conditions (Antón et al., 2008). Thus, the abundance of Salinibacter spp. usually ranges from 2 to 30% of the total cells counts, although in some cases, like in Tuz Lake, this value may be clearly underestimated, since the FISH probes used did not target the whole assemblage of Salinibacter sequences (Antón et al., 2008). In Santa Pola salterns, Salinibacter spp. is detected only in ponds with salinities above 22.4%, increasing their number with salinity (Antón et al., 2000). However, a direct proof of their metabolic activity in the highest salinity ponds (37% total salts) has not yet been obtained (Antón et al., 2008).
Figure 5. Distribution of Salinibacter clones or isolates around the world, indicating the detection methods used.
For the locations where FISH data are available, the abundance of S. ruber is provided. CR: crystallizer (adapted from Antón et al, 2008).
