Paulina Corral

Novel ether lipid cardiolipins in archaeal membranes of extreme haloalkaliphiles

The lipidome of two extremely haloalkaliphilic archaea, Natronococcus occultus and Natronococcus amylolyticus, has been examined by means of combined thin-layer chromatography and MALDI-TOF/MS analyses. The detailed investigation of lipid profiles has confirmed the presence of i) ether lipid phosphatidylglycerol and phosphatidylglycerophosphate methyl ester as main lipid components, ii) both C(20) and C(25) isopranoid chains in the lipid core and yielded new findings on membrane lipids of these unusual organisms. Besides some novel minor or trace phospholipids and glycolipids, data indicate the presence of ether lipid cardiolipin variants constituted by different combinations of C(20) and C(25) isopranoid chains, never before described in archaea. The role of C(25) isopranoid chains in the adaptation to high pH gradients in the presence of very high salt concentrations is discussed.

Natronococcus roseus sp. nov., a haloalkaliphilic archaeon from a hypersaline lake

A novel halophilic archaeon, strain CG-1(T), belonging to the genus Natronococcus was isolated from sediment of the soda lake Chagannor in Inner Mongolia, China. The colonies of this strain were pink pigmented, the intensity of the colour decreased when the cells grew at salt saturation levels. The cells were non-motile cocci and strictly aerobic. Hypotonic treatment did not cause cell lysis, even in distilled water. Strain CG-1(T) grew at 15-30.0 % (w/v) NaCl and at 30-50 °C and pH 8.0-11.0, with optimal growth occurring at 25-30 % (w/v) NaCl, 37-45 °C and pH 9-9.5. MgCl(2) was not required for growth. Strain CG-1(T) was most closely related to the type strains of Natronococcus amylolyticus Ah-36(T), Natronococcus jeotgali B1(T) and Natronococcus occultus SP4(T), with which it shared 98.4 %, 96.2 and 95.7 % 16S rRNA gene sequence similarity, respectively. The polar lipids consisted of C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and minor phospholipid components. No glycolipids were detected. The DNA G+C content of strain CG-1(T) was 62.1 mol%. DNA-DNA hybridization with N. amylolyticus DSM 10524(T), phylogenetically the most closely related species, was 39 %; this value showed that strain CG-1(T) constituted a different genospecies. The comparison of 16S rRNA gene sequences, detailed phenotypic characterization, polar lipid profile and DNA-DNA hybridization studies revealed that strain CG-1(T) belongs to the genus Natronococcus and constitutes a novel species for which the name Natronococcus roseus sp. nov. is proposed. The type strain is CG-1(T) (=CECT 7984(T)=IBRC-M 10656(T)=JCM 17958(T)).

Halorubrum aquaticum sp. nov., an archaeon isolated from hypersaline lakes

Two halophilic archaea, strains EN-2(T) and SH-4, were isolated from the saline lakes Erliannor and Shangmatala, respectively, in Inner Mongolia, China. Cells were strictly aerobic, motile rods. Colonies were red. Strains EN-2(T) and SH-4 were able to grow at 25-50 °C (optimum 35-40 °C), with 2.5-5.0 M NaCl (optimum 3.4 M NaCl) and at pH 6.0-9.0 (optimum pH 7.5). MgCl(2) was not required for growth. Cells lysed in distilled water and the lowest NaCl concentration that prevented cell lysis was 12 % (w/v). On the basis of 16S rRNA gene sequence analysis, strains EN-2(T) and SH-4 were closely related to Halorubrum cibi B31(T) (97.9 and 98.0 % similarity, respectively), Hrr. tibetense 8W8(T) (97.3 and 97.7 %), Hrr. alkaliphilum DZ-1(T) (96.8 and 97.1 %), Hrr. luteum CGSA15(T) (96.8 and 97.0 %) and Hrr. lipolyticum 9-3(T) (96.8 and 97.0 %). DNA-DNA hybridization showed that strains EN-2(T) and SH-4 did not belong to the same species as any of these strains (≤ 45 % DNA-DNA relatedness) but that they are members of the same species (>70 % DNA-DNA relatedness). Polar lipid analysis revealed that strains EN-2(T) and SH-4 contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diethers and several unidentified glycolipids. The DNA G+C content of both isolates was 62.1 mol%. It was concluded that strains EN-2(T) and SH-4 represent a novel species of the genus Halorubrum, for which the name Halorubrum aquaticum sp. nov. is proposed. The type strain is EN-2(T) ( = CECT 7174(T)  = CGMCC 1.6377(T)  = JCM 14031(T)).

Halorubrum persicum sp. nov., an extremely halophilic archaeon isolated from sediment of a hypersaline lake

An extremely halophilic archaeon belonging to the genus Halorubrum, strain C49T, was isolated from sediment of the hypersaline lake Aran-Bidgol in Iran. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain C49T was closely related to Halorubrum saccharovorum JCM 8865T (99.5 %) and other species of the genus Halorubrum. Studies based on multilocus sequence analysis revealed that strain C49T is placed among the species of Halorubrum; the strain constituted a defined branch in comparison with the type strains of species of Halorubrum, while the 16S rRNA gene sequence divergence could not define the status of the newly isolated strain. For optimum growth, strain C49T required 20 % (w/v) salts at pH 7.0 and 37 °C under aerobic conditions. Mg2+ was not required. The cells were pleomorphic rods, motile and stained Gram-variable. Colonies of the strain were pink. Hypotonic treatment with <12 % NaCl provoked cell lysis. The polar lipid pattern of strain C49T consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester derived from both C20C20 and C20C25 archaeol, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The DNA G+C content was 64.2 mol%. DNA-DNA hybridization studies and average nucleotide identity confirmed that strain C49T constitutes a distinct genospecies. Data obtained in this study show that strain C49T represents a novel species, for which the name Halorubrum persicum sp. nov. is proposed. The type strain is C49T ( = IBRC-M 10232T = JCM 30541T).

Horizontal gene transfer, dispersal and haloarchaeal speciation

The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria.

Natronorubrum sediminis sp. nov., an archaeon isolated from a saline lake.

Two novel haloalkaliphilic archaea, strains CG-6T and CG-4, were isolated from sediment of the hypersaline Lake Chagannor in Inner Mongolia, China. Cells of the two strains were pleomorphic, non-motile and strictly aerobic. They required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. They grew at pH 8.0-11.0, with optimum growth at pH 9.0. Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. The two strains had similar polar lipid compositions, possessing C20C20 and C20C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Comparison of 16S rRNA gene sequences and morphological features placed them in the genus Natronorubrum. 16S rRNA gene sequence similarities to strains of recognized species of the genus Natronorubrum were 96.2-93.8%. Detailed phenotypic characterization and DNA-DNA hybridization studies revealed that the two strains belong to a novel species in the genus Natronorubrum, for which the name Natronorubrum sediminis sp. nov. is proposed; the type strain is CG-6T (=CECT 7487T =CGMCC 1.8981T =JCM 15982T).

Halostagnicola bangensis sp. nov., an alkaliphilic haloarchaeon from a soda lake.

An extremely haloalkaphilic archaeon, strain T26(T), belonging to the genus Halostagnicola, was isolated from sediment of the soda lake Bange in the region of Tibet, China. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain T26(T) was closely related to Halostagnicola alkaliphila 167-74(T) (98.4 %), Halostagnicola larsenii XH-48(T) (97.5 %) and Halostagnicola kamekurae 194-10(T) (96.8 %). Strain T26(T) grew optimally in media containing 25 % (w/v) salts, at pH 9.0 and 37 °C in aerobic conditions. Mg(2+) was not required for growth. The cells were motile, pleomorphic and Gram-stain-variable. Colonies of this strain were pink pigmented. Hypotonic treatment caused cell lysis. The polar lipids of the isolate consisted of C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phospholipids components. Glycolipids were not detected, in contrast to the two neutrophilic species of this genus. The genomic DNA G+C content of strain T26(T) was 60.1 mol% and DNA-DNA hybridization showed a relatedness of 19 and 17 % with Halostagnicola alkaliphila CECT 7631(T) and Halostagnicola larsenii CECT 7116(T), respectively. The comparison of 16S rRNA gene sequences, detailed phenotypic characterization, polar lipid profile and DNA-DNA hybridization studies revealed that strain T26(T) belongs to the genus Halostagnicola, and represents a novel species for which the name Halostagnicola bangensis sp. nov. is proposed. The type strain is T26(T) ( = CECT 8219(T) = IBRC-M 10759(T) = JCM 18750(T)).

Genotypic and Lipid Analyses of Strains From the Archaeal Genus Halorubrum Reveal Insights Into Their Taxonomy, Divergence, and Population Structure

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge.

Halovenus salina sp. nov., an extremely halophilic archaeon isolated from a saltern

An extremely halophilic archaeon was isolated from a water sample of Isla Bacuta saltern in Huelva, Spain. Strain ASP54(T) is a novel red-pigmented, motile, rod-shaped, Gram-stain-negative and strictly aerobic haloarchaeon. Strain ASP54(T) grew in media containing 15-30% (w/v) salts and optimally with 25% (w/v) salts. It grew between pH 5.0 and 9.0 (optimally at pH 7.5) and at 20-40 °C (optimally at 37 °C). Phylogenetic analysis based on multi-locus sequence analysis (MLSA) and the comparison of 16S rRNA gene sequences revealed that strain ASP54(T) is most closely related to the genus Halovenus. The closest relatives were Halovenus aranensis EB27(T) (92.1% 16S rRNA gene sequence similarity), Halorientalis regularis TNN28(T) (92.1%), and Halorientalis persicus D108(T) (92.0%). The polar lipid pattern of strain ASP54(T) consisted of biphosphatidylglycerol, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl glucosyl diether and a minor-phospholipid. The predominant respiratory quinone was menaquinone-8 (MK-8) (83%), and a minor amount of MK-8(VIII-H2) (17%) was also detected. The G+C content of the genomic DNA of this strain was 63.1 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data presented in this study, strain ASP54(T) represents a novel species of the genus Halovenus, for which the name Halovenus salina sp. nov. is proposed. The type strain is ASP54(T) ( = CEC(T) 8749(T) = IBRC-M 10946(T) = JCM 30072(T)).