Influence of NaCl concentration on microbiologically influenced corrosion of carbon steel by halophilic archaeon Natronorubrum tibetense.

The influence of NaCl concentration on microbiologically influenced corrosion (MIC) of Q235 carbon steel by the halophilic archaeon Natronorubrum tibetense was investigated by immersion tests and electrochemical measurements. An increase in NaCl concentration from 0 g/mL to 0.1 g/mL promoted the anodic dissolution of carbon steel and accelerated its corrosion, but MIC did not occur. A further increase in NaCl concentration to 0.2 g/mL led to MIC in inoculated medium, and the occurrence of the MIC resulted in further aggravation of carbon steel corrosion. Once the NaCl concentration reached 0.3 g/mL, the high concentration of chloride ions greatly interfered with the adsorption of dissolved oxygen and the attachment of N. tibetense cells to the surface of carbon steel, thus reducing the corrosion rate of carbon steel and inhibiting the MIC.

Functional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP).

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.

Efficient non-cytotoxic fluorescent staining of halophiles.

Research on halophilic microorganisms is important due to their relation to fundamental questions of survival of living organisms in a hostile environment. Here we introduce a novel method to stain halophiles with MitoTracker fluorescent dyes in their growth medium. The method is based on membrane-potential sensitive dyes, which were originally used to label mitochondria in eukaryotic cells. We demonstrate that these fluorescent dyes provide high staining efficiency and are beneficial for multi-staining purposes due to the spectral range covered (from orange to deep red). In contrast with other fluorescent dyes used so far, MitoTracker does not affect growth rate, and remains in cells after several washing steps and several generations in cell culture. The suggested dyes were tested on three archaeal (Hbt. salinarum, Haloferax sp., Halorubrum sp.) and two bacterial (Salicola sp., Halomonas sp.) strains of halophilic microorganisms. The new staining approach provides new insights into biology of Hbt. salinarum. We demonstrated the interconversion of rod-shaped cells of Hbt. salinarium to spheroplasts and submicron-sized spheres, as well as the cytoplasmic integrity of giant rod Hbt. salinarum species. By expanding the variety of tools available for halophile detection, MitoTracker dyes overcome long-standing limitations in fluorescence microscopy studies of halophiles.

Diversity of the cell-wall associated genomic island of the archaeon Haloquadratum walsbyi.

BACKGROUND: Haloquadratum walsbyi represents up to 80% of cells in NaCl-saturated brines worldwide, but is notoriously difficult to maintain under laboratory conditions. In order to establish the extent of genetic diversity in a natural population of this microbe, we screened a H. walsbyi enriched metagenomic fosmid library and recovered seven novel version of its cell-wall associated genomic island. The fosmid inserts were sequenced and analysed. RESULTS: The novel cell-wall associated islands delineated two major clades within H. walsbyi. The islands predominantly contained genes putatively involved in biosynthesis of surface layer, genes encoding cell surface glycoproteins and genes involved in envelope formation. We further found that these genes are maintained in the population and that the diversity of this region arises through homologous recombination but also through the action of mobile genetic elements, including viruses. CONCLUSIONS: The population of H. walsbyi in the studied saltern brine is composed of numerous clonal lineages that differ in surface structures including the cell wall. This type of variation probably reflects a number of mechanisms that minimize the infection rate of predating viruses.

Halorussus ruber sp. nov., isolated from an inland salt lake of China.

Halophilic archaeal strain YC25(T) was isolated from Yuncheng salt lake in Shanxi, China. Cells of strain YC25(T) were observed to be pleomorphic rods, stained Gram-negative, and formed red-pigmented colonies on solid media. Strain YC25(T) was found to be able to grow at 25-50 °C (optimum 37 °C), at 1.4-4.8 M NaCl (optimum 1.7 M), at 0-1.0 M MgCl2 (optimum 0.01 M), and at pH 5.5-9.0 (optimum pH 6.5). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was found to be 8 % (w/v). The major polar lipids of the strain were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), sulfated mannosyl glucosyl diether (S-DGD-1), galactosyl mannosyl glucosyl diether (TGD-1), mannosyl glucosyl diether (DGD-1), and an unknown diglycosyl diether (DGD-2) chromatographically identical to those of Halorussus rarus CGMCC 1.10122(T). The 16S rRNA gene and rpoB' gene of strain YC25(T) were phylogenetically related to the corresponding genes of Halorussus rarus CGMCC 1.10122(T) (94.3-95.4 and 91.5 % nucleotide identity, respectively). The DNA G+C content of strain YC25(T) was determined to be 63.3 mol%. The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain YC25(T) (=CGMCC 1.12122(T) = JCM 18363(T)) represents a new species of Halorussus, for which the name Halorussus ruber sp. nov. is proposed.

Halohasta litorea gen. nov. sp. nov., and Halohasta litchfieldiae sp. nov., isolated from the Daliang aquaculture farm, China and from Deep Lake, Antarctica, respectively.

Two halophilic archaeal strains, R30(T) and tADL(T), were isolated from an aquaculture farm in Dailing, China, and from Deep Lake, Antarctica, respectively. Both have rod-shaped cells that lyse in distilled water, stain Gram-negative and form red-pigmented colonies. They are neutrophilic, require >120 g/l NaCl and 48-67 g/l MgCl(2) for growth but differ in their optimum growth temperatures (30 °C, tADL(T) vs. 40 °C, R30(T)). The major polar lipids were typical for members of the Archaea but also included a major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1). The 16S rRNA gene sequences of the two strains are 97.4 % identical, show most similarity to genes of the family Halobacteriaceae, and cluster together as a distinct clade in phylogenetic tree reconstructions. The rpoB' gene similarity between strains R30(T) and tADL(T) is 92.9 % and less to other halobacteria. Their DNA G + C contents are 62.4-62.9 mol % but DNA-DNA hybridization gives a relatedness of only 44 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe two new species of a novel genus, represented by strain R30(T) (= CGMCC 1.10593(T) = JCM 17270(T)) and strain tADL(T) (= JCM 15066(T) = DSMZ 22187(T)) for which we propose the names Halohasta litorea gen. nov., sp. nov. and Halohasta litchfieldiae sp. nov., respectively.

Archaebacterial lipid membranes as models to study the interaction of 10-N-nonyl acridine orange with phospholipids.

The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms.

Haloquadratum walsbyi gen. nov., sp. nov., the square haloarchaeon of Walsby, isolated from saltern crystallizers in Australia and Spain.

Strains C23T and HBSQ001 were isolated from solar salterns and are novel square-shaped, aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media containing 18 % salts at around neutral pH. Mg2+ is not required. The DNA G+C content of both isolates was 46.9 mol% and DNA-DNA cross-hybridization showed a relatedness of 80 %. Their 16S rRNA gene sequences showed only 2 nucleotide differences (99.9 % identity) and phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halogeometricum borinquense PR 3T (91.2 % sequence identity). The major polar glycolipid of both isolates was the sulfated diglycosyl diether lipid S-DGD-1. Electron cryomicrosopy of whole cells revealed similar internal structures, such as gas vesicles and polyhydroxyalkanoate granules, but the cell wall of isolate HBSQ001 displayed a more complex S-layer compared with that of isolate C23T. The phenotypic characterization and phylogenetic data support the placement of isolates C23T and HBSQ001 in a novel species in a new genus within the Halobacteriaceae, for which we propose the name Haloquadratum walsbyi gen. nov., sp. nov. The type strain of Haloquadratum walsbyi is C23T (=JCM 12705T=DSM 16854T).

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii.

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.

Halorubrum orientale sp. nov., a halophilic archaeon isolated from Lake Ejinor, Inner Mongolia, China.

A motile, pleomorphic, red-pigmented archaeon, strain EJ-52T, was isolated from water from Lake Ejinor, a saline lake in Inner Mongolia, China. Analysis of the almost-complete 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum, being most closely related to Halorubrum saccharovorum ATCC 29252T (96.1% sequence similarity), Halorubrum lacusprofundi JCM 8891T (95.9%), Halorubrum tibetense AS 1.3239T (95.2%), Halorubrum alcaliphilum AS 1.3528T (95.2%) and Halorubrum vacuolatum JCM 9060T (95.1%). The polar lipids of strain EJ-52T were C20C20 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol phosphate methyl ester and a sulfated diglycosyl diether. Strain EJ-52T requires at least 2.5 M NaCl for growth and grows optimally at 3.4 M NaCl. The strain grows at 25-50 degrees C, with optimal growth occurring at 35-45 degrees C. Mg2+ is not required. The DNA G+C content is 64.2 mol%. On the basis of the data obtained in this study, strain EJ52T represents a novel species, for which the name Halorubrum orientale sp. nov. is proposed. The type strain is EJ-52T (=CECT 7145T=JCM 13889T=CGMCC 1.6295T).

Natrinema ejinorense sp. nov., isolated from a saline lake in Inner Mongolia, China.

A Gram-negative, non-motile, neutrophilic, pleomorphic and extremely halophilic archaeon, strain EJ-57T, was isolated from saline Lake Ejinor in Inner Mongolia, China. Strain EJ-57T was able to grow at 25-50 degrees C, required at least 1.8 M NaCl for growth (optimum at 3.4 M NaCl) and grew over a pH range from 6.0 to 8.5 (optimum at pH 7.0). Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. Analysis of the almost complete 16S rRNA gene sequence indicated that the isolate represented a member of the genus Natrinema in the family Halobacteriaceae. Strain EJ-57T was most closely related to Natrinema versiforme JCM 10478T (96.2% sequence similarity), Natrinema pallidum NCIMB 777T (95.9% sequence similarity), Natrinema altunense JCM 12890T (95.8% sequence similarity) and Natrinema pellirubrum NCIMB 786T (95.5 % sequence similarity). However, DNA-DNA hybridization experiments showed that strain EJ-57T was not related to these species, with levels of DNA-DNA relatedness equal to or below 39%. The major polar lipids of the isolate were C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and the disulfated glycolipid S2-DGA-1. The G+C content of the genomic DNA was 64.7 mol%. Comparative analysis of phenotypic characteristics between strain EJ-57T and recognized Natrinema species supported the conclusion that EJ-57T represents a novel species within this genus, for which the name Natrinema ejinorense sp. nov. is proposed. The type strain is EJ-57T (=CECT 7144T=JCM 13890T=CGMCC 1.6202T).

Halostagnicola larsenii gen. nov., sp. nov., an extremely halophilic archaeon from a saline lake in Inner Mongolia, China.

Strain XH-48(T) was isolated from the sediment of Lake Xilinhot, a saline lake in Inner Mongolia (China). The organism is pleomorphic, neutrophilic and requires at least 2.5 M (15 %) NaCl, but not MgCl(2), for growth; it exhibits optimal growth at 3.4 M (20 %) NaCl. The G+C content of its DNA is 61 mol%. 16S rRNA gene sequence analysis revealed that strain XH-48(T) is a member of the family Halobacteriaceae, but there were low levels of similarity with other members of this family. The highest sequence similarity values (94.5 and 93.3 %) were obtained with the 16S rRNA genes of Natrialba aegyptiaca and Natrialba asiatica, respectively. Polar lipid analyses revealed that strain XH-48(T) contains phosphatidylglycerol and phosphatidylglyceromethylphosphate, derived from both C(20)C(20) and C(20)C(25) glycerol diethers, and two unidentified glycolipids. On the basis of the data obtained, the novel isolate cannot be classified within any recognized genus. Strain XH-48(T) should be placed within a novel genus and species within the family Halobacteriaceae, order Halobacteriales, for which the name Halostagnicola larsenii gen. nov., sp. nov. is proposed. The type strain of Halostagnicola larsenii is strain XH-48(T) (=DSM 17691(T)=CGMCC 1.5338(T)=JCM 13463(T)=CECT 7116(T)).

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer.

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.

Natrinema altunense sp. nov., an extremely halophilic archaeon isolated from a salt lake in Altun Mountain in Xinjiang, China.

A novel extremely halophilic strain, AJ2(T), was isolated from Ayakekum salt lake located in the Altun Mountain National Nature Reserve in Xinjiang, China. This isolate was neutrophilic, motile and grew in a wide range of MgCl(2) concentrations (0.005-1.0 M). The major polar lipids of the isolate were C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. A comprehensive 16S rRNA gene sequence analysis revealed that the isolate shared 96.6-97.7 % sequence identity with Natrinema species. The isolate, however, could be genetically differentiated from these species by DNA-DNA hybridization analysis and on the basis of its physiological properties. On the basis of the polyphasic evidence, strain AJ2(T) (= AS 1.3731(T) = JCM 12890(T)) represents the type strain of a novel species, for which the name Natrinema altunense sp. nov. is proposed.

Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon from Lake Zabuye in Tibet, China.

A novel haloalkaliphilic archaeon, strain 8W8T, was isolated from Lake Zabuye, on the Tibetan Plateau, China. On the basis of 16S rRNA gene sequence analysis, strain 8W8T was shown to belong to the genus Halorubrum and was related to Halorubrum vacuolatum (96.7% sequence similarity), Halorubrum saccharovorum (96.0%), Halorubrum lacusprofundi (95.4%) and Halorubrum sodomense (95.3%). The phylogenetic distance from any species within the other genera of Halobacteriales was lower than 90%. The major polar lipids of strain 8W8T were C20C20 and C20C25 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol phosphate methyl ester. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 8W8T from the eight Halorubrum species with validly published names. Therefore, strain 8W8T represents a novel species, for which the name Halorubrum tibetense sp. nov. is proposed, with the type strain 8W8T (=AS 1.3239T=JCM 11889T).

FtsZ ring: the eubacterial division apparatus conserved in archaebacteria.

FtsZ is a tubulin-like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.

Phototactic behaviour of the archaebacterial Natronobacterium pharaonis.

Natronobacterium pharaonis can react tactically to photo- and chemostimuli. It moves by rotation of a flagellar bundle which is monopolarly inserted. Under sufficient oxygen supply the photophobic response of N. pharaonis has been measured. The resulting action spectrum matches the absorption spectrum of the purified retinylidene protein psR-II. Retical synthesis could be inhibited by nicotine. Cells grown in the presence of nicotine show a strongly reduced photoresponse, which could be restored by addition of retinal. These data identify psR-II as the receptor for negative phototaxis.

Ch2, a novel halophilic archaeon from an Australian solar saltern.

A novel halophilic archaeon, strain Ch2, was isolated from a marine solar saltern in Geelong, Australia. The fact that this organism had a dam-methylated genome suggested that it is closely related to the taxon that includes Halobacterium saccharovorum, Halobacterium sodomense, and Halobacterium trapanicum. A sequence analysis of the 16S rRNA gene (Ch2 has three copies of this gene) showed that Ch2 is phylogenetically equidistant from the genera Haloarcula and Haloferax and closely related to H. saccharovorum. The susceptibility of both Ch2 and H. saccharovorum to the recently isolated halophage HF2 supported the hypothesis that these two organisms are closely related.

Survival strategies of microorganisms in extreme saline environments.

Halophilic representatives are found in all main lines of evolutionary descendence of microbes: in archaebacteria, Gram-negative and Gram-positive eubacteria, and also in eucaryotes. In principle all halophilic microorganisms have to adapt their surface and membrane structures to their highly ionic environments. Concerning their intracellular compartment two different strategies have been developed: Inorganic ions are largely excluded in some microorganisms while such ions are actively accumulated in others. In particular the second group of organisms has to adapt the whole metabolic machinery to the highly ionic conditions of several molar salts, whereas in the first group only the outer surface of the cytoplasmic membrane and the extracytoplasmic structures are in contact with high concentrations of inorgainic ions. In this latter group, a variety of organic solutes is accumulated in response to increases of the salinity of the environment.

Box-shaped halophilic bacteria.

Three morphologically similar strains of halophilic, box-shaped procaryotes have been isolated from brines collected in the Sinai, Baja California (Mexico), and southern California (United States). Although the isolates in their morphology resemble Walsby's square bacteria, which are a dominant morphological type in the Red Sea and Baja California brines, they are probably not identical to them. The cells show the general characteristics of extreme halophiles and archaebacteria. They contain pigments similar to bacteriorhodopsin which apparently mediate light-driven ion translocation and photophosphorylation.