Novel sulfonolipid in the extremely halophilic bacterium Salinibacter ruber

Salinibacter ruber is an extremely halophilic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria. Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of S. ruber shows a characteristic peak at m/z 660 as the most prominent peak in the high-mass range of the spectrum. A novel sulfonolipid, giving rise to the molecular ion [M-H]- of m/z 660, has been identified. The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-O-(13'-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus Cytophaga and closely related genera (W. Godchaux and E. R. Leadbetter, J. Bacteriol. 153:1238-1246, 1983) and of diatoms (R. Anderson, M. Kates, and B. E. Volcani, Biochim. Biophys. Acta 528:89-106, 1978). Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at m/z 660 the lipid signature of Salinibacter. This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of Salinibacter within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.     

Sugar metabolism in the extremely halophilic bacterium Salinibacter ruber

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by a small number of sugars (glucose, maltose, starch at 1 g l(-1)). Glucose consumption starts after other substrates have been depleted. Glucose metabolism proceeds via a constitutive, salt-inhibited hexokinase and a constitutive salt-dependent nicotinamide adenine dinucleotide phosphate (NADP)-linked glucose-6-phosphate dehydrogenase. Glucose dehydrogenase and fructose-1,6-bisphosphate aldolase activity could not be detected. It is therefore suggested that Salinibacter metabolizes glucose by the classic Entner-Doudoroff pathway and not by the Embden-Meyerhof glycolytic pathway or by the modified Entner-Doudoroff pathway present in halophilic Archaea of the family Halobacteriaceae, in which the phosphorylation step is postponed. However, activity of 2-keto-3-deoxy-6-phosphogluconate aldolase could not be detected in extracts of Salinibacter cells, whether or not grown in the presence of glucose.     

Intraspecific comparative analysis of the species Salinibacter ruber

Salinibacter ruber is the first extremely halophilic member of the Bacteria domain of proven environmental relevance in hypersaline brines at or approaching NaCl saturation, that has been brought to pure culture. A collection of 17 strains isolated from five different geographical locations (Mallorca, Alicante, Ebro Delta, Canary Islands, and Peruvian Andes) were studied following the currently accepted taxonomic approach. Additionally, random amplification of genomic DNA led to the phenetic analysis of the intraspecific diversity. Altogether the taxonomic study indicated that S. ruber remained highly homogeneous beyond any geographical barrier. However, genomic fingerprints indicated that populations from different isolation sites could still be discriminated.     

Glycerol metabolism in the extremely halophilic bacterium Salinibacter ruber

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by glycerol. In contrast to glucose consumption, which starts only after more easily degradable substrates present in yeast extract have been depleted, glycerol is consumed during the earliest growth phases. When U-(14)C-labeled glycerol was added to the culture, up to 25% of the radioactivity was incorporated by the cells. Glycerol kinase activity was detected only in cells grown in the presence of glycerol (up to 90 nmol mg protein(-1) min(-1)). This enzyme functioned over salt concentrations from 0.6 to 2.8 M KCl. No significant activity of NAD-dependent glycerol dehydrogenase was found. It is suggested that Salinibacter may use glycerol as one of its principal substrates in its habitat, the saltern crystallizer ponds.     

Photoactive yellow protein from the halophilic bacterium Salinibacter ruber

A gene for photoactive yellow protein (PYP) was identified from the genome sequence of the extremely halophilic aerobic bacterium Salinibacter ruber (Sr). The sequence is distantly related to the prototypic PYP from Halorhodospira halophila (Hh) (37% identity) and contains most of the amino acid residues identified as necessary for function. However, the Sr pyp gene is not flanked by its two biosynthetic genes as in other species. To determine as to whether the Sr pyp gene encodes a functional protein, we cloned and expressed it in Escherichia coli, along with the genes for chromophore biosynthesis from Rhodobacter capsulatus. The Sr PYP has a 31-residue N-terminal extension as compared to other PYPs that appears to be important for dimerization; however, truncation of these extra residues did not change the spectral and photokinetic properties. Sr PYP has an absorption maximum at 431 nm, which is at shorter wavelengths than the prototypical Hh PYP (at 446 nm). It is also photoactive, being reversibly bleached by either blue or white light. The kinetics of dark recovery is slower than any of the PYPs reported to date (4.27 x 10(-4) s(-1) at pH 7.5). Sr PYP appears to have a normal photocycle with the I1 and I2 intermediates. The presence of the I2' intermediate is also inferred on the basis of the effects of temperature and alchohol on recovery. Sr PYP has an intermediate spectral form in equilibrium with the 431 nm form, similar to R. capsulatus PYP and the Y42F mutant of Hh PYP. Increasing ionic strength stabilizes the 431 nm form at the expense of the intermediate spectral form, and the kinetics of recovery is accelerated 6.4-fold between 0 and 3.5 M salt. This is observed with ions from both the chaotropic and the kosmotropic series. Ionic strength also stabilizes PYP against thermal denaturation, as the melting temperature is increased from 74 degrees C in buffer alone to 92 degrees C in 2 M KCl. Sr accumulates KCl in the cytoplasm, like Halobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP is an example of a halophilic protein that requires KCl to electrostatically screen the excess negative charge and stabilize the tertiary structure.     

Spectral tuning in sensory rhodopsin I from Salinibacter ruber

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ～560 nm; SRII, ～500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.     

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.     

Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber

Since its discovery in 1998, representatives of the extremely halophilic bacterium Salinibacter ruber have been found in many hypersaline environments across the world, including coastal and solar salterns and solar lakes. Here, we review the available information about the distribution, abundance and diversity of this member of the Bacteroidetes.     

Insights into Head-Tailed Viruses Infecting Extremely Halophilic Archaea

Extremophilic archaea, both hyperthermophiles and halophiles, dominate in habitats where rather harsh conditions are encountered. Like all other organisms, archaeal cells are susceptible to viral infections, and to date, about 100 archaeal viruses have been described. Among them, there are extraordinary virion morphologies as well as the common head-tailed viruses. Although approximately half of the isolated archaeal viruses belong to the latter group, no three-dimensional virion structures of these head-tailed viruses are available. Thus, rigorous comparisons with bacteriophages are not yet warranted. In the present study, we determined the genome sequences of two of such viruses of halophiles and solved their capsid structures by cryo-electron microscopy and three-dimensional image reconstruction. We show that these viruses are inactivated, yet remain intact, at low salinity and that their infectivity is regained when high salinity is restored. This enabled us to determine their three-dimensional capsid structures at low salinity to a ∼10-Å resolution. The genetic and structural data showed that both viruses belong to the same T-number class, but one of them has enlarged its capsid to accommodate a larger genome than typically associated with a T=7 capsid by inserting an additional protein into the capsid lattice.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Delta9cis and C18:1 Delta11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium

The moderately halophilic bacterium Vibrio costicola accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na⁺ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.     

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.     

The groESL Operon of the Halophilic Lactic Acid Bacterium Tetragenococcus halophila

The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45°C), and 4-fold by high NaCl (3-4 M). The Bacillus subtilis σ^A-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.     

Role of Central Metabolism in the Osmoadaptation of the Halophilic Bacterium Chromohalobacter salexigens

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-¹³C]-, [2-¹³C]-, [6-¹³C]-, and [U-¹³C₆]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.     

Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp.

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.     

Repair of Oxidized Bases in the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans

Deinococcus radiodurans is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that D. radiodurans possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase–a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the Escherichia coli Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.     

Enzyme Characteristics of Extremely Halophilic Archaeal Community in Tuzkoy Salt Mine,Turkey

Tuzkoy is a large and unpolluted salt mine in Central Anatolia, Turkey. High quality salt in this mine may be supplied to the table salt market after a simple process instead of complicated refining. However, the extremely halophilic microbial content of the salt has not yet been investigated which may result in detrimental effects in industrial processes such the food and leather industries, if unprocessed salt is directly used. Therefore, to identify and characterize the microbial contaminants in Tuzkoy salt, chemical and microbial analyses were conducted on salt crystal samples collected from three different locations of the mine. Generally 10⁵–10⁶ colony forming units of extremely halophilic microorganisms were detected per gram of salt samples. Twelve colonies were selected randomly for further characterization. Phenotypic characterization, lipid analysis, antibiotic susceptibility tests and positive PCR amplification results with Archaea-specific primers confirmed that all strains were halophilic Archaea belong to family Halobacteriaceae. According to denaturing gradient gel electrophoresis (DGGE) results, 10 comparatively different strains were selected for DNA sequencing. DNA sequences and phylogenetic analyses revealed that the isolated strains were mainly species of the genera Halobacterium, Haloarcula, Natrinema and Halorubrum. In addition enzymatic activity tests were also conducted to evaluate the salt quality for industrial applications. Results of gelatinase, caseinase, amylase, cellulase and lipase activity tests revealed that the isolated strains produced hydrolytic enzymes, which could cause deterioration in salt-treated food and hide. It may be pointed out that cellulase activity in halophilic Archaea has not been reported previously. Moreover, β-galactosidase activity has been reported in some Haloferax and Halorubrum species but not in the genus Halobacterium. This revised version was published online in July 2006 with corrections to the Cover Date.