Search

The current study focuses on the tolerance of a strain of Halobacterium salinarum isolated from Sfax solar saltern (Tunisia) towards cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn), and copper (Cu) by using agar dilution methods in complex and minimal media. The results showed the least inhibitory metals based on Minimum Inhibitory Concentrations (MICs) were lead (MIC = 4.5 mM), cadmium (MIC = 4 mM), and nickel (MIC = 2.5 mM) in complex medium. The MICs of these metals were more inhibitory (MIC < 2 mM) in the other tested media. The archaeal strain revealed a high sensitivity for copper and zinc, with MICs below 0.5 mM for both metals. Growth kinetics in complex and minimal media showed the strain to be more sensitive to the metals in liquid media than in solid media. The growth kinetic assays indicated the presence of selected heavy metals resulted in a lower growth rate and lower total cell mass relative to the control. Despite that cadmium and lead are nonessential and have no nutrient value, they were the most tolerated metals by H. salinarum strain. In addition, pigment intensity in the strain was inhibited by the presence of the heavy metals relative to the control.

Although tractable model organisms are essential to characterize the molecular mechanisms of evolution and adaptation, the ecological relevance of their behavior is not always clear because certain traits are easily lost during long-term laboratory culturing. Here, we demonstrate that despite their long tenure in the laboratory, model organisms retain "ecological memory" of complex environmental changes. We have discovered that Halobacterium salinarum NRC-1, a halophilic archaeon that dominates microbial communities in a dynamically changing hypersaline environment, simultaneously optimizes fitness to total salinity, NaCl concentration, and the [K]/[Mg] ratio. Despite being maintained under controlled conditions over the last 50 years, peaks in the three-dimensional fitness landscape occur in salinity and ionic compositions that are not replicated in laboratory culturing but are routinely observed in the natural hypersaline environment of this organism. Intriguingly, adaptation to variations in ion composition was associated with differential regulation of anaerobic metabolism genes, suggesting an intertwined relationship between responses to oxygen and salinity. Our results suggest that the ecological memory of complex environmental variations is imprinted in the networks for coordinating multiple cellular processes. These coordination networks are also essential for dealing with changes in other physicochemically linked factors present during routine laboratory culturing and, hence, retained in model organisms.

Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms could have potentially flourished in these settings. If so, biomolecules found in microorganisms adapted to high salinity and basic pH environments on Earth may be reliable biomarkers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman (RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore carotenoid molecules in the cell membrane with respect to the various protein and lipid cellular components. RR spectra acquired from all halophilic archaea investigated contained major features at approximately 1000, 1152, and 1505 cm(-1). The bands at 1505 cm(-1) and 1152 cm(-1) are due to in-phase C=C (nu(1) ) and C-C stretching ( nu(2) ) vibrations of the polyene chain in carotenoids. Additionally, in-plane rocking modes of CH(3) groups attached to the polyene chain coupled with C-C bonds occur in the 1000 cm(-1) region. We also investigated the RR spectral differences between bacterioruberin and bacteriorhodopsin as another potential biomarker for hypersaline environments. By comparison, the RR spectrum acquired from bacteriorhodopsin is much more complex and contains modes that can be divided into four groups: the C=C stretches (1600-1500 cm(-1)), the CCH in-plane rocks (1400-1250 cm(-1)), the C-C stretches (1250-1100 cm(-1)), and the hydrogen out-of-plane wags (1000-700 cm(-1)). RR spectroscopy was shown to be a useful tool for the analysis and remote in situ detection of carotenoids from halophilic archaea without the need for large sample sizes and complicated extractions, which are required by analytical techniques such as high performance liquid chromatography and mass spectrometry.

Halite crystals were selected from a 186 m subsurface core taken from the Badwater salt pan, Death Valley, California to ascertain if halophilic Archaea and their associated 16S rDNA can survive over several tens of thousands of years. Using a combined microscope microdrill/micropipette system, fluids from brine inclusions were aseptically extracted from primary, hopper texture, halite crystals from 8 and 85 metres below the surface (mbls). U-Th disequilibrium dating indicates that these halite layers were deposited at 9,600 and 97,000 years before present (ybp) respectively. Extracted inclusions were used for polymerase chain reaction (PCR) analysis with haloarchaea-specific 16S rDNA primers or placed into haloarchaea culture medium. Enrichment cultures were obtained from 97 kyr halite crystal inclusion fluid and haloarchaea-containing prepared crystals (positive controls), whereas inclusions from crystals of 9.6 kyr halite and the haloarchaea-free halite crystals (negative controls) resulted in no growth. Phylogenetic analysis (16S rDNA) of the 97 kyr isolate, designated BBH 001, revealed a homology of 100% with Halobacterium salinarum. DNA-DNA hybridization experiments confirmed that BBH 001 was closely related to H. salinarum (81-75% hybridization) and its ascription to this haloarchaea species. The described method of retrieving particle-containing brine from fluid inclusions offers a robust approach for assessing the antiquity of microorganisms associated with evaporites.

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

A phylogenetic analysis of 69 halobacterial 16S rRNA gene sequences has been carried out, integrating data from new isolates, previously described halobacteria and cloned sequences from uncultivated halobacteria. Halobacterium halobium NCIMB 777, Halobacterium trapanicum NCIMB 784 and Halobacterium salinarium NCIMB 786, together with several other strains (strains T5.7, L11 and Halobacterium trapanicum NCIMB 767) constitute a distinct lineage with at least 98.2% sequence similarity. These strains have been incorrectly assigned to the genus Halobacterium. Therefore, based on a variety of taxonomic criteria, it is proposed that Halobacterium salinarium NCIMB 786 is renamed as Natrinema pellirubrum nom. nov., the type species of the new genus Natrinema gen. nov., and that Halobacterium halobium NCIMB 777 and Halobacterium trapanicum NCIMB 784 are renamed as a single species, Natrinema pallidum nom. nov. It was notable that halobacteria closely related to the proposed new genus have been isolated from relatively low-salt environments.

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.