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The aim of this study was to classify certain environmental haloarchaea and methanoarchaea using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and to expand the archaeal mass spectral database. A total of 69 archaea were collected including type strains and samples isolated locally from different environments. For extraction of the haloarchaeal total cell peptides/proteins, a simple method of acetonitrile extraction was developed. Cluster analysis conducted with the MALDI-TOF MS data overcame the high divergence in intragenomic 16S rRNA sequences in haloarchaea and clearly distinguished Methanohalophilus mahii from M. portucalensis. Putative biomarkers that can distinguish several particular archaeal genera were also assigned. In conclusion, this study expands the mass spectral database of peptide/protein fingerprints from bacteria and fungi to the archaea domain and provides a rapid identification platform for environmental archaeal samples.

Extensive diversity in features of aromatic amino acid biosynthesis and regulation has become recognized in eubacteria, but almost nothing is known about the extent to which such diversity exists within the archaebacteria. Methanohalophilus mahii, a methylotrophic halophilic methanogen, was found to synthesize l-phenylalanine and l-tyrosine via phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Enzymes capable of using l-arogenate as substrate were not found. Prephenate dehydrogenase was highly sensitive to feedback inhibition by l-tyrosine and could utilize either NADP+ (preferred) or NAD+ as cosubstrate. Tyrosine-pathway dehydrogenases having the combination of narrow specificity for a cyclohexadienyl substrate but broad specificity for pyridine nucleotide cofactor have not been described before. The chorismate mutase enzyme found is a member of a class which is insensitive to allosteric control. The most noteworthy character state was prephenate dehydratase which proved to be subject to multimetabolite control by feedback inhibitor (l-phenylalanine) and allosteric activators (l-tyrosine, l-tryptophan, l-leucine, l-methionine and l-isoleucine). This interlock type of prephenate dehydratase, also known to be broadly distributed among the gram-positive lineage of the eubacteria, was previously shown to exist in the extreme halophile, Halobacterium vallismortis. The results are consistent with the conclusion based upon 16S rRNA analyses that Methanomicrobiales and the extreme halophiles cluster together.

Methanohalophilus mahii SLP andMethanohalophilus halophilus Z-7982, two closely-related, moderately halophilic, methylotrophic methanogens, were tested for their adaptation to saline conditions. They grew in a wider range of salinities than previously reported, in a defined medium with as little as 0.1 M NaCl, and with a high as 4.0 M NaCl forM. halophilus and 4.5 M NaCl forM. mahii. Fastest growth occurred with 1.5 M NaCl forM. mahii and 1.0 M NaCl forM. halophilus. M. mahii also grew in media in which NaCl was replaced by sucrose or KCl as osmolytes up to the osmolal equivalent of 2 and 2.5 M NaCl (these media contained other sodium salts totaling about 0.1 M Na+). In media with either sucrose of KCl replacing NaCl,M. mahii grew fastest at osmolalities approximately equiosmolal to 1 M NaCl.M. mahii not only grew well at a wide range of osmosities, it also tolerated rapid shifts in osmolality. Cells subjected to a rapid 10-fold hypertonic shift resumed growth without a prolonged lag. When cells were subjected to a rapid 10-fold hypotonic shift, 90% of cells lysed, but the remaineder continued to swell with little further lysis during the next 45 min. Surviving cells resumed growth.Methanohalophilus strains grown in defined medium had low cytosolic Na+ concentrations; K+ concentrations were as high as 0.35 M. Organic osmotica in the cytosol include glycine betaine and larger amounts of N,N-dimethylglycine.

Methanohalophilus mahiiis the type species of the genusMethanohalophilus, which currently comprises three distinct species with validly published names.Mhp. mahiirepresents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLPTwas isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of theGenomic Encyclopedia of Bacteria and Archaeaproject. A comparison of the reconstructed energy metabolism in the halophilic speciesMhp. mahiiwith other representatives of theMethanosarcinaceaereveals some interesting differences to freshwater species.

Trimethyl glycine (glycine betaine) was detected by 13C nuclear magnetic resonance spectroscopy at high intracellular concentrations in several methanogens (Methanogenium cariaci, "Methanogenium anulus" AN9, Methanohalophilus zhilinae, Methanohalophilus mahii, and Methanococcus voltae) grown on marine media containing yeast extract. 13C labeling studies with Methanogenium cariaci suggested that the betaine which accumulated inside the cells was not synthesized de novo but was transported in from the medium. Proof of such a transport system was provided by growing Methanogenium cariaci on yeast-free medium supplemented with betaine. Under these conditions, betaine was the dominant osmoregulator.

We demonstrated the ability of several methylotrophic methanogens to degrade dimethylsulfide and methane thiol to hydrogen sulfide, methane, and carbon dioxide. This is the first report of the growth of a pure culture of methanogen on methane thiol. Methanolobus siciliae HI350, a methylotrophic methanogen isolated from an oil well, was grown routinely on trimethylamine. When this culture was inoculated into a medium with 5 mM dimethyl sulfide, it began producing methane and hydrogen sulfide after a lag of several weeks. Methane production was slow, with an apparent microbial growth rate of 0.0033 h-1 (about 3% as fast as growth on trimethylamine or methanol). The lag was shorter when 2 mM methane thiol was substrate. When a culture of M. siciliae HI350 growing on dimethylsulfide was subcultured on dimethylsulfide, the lag disappeared and growth rate was higher (0.087 h-1). Dimethylsulfide-grown cultures also had no lag when transferred to media with methane thiol. Studies of cell-free extracts suggested that enzymes for the degradation of dimethylsulfide and methane thiol were inducible, whereas those for the degradation of methanol and trimethylamine were constitutive. Degradation of dimethylsulfide or methane thiol was complete, and stoichiometric quantities of methane and hydrogen sulfide were formed. Most surprisingly, this strain could be adapted to grow with high concentrations of dimethylsulfide or methane thiol, as high as 30 mM. Other methanogens which have been reported to catabolize dimethylsulfide are Methanolobus siciliae T4/M, Methanohalophilus zhilinaeae WeN5, Methanohalophilus oregonensis WALL, and strain GS-16. The M. siciliae strains and strain GS-16 were also able to use methane thiol as their catabolic substrate (other strains were not tested). We tested other methylotrophic methanogens for their ability to use dimethylsulfide at concentrations which did not inhibit their ability to degrade trimethylamine and found the following cultures unable to catabolize dimethylsulfide: Methanococcoides methylutens TMA-10, Methanolobus tindarius Tindari 3, Methanolobus vulcani PL-12/M, Methanohalophilus mahii SLP, Methanohalophilus halophilus Z-7982, Methanosarcina mazeii LYC, and Methanosarcina mazeii C 16.

We report here the complete genome sequence (2.08 Mb) of Methanohalophilus portucalensis strain FDF-1 T , a halophilic methylotrophic methanogen isolated from the sediment of a saltern in Figeria da Foz, Portugal. The average nucleotide identity and DNA-DNA hybridization analyses show that Methanohalophilus mahii , M. halophilus , and M. portucalensis are three different species within the Methanosarcinaceae family.