The streptothricin acetyltransferase (sat) gene as a positive selectable marker for methanogenic archaea

ABSTRACT A repertoire of sophisticated genetic tools has significantly enhanced studies of Methanosarcina genera, yet the lack of multiple positive selectable markers has limited the types of genetic experiments that can be performed. In this study, we report the development of an additional positive selection system for Methanosarcina that utilizes the antibiotic nourseothricin and the Streptomyces rochei streptothricin acetyltransferase (sat) gene, which may be broadly applicable to other groups of methanogenic archaea. Nourseothricin was found to inhibit growth of four different methanogen species at concentrations ≤300 μg/ml in liquid or on solid media. Selection of nourseothricin resistant transformants was possible in two genetically tractable Methanosarcina species, M. acetivorans and M. barkeri, using the sat gene as a positive selectable marker. Additionally, the sat marker was useful for constructing a gene deletion mutant strain of M. acetivorans, emphasizing its utility as a second positive selectable marker for genetic analyses of Methanosarcina genera. Interestingly, two human gut-associated methanogens Methanobrevibacter smithii and Methanomassillicoccus luminyensis were more sensitive to nourseothricin than either Methanosarcina species, suggesting the nourseothricin-sat gene pair may provide a robust positive selection system for development of genetic tools in these and other methanogens.

Prokaryote community dynamics in anaerobic co-digestion of swine manure, rice straw and industrial clay residuals

The aim of this study was to analyze the effect of the addition of rice straw and clay residuals on the prokaryote methane-producing community structure in a semi-continuously stirred tank reactor fed with swine manure. Molecular techniques, including terminal restriction fragment length polymorphism and a comparative nucleotide sequence analyses of the prokaryotic 16S rRNA genes, were performed. The results showed a positive effect of clay addition on methane yield during the co-digestion of swine manure and rice straw. At the digestion of swine manure, the bacterial phylum Firmicutes and the archaeal family Methanosarcinaceae, particularly Methanosarcina species, were predominant. During the co-digestion of swine manure and rice straw the microbial community changed, and with the addition of clay residual, the phylum Bacteroidetes predominated. The new nutritional conditions resulted in a shift in the archaeal family Methanosarcinaceae community as acetoclastic Methanosaeta species became dominant.

The ether lipids of Methanosarcina mazei and other Methanosarcina species, compared by fast atom bombardment mass spectrometry

The total lipids of Methanosarcina mazei accounted for 4.0% of the cell dry mass, and 90% of these were polar lipids. Nearly all of the polar fraction consisted of diether (2,3-di-O-phytanyl-sn-glycerol) and 3-hydroxydiether analogs of phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine. Several highly fluorescent trace components in the lipid extracts were purified and partially characterized by mass spectrometry. Fast atom bombardment mass spectrometry of total lipid extracts provided data to establish a close relationship among the polar lipids present in Methanosarcina mazei, Methanosarcina thermophila, Methanosarcina acetivorans, Methanosarcina barkeri Fusaro, and Methanosarcina barkeri MS.Key words: diether lipids, hydroxydiether lipids, fast atom bombardment mass spectrometry, Methanosarcina, archaeobacteria.