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Growing cultures of the green obligate photolithotroph, Chlorobaculum parvum DSM 263T (formerly Chlorobium vibrioforme forma specialis thiosulfatophilum NCIB 8327), oxidized sulfide quantitatively to elemental sulfur, with no sulfate formation. In the early stages of growth and sulfide oxidation, the sulfur product became significantly enriched with ³⁴S, with a maximum δ³⁴S above +5‰, while the residual sulfide was progressively depleted in ³⁴S to δ³⁴S values greater than -4‰. As oxidation proceeded, the δS of the sulfur declined to approach that of the initial sulfide when most of the substrate sulfide had been converted to sulfur in this closed culture system. No significant formation of sulfate occurred, and the substrate sulfide and elemental sulfur product accounted for all the sulfur provided throughout oxidation. The mean isotope fractionation factors (e) for sulfide and sulfur were equivalent at e values of -2.4‰ and +2.4‰ respectively. The significance of the experimentally-observed fractionation to the ³⁴S/³²S ratios seen in natural sulfur-containing minerals is considered.
The class Acidithiobacillia was established using multiprotein phylogenetic analysis of all the available genomes of the genus Acidithiobacillus (comprising Family I, the Acidithiobacillaceae, of the Acidithiobacillales, the order created for Bergey’s Manual of Systematic Bacteriology), and for representative genomes of all available bacterial orders. The Acidithiobacillales contain a second family, the Thermithiobacillaceae, represented by Thermithiobacillus tepidarius, and created on the basis of nearest neighbour 16S ribosomal RNA gene sequence similarities. This could not be included in the original multiprotein analysis as no genome sequence for Thermithiobacillus was available. The publication of the genome sequence of Thermithiobacillus tepidarius in 2013 has enabled phylogenetic assessment of this organism by comparative multigenome analysis. This shows definitively that Thermithiobacillus is a member of the class Acidithiobacillia, distinct from the Acidithiobacillus genus, and confirms it to represent a second family within the Acidithiobacillia.
The phylogenetic significance of the diversity of key enzymes of methylotrophic and autotrophic metabolism is discussed. Primers for these key enzymes were designed using gene sequences encoding methanol dehydrogenase (mxaF; using subsets from database sequences for 22 Bacteria), hydroxypyruvate reductase (hpr; 36 sequences), methylamine dehydrogenase (mauA; 12 sequences), methanesulfonate monooxygenase (msmA; four sequences), and the ccbL and cbbM genes of ribulose bisphosphate carboxylase (26 and 23 sequences). These were effective in amplifying the correct gene products for the target genes in reference organisms and in test organisms not previously shown to contain the genes, as well as in some methylotrophic Proteobacteria isolated from the human mouth. The availability of the new primers increases the probability of detecting diverse examples of the genes encoding these key enzymes both in natural populations and in isolated bacterial strains.
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