Production of volatile sulfur compounds in cheese is associated with desirable flavors. The direct source of these compounds has been assumed to arise from the metabolism of methionine and cysteine. However, the methionine concentration in cheese rises above the amount found in casein during aging, suggesting that alternative sulfur sources are present in milk. This led us to hypothesize that lactococci may acquire sulfur from the inorganic sulfur pool of milk, in addition to methionine and cysteine, to generate volatile sulfur compounds during cheese ripening. A turbidimetric method to determine total sulfate content in milk samples was developed. The average sulfate content of milk was determined to be ~49 mg/L ± 2.0 mg/L. The limit of detection of the test was ~2.5 mg/L in Tris buffer and ~10 mg/L in milk. Skim milk samples had significantly higher total sulfate content as compared to whole milk samples. Transport of sulfate by three strains of Lactococcus sp. was studied after we determined that milk had small, but measurable amounts of inorganic sulfate. A decrease in the environmental pH increased sulfate transport. The maximum transport occurred during exponential cellular growth phase. All strains tested had the ability to transport much more sulfate than is native in milk. The last phase of study was to determine the metabolic fate of sulfate. Incorporation of radio-labeled sulfate into cellular protein was studied by two-dimensional gel-electrophoresis of crude cellular lysate followed by auto-radiography. Production of volatile sulfur compounds from inorganic sulfur was determined with analysis of the head space gas with gas chromatography and scintillation counting. The incorporation of radio-labeled sulfur from sulfate was not detected in proteins on two-dimensional gels. Detectable volatile sulfur compounds were found only in the case of gas chromatographic analysis of ML3 head space. However, radio-labeled volatile sulfur was detected in the head space of all the three strains with scintillation counting. This study defined that lactococci can fix inorganic sulfur into volatile sulfur compounds in small amounts.

Inorganic sulfur-oxidising Bacteria are present throughout the Proteobacteria and inhabit all environments of Earth. Despite these facts they are still poorly understood in terms of taxonomy, physiology, biochemistry and genetics. Using phylogenetic and chemotaxonomic analysis two species that were erroneously classified as Thiobacillus trautweinii spp. in 1921 and 1934 are in fact novel chemolithoheterotrophic species for which the names Pseudomonas trautweiniana sp. nov. and Achromobacter starkeyanus sp. nov. are proposed, respectively. These species were found to oxidise thiosulfate in a “fortuitous” manor when grown in continuous culture and increases in maximum theoretical growth yield (YMAX) and maximum specific growth rate (μMAX) were observed. Cytochrome c linked thiosulfate-dependent ATP production was confirmed in both species, confirming “true” chemolithoheterotrophy. Evidence is presented that the ATP concentration governs the benefits of chemolithoheterotrophy. There were significant changes in enzyme activities, including enzymes of the TCA cycle that might be affecting amino acid synthesis. This is strong evidence that chemolithoheterotrophy gives a strong physiological boost and evolutionary advantage over strictly heterotrophic species. An autotrophic species that was historically placed in Thiobacillus was also shown to be a novel species for which the name Thermithiobacillus parkerianus sp. nov. is proposed. The enzyme profiles of Thermithiobacillus parkerianus differed significantly between different inorganic sulfur growth substrates and was the first time all TCA cycle enzymes were assayed in a member of the Acidithiobacillia. The properties of thiosulfate dehydrogenase varied significantly between Pseudomonas sp. Strain T, Achromobacter sp. Strain B and Thermithiobacillus sp. ParkerM both in terms of optimal parameters and the effect of inhibitors. This evidence adds to the increasing body of work indicating there to be at least two thiosulfate dehydrogenases present in the Bacteria.