Identification, Expression and Activity of Candidate Nitrite Reductases From Orange Beggiatoaceae, Guaymas Basin

Abstract

Orange filamentous <i>Beggiatoaceae</i> form massive microbial mats on hydrothermal sediments in Guaymas Basin; these bacteria are considered to oxidize sulfide with nitrate and nitrite as electron acceptors. From a previously analyzed genome of an orange <i>Beggiatoaceae</i> filament, three candidate genes for enzymes with nitrite-reducing function - an orange octaheme cytochrome, a <i>nirS</i> nitrite reductase, and a nitrite/tetrathionate-reducing octaheme cytochrome - were cloned and expressed in <i>Escherichia coli</i>. The expressed and purified orange cytochrome showed reduced nitrite-reducing activity compared to the multifunctional native protein obtained from microbial mats. The <i>nirS</i> gene product showed <i>in vitro</i> but no in-gel nitrite-reducing activity; and the nitrite/tetrathionate-reducing octaheme cytochrome was capable of reducing both nitrite and tetrathionate <i>in vitro</i>. Phylogenetic analysis shows that the orange <i>Beggiatoaceae nirS</i>, in contrast to the other candidate nitrite reductases, does not form monophyletic lineages with its counterparts in other large sulfur-oxidizing bacteria, and most likely represents a recent acquisition by lateral gene transfer. The nitrite/tetrathionate-reducing enzyme of the orange <i>Beggiatoaceae</i> is related to nitrite- and tetrathionate reductases harbored predominantly by Gammaproteobacteria, including obligate endosymbionts of hydrothermal vent tubeworms. Thus, the orange Guaymas Basin <i>Beggiatoaceae</i> have a repertoire of at least three different functional enzymes for nitrite reduction. By demonstrating the unusual diversity of enzymes with a potential role in nitrite reduction, we show that bacteria in highly dynamic, sulfide-rich hydrothermal vent habitats adapt to these conditions that usually prohibit nitrate and nitrite reduction. In the case of the orange Guaymas <i>Beggiatoaceae</i>, classical denitrification appears to be replaced by different multifunctional enzymes for nitrite and tetrathionate reduction; the resulting ecophysiological flexibility provides a new key to the dominance of these <i>Beggiatoaceae</i> in hydrothermal hot spots.