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

Abstract

Orange filamentous Beggiatoaceae 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 Beggiatoaceae filament, three candidate genes for enzymes with nitrite-reducing function - an orange octaheme cytochrome, a nirS nitrite reductase, and a nitrite/tetrathionate-reducing octaheme cytochrome - were cloned and expressed in Escherichia coli. The expressed and purified orange cytochrome showed reduced nitrite-reducing activity compared to the multifunctional native protein obtained from microbial mats. The nirS gene product showed in vitro but no in-gel nitrite-reducing activity; and the nitrite/tetrathionate-reducing octaheme cytochrome was capable of reducing both nitrite and tetrathionate in vitro. Phylogenetic analysis shows that the orange Beggiatoaceae nirS, 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 Beggiatoaceae is related to nitrite- and tetrathionate reductases harbored predominantly by Gammaproteobacteria, including obligate endosymbionts of hydrothermal vent tubeworms. Thus, the orange Guaymas Basin Beggiatoaceae 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 Beggiatoaceae, 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 Beggiatoaceae in hydrothermal hot spots.

Keywords: Beggiatoaceae; Guaymas Basin; cytochrome; nitrite reductase; tetrathionate reductase.

FIGURE 1

In situ close-up video still photo of an orange Beggiatoaceae mat in Guaymas Basin, taken by the bottom-facing Alvin camera a few centimeters above the seafloor during Alvin dive 4872 in the Cathedral Hill area of Guaymas Basin (27°N00.70/111°W24.25). The image shows the complex three-dimensional structure of a mat, arranged around several small hydrothermal outflow areas marked by white sulfur deposits, visible in the center of the image. The Individual orange filaments have approx. 40 μm diameter.

FIGURE 2

Genomic arrangement and context of target genes (ORFs BOGUAY_2967, 2386 and 0691), with arrows denoting the position of PCR primers pairs including and excluding flanking regions Genomic maps are derived from the GOLD Project ID# Gp0006298 on the IMG database. (A) A subset of the 41659 bp BOGUAY_Contig_00024 contains BOGUAY_0691 gene which translates into a 511aa protein. Primer 5′ ends are shown for pair 3, 0691 ExpF (a, 225 bp upstream of translational start site) and 0691 ExpR (b, ends 38 bp downstream of translational stop codon); and pair 6, 0691F (c, 5′-end at the start codon) and 0691R (d; 5′-end at the end of the gene, excluding the stop codon). (B) A subset of the 12025 bp BOGUAY_Contig_00500 contains the BOGUAY_2967 gene, which translates into a 671aa protein. Primer 5′ ends are shown for primer pair 1, 2967 ExpF (a; 184 bp upstream of translational start site) and 2967 ExpR (b; ends 281 bp downstream of translational stop codon); and for primer pair 4, 2967F (c, 5′-end at the start codon) and 2967 R (d; 5′-end at the end of the gene, excluding the stop codon). (C) A subset of the 15900 bp BOGUAY_Contig_01341 contains the BOGUAY_2386 gene, which translates into a 582aa protein. Primer 5′ ends are shown for pair 2, 2386 ExpF (a, 40 bp upstream of translational start site), and 2386 ExpR (b, ends 91 bp downstream of translational stop codon); and for primer pair 5, 2386F (c, 5′-end at the start codon) and 2386R (d, position 5′-end at the end of the gene, excluding the stop codon).

FIGURE 3

Maximum Likelihood phylogeny for the orange octaheme cytochrome (BOGUAY_0691), based on aligned protein sequences obtained by translating the gene. The scale bar shows the number of amino acid substitutions per site. Tree topology was tested with 1000 bootstrap replicates, and branching points are annotated with the percentages of recovering each node. Taxon labels start with IMG Gene ID numbers, followed by species, strain, or sequence/phylotype designations, and concluded with genomic ID and contig number as in IMG, unless omitted for sequence entries sharing the same origin.

FIGURE 4

Maximum Likelihood phylogeny for candidate nitrite reductase NirS (BOGUAY_2967), based on aligned protein sequences obtained by translating the gene. Scale bar, bootstrap and taxon label annotation are the same as in Figure 3.

FIGURE 5

Maximum Likelihood phylogeny for candidate octaheme nitrite/tetrathionate reductase (BOGUAY_2386), based on aligned protein sequences obtained by translating the gene. Scale bar, bootstrap and taxon label annotation are the same as in Figure 3.

FIGURE 6

In-gel nitrite reduction assay for expressed protein of BOGUAY_2386. (A) Coomassie total protein stain; the arrow identifies the 67 kda marker protein. (B) Nitrite reduction gel after 2 h development of the Methyl Viologen (MV) destaining reaction in anaerobe chamber followed by 1 min exposure to atmospheric oxygen. (C) Nitrite reduction gel after 2 h development in anaerobe chamber and 30 min exposure to atmospheric oxygen. The expected molecular weight for the monomer is 65.1 kba, which matches the position of the lowermost band close to the 67 kba ladder protein (A). Additional bands of higher molecular weight that show nitrite-reducing enzymatic activity are presumably dimers and – near the top of the lane – non-migrating multimers that may reflect partial hydrophobicity of the protein. The monomer and the multimers are marked with arrows in all three panels. L, Ladder; FT, Flow through of cell lysate; W1, Wash 1; W2, Wash 2; E1u, Elution 1 of uninduced protein purification; E1, first elution of induced protein purification; E2, second elution of induced protein purification; E3, third elution 3 of induced protein purification; E4, Fourth elution 4 of induced protein purification. For orientation, a thin white line is placed between the E1u and E1 lane in all gels.