Lateral transfer of the denitrification pathway genes among Thermus thermophilus strains

Abstract

Nitrate respiration is a common and strain-specific property in Thermus thermophilus encoded by the nitrate respiration conjugative element (NCE) that can be laterally transferred by conjugation. In contrast, nitrite respiration and further denitrification steps are restricted to a few isolates of this species. These later steps of the denitrification pathway are under the regulatory control of an NCE-encoded transcription factor, but nothing is known about their coding sequences or its putative genetic linkage to the NCE. In this study we examine the genetic linkage between nitrate and nitrite respiration through lateral gene transfer (LGT) assays and describe a cluster of genes encoding the nitrite-nitric oxide respiration in T. thermophilus PRQ25. We show that the whole denitrification pathway can be transferred from the denitrificant strain PRQ25 to an aerobic strain, HB27, and that the genes coding for nitrite and nitric oxide respiration are encoded near the NCE. Sequence data from the draft genome of PRQ25 confirmed these results and allowed us to describe the most compact nor-nir cluster known thus far and to demonstrate the expression and activities of the encoded enzymes in the HB27 denitrificant derivatives obtained by LGT. We conclude that this NCE nor-nir supercluster constitutes a whole denitrification island that can be spread by lateral transfer among Thermus thermophilus strains.

FIG. 1.

LGT of the denitrification pathway. (A) Nitrite detection after 48 h of anaerobic incubation in the presence of nitrate 40 mM (column N), nitrite 5 mM (column Ni), or without electron acceptor (column C). Note that nitrate reduction results in the production of high nitrite concentrations (dark color), whereas nitrite consumption is detected as the lack of color. (B) SDS-PAGE of membrane proteins from the indicated strains stained with Coomassie blue. Strains: wild-type HB27 (lane wt); PRQ25 (lane PR); HB27 derivatives HB27nkat (lane nk), HB27dkat (lane dk), HB27d (lane d), and HB27C15 (lane C15).

FIG. 2.

Denitrification in HB27 derivatives. (A) Anaerobic growth of the indicated strains on nitrate (40 mM) after 48 h at 70°C. (B) Anaerobic growth with nitrite (10 mM) after 48 h at the same temperature. (C) N₂O production by the indicated strains after 5 days of anaerobic growth with nitrate (10 mM). Strains are named as in Fig. 1. The standard deviations are indicated.

FIG. 3.

Denitrification genes and proteins in HB27 derivatives. (A) Gene map of the nor-nir cluster and the surrounding genetic environment. (B) PCR amplification of narC, nirS, norC, and groE (housekeeping gene) using similar amounts of total DNA from the indicated strains as templates and the primer pair indicated in Table 1. (C) Western blots to detect NarG (nitrate reductase alpha subunit), NirS (putative cytochrome cd₁ nitrite reductase), NorC (putative nitric oxide reductase cytochrome c subunit), and DnrT (NCE transcription factor) on the indicated strains grown under anaerobic conditions with nitrate for 16 h. The strains are labeled as in Fig. 1.