The bacterial strains JAM1T and GP59 of the species Methylophaga nitratireducenticrescens differ in their expression profiles of denitrification genes in oxic and anoxic cultures
Authors: Lestin Livie , Villemur Richard 
Published: October 2024

Background
Strain JAM1T and strain GP59 of the methylotrophic, bacterial species Methylophaga nitratireducenticrescens were isolated from a microbial community of the biofilm that developed in a fluidized-bed, methanol-fed, marine denitrification system. Despite of their common origin, both strains showed distinct physiological characters towards the dynamics of nitrate (${\mathrm{NO}}_{3}^{-}$) reduction. Strain JAM1T can reduce ${\mathrm{NO}}_{3}^{-}$ to nitrite (${\mathrm{NO}}_{2}^{-}$) but not ${\mathrm{NO}}_{2}^{-}$ to nitric oxide (NO) as it lacks a NO-forming ${\mathrm{NO}}_{2}^{-}$ reductase. Strain GP59 on the other hand can carry the complete reduction of ${\mathrm{NO}}_{3}^{-}$ to N2. Strain GP59 cultured under anoxic conditions shows a 24-48h lag phase before ${\mathrm{NO}}_{3}^{-}$ reduction occurs. In strain JAM1T cultures, ${\mathrm{NO}}_{3}^{-}$ reduction begins immediately with accumulation of ${\mathrm{NO}}_{2}^{-}$. Furthermore, ${\mathrm{NO}}_{3}^{-}$ is reduced under oxic conditions in strain JAM1T cultures, which does not appear in strain GP59 cultures. These distinct characters suggest differences in the regulation pathways impacting the expression of denitrification genes, and ultimately growth.

Methods
Both strains were cultured under oxic conditions either with or without ${\mathrm{NO}}_{3}^{-}$, or under anoxic conditions with ${\mathrm{NO}}_{3}^{-}$. Transcript levels of selected denitrification genes (nar1 and nar2 encoding ${\mathrm{NO}}_{3}^{-}$ reductases, nirK encoding ${\mathrm{NO}}_{2}^{-}$ reductase, narK12f encoding ${\mathrm{NO}}_{3}^{-}$/${\mathrm{NO}}_{2}^{-}$transporter) and regulatory genes (narXL and fnr) were determined by quantitative reverse transcription polymerase chain reaction. We also derived the transcriptomes of these cultures and determined their relative gene expression profiles.

Results
The transcript levels of nar1 were very low in strain GP59 cultured under oxic conditions without ${\mathrm{NO}}_{3}^{-}$. These levels were 37 times higher in strain JAM1T cultured under the same conditions, suggesting that Nar1 was expressed at sufficient levels in strain JAM1T before the inoculation of the oxic and anoxic cultures to carry ${\mathrm{NO}}_{3}^{-}$ reduction with no lag phase. Transcriptomic analysis revealed that each strain had distinct relative gene expression profiles, and oxygen had high impact on these profiles. Among denitrification genes and regulatory genes, the nnrS3 gene encoding factor involved in NO-response function had its relative gene transcript levels 5 to 10 times higher in strain GP59 cultured under oxic conditions with ${\mathrm{NO}}_{3}^{-}$ than those in both strains cultured under oxic conditions without ${\mathrm{NO}}_{3}^{-}$. Since NnrS senses NO, these results suggest that strain GP59 reduced ${\mathrm{NO}}_{3}^{-}$ to NO under oxic conditions, but because of the oxic environment, NO is oxidized back to ${\mathrm{NO}}_{3}^{-}$ by flavohemoproteins (NO dioxygenase; Hmp), explaining why ${\mathrm{NO}}_{3}^{-}$ reduction is not observed in strain GP59 cultured under oxic conditions.

Conclusions
Understanding how these two strains manage the regulation of the denitrification pathway provided some clues on how they response to environmental changes in the original biofilm community, and, by extension, how this community adapts in providing efficient denitrifying activities.