The contribution of the NarB and NarGHI enzymes to nitrate reduction in Mycobacterium smegmatis

Abstract

Background: Nitrate reduction in bacteria is an essential step in the nitrogen cycle. For this, the reduction of nitrate to nitrite is catalyzed by a variety of nitrate reductase enzymes. In the pathogen Mycobacterium tuberculosis, nitrate reduction is driven by the NarGHI respiratory and assimilatory nitrate reductase. In addition to this enzyme, Mycobacterium smegmatis carries a second putative narB-encoded nitrate reductase and the contribution of this enzyme to nitrate reduction remains unknown. Herein, we set out to investigate this. Results: To assess the relative contribution of NarGHI and NarB, the corresponding gene loci we deleted using two-step allelic replacement, individually and in combination, followed by investigation of nitrate reduction using the Griess assay. However, previous reports demonstrated that this assay was unable to report on nitrate reduction in M. smegmatis, as it yielded no detectable levels of the nitrite product. To address this, we modified the assay through the addition of zinc, which reduces nitrate remaining in the reaction to nitrite thus allowing for assessment of nitrate depletion. This then serves as a surrogate for nitrate reductase activity. The mutant strains lacking narB and/or narGHJI retained the ability to reduce nitrate at levels comparable to the wild type. We further investigated nitrate assimilation and all strains defective for these enzymes were able to grow on nitrate as the sole nitrogen source. Conclusions: Collectively, these data confirm that NarB and NarGHI are individually and collectively dispensable for both respiratory and assimilatory nitrate reduction in M. smegmatis. Furthermore, we identified MSMEG_4206 as a putative, previously unannotated, nitrate reductase in this organism.

Variation in Bacterial Community Structure Under Long-Term Fertilization, Tillage, and Cover Cropping in Continuous Cotton Production

“…Spearman’s correlation was performed among bacteria at genus level known to be associated with N fixation, nitrification, and denitrification in each sampling date, and significant correlations ( p < 0.05) were corrected using Benjamini-Hochberg (BH) method ( Benjamini, 1995 ). All functional taxa mentioned in this study were found by genus in our samples as reported in previous studies ( Pagan et al, 1975 ; Pichinoty et al, 1976 ; Carlson and Ingraham, 1983 ; Davey and Marchant, 1983 ; Awonaike et al, 1990 ; Gee et al, 1990 ; Urakami et al, 1995 ; Baumann et al, 1996 ; Albrecht et al, 1997 ; Schramm et al, 1999 ; Hurek and Reinhold-Hurek, 2003 ; Verbaendert et al, 2011 ; Kostka et al, 2012 ; Cua and Stein, 2014 ; Sun et al, 2016 ; Fukami et al, 2018 ; Cardoso and Kana, 2019 ; Liu et al, 2019 ; Zhang H. et al, 2019 ; Spieck et al, 2020 ).…”