Fig 1.
Nitrate reductase forms part of the Nitrogen cycle.
(A) A schematic representation of an example of the Nitrogen cycle in prokaryotes is shown. Different prokaryotic organisms are able to catalyze each of the reactions depicted. Figure adapted from [5]. (B) Nitrate assimilation pathway in M. smegmatis. Genes encoding proteins for each step of the pathway are shown. *MSMEG_4206 is annotated as a pseudogene due to a frameshift. ¥Five additional homologues of unknown function are present in the genome. Gene annotations were obtained from Mycobrowser and reference [6].
Fig 2.
MSMEG_4206 is not a pseudogene and has a distinct domain architecture.
(A) A schematic representation of the domain architecture of NarB (Accession: A0QW69) which is commonly found in bacterial NR enzymes. (B) Sequencing of the forward and reverse strand in the region of MSMEG_4206 predicted to contain a frameshift revealed that the frameshift is not present. (C) The distinct domain architecture of MSMEG_4206, as shown on InterPro (https://www.ebi.ac.uk/interpro/protein/). (D) The expression of each putative NR-encoding gene was measured in the presence of different nitrogen sources. The averages of three independent experiments with standard errors are shown. The Student’s t-test was performed for statistical analysis. *P< 0.005.
Table 1.
M. smegmatis genes encoding proteins with the same domain architecture as NarB.
Fig 3.
MSMEG_4206 is an assimilatory nitrate reductase.
(A) Growth analysis of mutant strains in MPLN with nitrate as the sole nitrogen source. The average of three independent experiments was plotted for each curve and standard errors are depicted. **** Statistical significance was determined by 2way-ANOVA (B) The conventional Griess assay involves the visualization of samples following the addition of the Griess reagents. A pink colour represents positivity for nitrate reduction, while no colour change is generally considered negative. In the modified Griess assay, a second step is included for samples that had no colour change during the conventional assay. Upon the addition of zinc, a colour change to pink is indicative of no NR activity and the sample is then classified as NR negative. No colour change after the addition of zinc represents complete nitrate utilization and thus NR positivity. (C) The modified Griess assay showed that MSMEG_4206 is required for nitrate utilization during aerobic growth. The average of three independent experiments was plotted for each panel and standard errors are depicted. ND: Not detected.
Fig 4.
NR activity is not required for the survival of M. smegmatis under anaerobic conditions.
(A) Anaerobic survival of the mutant strains was determined by enumeration of CFU’s after 6 days of anaerobiosis in the presence of nitrate and no differences were observed. (B) The modified Griess assay revealed that all the strains tested are unable to utilize nitrate under anaerobic conditions. The average of three independent experiments was plotted for each panel and standard errors are depicted. **** Statistical significance was determined by 2way-ANOVA. ND: Not detected.