Identification of a respiratory-type nitrate reductase and its role for survival of Mycobacterium smegmatis in Wayne model

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

Download Full-text

Mycobacterium smegmatis does not display functional redundancy in nitrate reductase enzymes

PLoS ONE ◽

10.1371/journal.pone.0245745 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245745

Author(s):

Nicole C. Cardoso ◽

Andrea O. Papadopoulos ◽

Bavesh D. Kana

Keyword(s):

Nitrate Reductase ◽

Mycobacterium Smegmatis ◽

Reductase Activity ◽

Nitrate Assimilation ◽

Anaerobic Conditions ◽

Relative Contribution ◽

Allelic Replacement ◽

Encoding Genes ◽

Aerobic And Anaerobic ◽

Aerobic And Anaerobic Conditions

Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.

Download Full-text

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

10.21203/rs.2.9260/v1 ◽

2019 ◽

Author(s):

Nicole Collette Cardoso ◽

Bavesh Davandra Kana

Keyword(s):

Nitrate Reductase ◽

Nitrate Reduction ◽

Mycobacterium Smegmatis ◽

Reductase Activity ◽

Nitrate Assimilation ◽

Wild Type ◽

Relative Contribution ◽

Allelic Replacement ◽

Mutant Strains ◽

Assimilatory Nitrate Reduction

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.

Download Full-text