Part:BBa_K4816011

Nitrite Reductase (NirS) from Paracoccus denitrificans PD1222

Figure 1. Nitrite reductase (NirS) in cyan showed as a cartoon representation and surface. The image was taken using the pdb accession 6TPO in Pymol.

Biological Background

Denitrification is a vital component of the nitrogen cycle, a biological process that converts nitrates and nitrites into nitrogen gas, ultimately returning nitrogen to the atmosphere. This cycle is primarily orchestrated by various types of bacteria, and denitrification can occur under two distinct conditions: aerobic and anaerobic.

Aerobic denitrification, in particular, is facilitated by specific bacteria that contain a set of four critical enzymes located in the periplasm. These enzymes play pivotal roles in the denitrification process:

• Nitrate Reductase (NapA) BBa_K4816009
• Nitrite Reductase (NirS) BBa_K4816011
• Nitric Oxide Reductase (NorB) BBa_K4816010
• Nitrous Oxide Reductase (NosZ) BBa_K4816012

The Denitrifying cycle consists of a series of reactions that lead to the production of nitrogen gas, the biochemical pathway is a four-step process where first nitrate (NO3-) is reduced to nitrite (NO2-) by nitrogen reductase, the catalytic active part expressed by the gene napA, then nitrite is further reduced to nitric oxide (NO) by nitrite reductase (nirS), nitric oxide is reduced to nitrous oxide (N2O), which is a potent greenhouse gas, by the enzyme nitric oxide reductase (norB), finally the limiting step of the pathway is the reduction of nitrous oxide into nitrogen gas (N2) by the enzyme nitrous oxide reductase (nosZ). As shown in equation 1.

Equation 1: Denitrifying cycle shown as a serie of biochemical equilibrium reactions.

One notable bacterium frequently employed in denitrification studies is Paracoccus denitrificans. The preference for Paracoccus denitrificans over organisms like Pseudomonas aeruginosa is often due to the latter's pathogenic nature. Paracoccus denitrificans, on the other hand, is non-pathogenic and can serve as a safer model for denitrification research.

Additionally, Paracoccus denitrificans holds further significance in the context of synthetic biology. This bacterium is more closely related to cyanobacteria, photosynthetic organisms capable of harnessing solar energy to convert carbon dioxide into organic compounds. This connection suggests the potential for future applications in synthetic biology, where Paracoccus denitrificans enzyme may be engineered to enhance environmental sustainability and resource utilization using Cyanobacteria chasis.

To get more information about the project visit the wiki iGEM Bonn-Rheinbach 2023: NitraNix

Gene Isolation

Paracoccus denitrificans PD1222 gene encoding for NirS part of the chromosome 1 (NCBI ID: CP000489.1) located between base pairs (2503999 and 2505789). NirS is the active subunit of the nitrite reductase. This modified gene contains two unique restriction sites, NdeI flanking the 5' terminus and BlpI flanking the 3' terminus. Using the primers foward BBa_K4816003 and reversed BBa_K4816004 to obtain the gene of interest by PCR from Paracoccus denitrificans PD1222 plasmid DNA. The restriction site addition allows the insertion into different vectors. In this case, we use them to insert in the plasmid pet 23 a (+) to further protein expression and isolation. Important to notice the addition of the His-Tag in the C-Terminus of the protein, introduced by the primer.

Figure 2. Representation of NirS gene with all the unique cutting sites. Note the presence of XhoI in position 127.

Usage and Biology

NirS was obtained by PCR amplification of genomic DNA at 53°c obtaining a prominent band at nearly 2kb. Multiple repeats were made in order to corroborate the presence of NirS inside the vector of interest pet 23 a+ to validate the right insertion.

Figure 3. Gel electrophoresis of PCR products, with corresponding primers’ ideal annealing temperatures. Using 1KB ladder plus from neb.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 505
  Illegal EcoRI site found at 1446
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 505
  Illegal EcoRI site found at 1446
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 505
  Illegal EcoRI site found at 1446
  Illegal BglII site found at 275
  Illegal BglII site found at 1493
  Illegal BamHI site found at 123
  Illegal XhoI site found at 139
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 505
  Illegal EcoRI site found at 1446
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 505
  Illegal EcoRI site found at 1446
  Illegal NgoMIV site found at 405
  Illegal NgoMIV site found at 625
  Illegal NgoMIV site found at 877
  Illegal NgoMIV site found at 1585
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 835
  Illegal SapI site found at 1425