Analyzing the Function of NucS in M. smegmatis
Abstract
Understanding the functions of certain proteins may aid the world in its efforts to combat the global health threat of antimicrobial resistance.
Antimicrobials are critical to the treatment of bacterial infections. However, many bacteria have developed antimicrobial resistance (AMR), which was declared among the ten most critical threats to public health by the World Health Organization. NucS is a protein that repairs resistance-causing mismatches in mycobacteria, and further understanding of its function may provide insight into AMR in mycobacteria, including in M. tuberculosis.
This project aims to determine the function of the five amino acid sequence at the C-terminal of NucS. In this project, NucS was tagged with fluorescent proteins by constructing the plasmid pEW2, containing mCherry. Plasmid pEW2 was then then integrated into the genome of M. smegmatis. Afterwards, the bacteria were observed under a fluorescence microscope and grown on rifampicin plates. Though foci were seen, they were in different locations in the cell. It was subsequently found that the bacteria were mutagenic. Such a result may suggest that the five C-terminal amino acids serve a purpose in addition to binding to DnaN, though more research will be needed.
The plasmid made in this investigation can be used to further investigate the amino acids, locations, and interactions of NucS, such as during different cell processes or when an amino acid or the promoter of NucS is changed.
Graphical Abstract
Problem Statement
Though the NucS protein is important to mismatch repair in mycobacteria, questions still remain on its functions and interactions.
Overall Aim
This project aims to determine the function of the five amino acid sequence at the C-terminal of NucS and to investigate the interaction between NucS and DnaN.
Background
Antimicrobials are critical in the treatment of bacterial infections and thus the preservation of global health. However, many bacteria can develop antimicrobial resistance (AMR) (Davies & Davies, 2010). AMR can be conferred in many ways, though the most common method involves mutations (Annunziato, 2019). The increase in AMR poses a major risk to global health, with death rates caused by AMR predicted to skyrocket to 10 million deaths annually within the next three decades (Ten Health Issues WHO Will Tackle This Year, 2019).
NucS has been shown to prevent mutations in mycobacteria, a genus which includes mycobacterium tuberculosis. More understanding of its precise function may elucidate further directions for protecting and promoting DNA repair in mycobacteria.
Investigating the interactions of NucS with other proteins in the repair pathway is one such inquiry that would shed light on its function (Cebrián-Sastre et al., 2021). Such interactions may be observed by tagging NucS.
Procedure Infographic
Procedure
To tag NucS with mCherry, a plasmid, pEW2, needed to be constructed from a mCherry insert and a backbone vector derived from pKM469. We cut both our insert and backbone at their HindIII and XbaI sites to form overhangs, or sticky ends, in their DNA. Finally, we ligated the cut insert and plasmid to form pEW2, the plasmid we would use to tag NucS.
Following the formation of pEW2, we electroporated pEW2 into E. coli to clone it. As our intended plasmid had chloramphenicol resistance, we selected for bacteria with the plasmid by plating on chloramphenicol. We then purified candidates for sequencing using the Qiagen plasmid purification kit and sent them for sequencing. Next, we used the ORBIT method of recombineering (for more information on the ORBIT process, see Murphy et al., 2018) to integrate the correct plasmid into the chromosome of M. smegmatis before the replisome tag. Successful colonies were selected for by plating on hygromycin. PCRs were performed on selected colonies on the plate and the junctions formed were both run on a gel and sent for sequencing to ensure successful tagging of NucS.
To determine mutagenicity, the M. smegmatis strains containing NucS tagged with mCherry were plated on rifampicin, and M. smegmatis strains with deletions in the nucS gene and wild-type strains of M. smegmatis were plated as the positive and negative controls, respectively.
To determine if and where the mCherry-tagged NucS could be seen in the cell, we utilized fluorescence microscopy, with untagged M. smegmatis as our control.
Figure 1
Figure 1
PCRed junctions from chromosome of M. smegmatis after integration of the plasmid. All three samples contained the correct sequence.
Figure 2
Figure 2
Three rifampicin plate examples. From left to right, they are a wild type strain, a strain with NucS tagged with mCherry, and a strain with a deletion in nucS. The wild type plate has 17 colonies, the mCherry plate has 101 colonies, and the deletion strain has 111 colonies.
Figure 3
Figure 3
Six samples each of a wild type M. smegmatis strain, a strain containing NucS tagged with mCherry, and a strain containing a deletion in nucS were plated on rifampicin. Their average percent frequencies of mutation were then calculated and graphed.
Analysis
As indicated by our graph, there was a significant difference between the frequency of mutation of our wild type and our NucS-mCherry strains, but not between our ΔNucS and mCherry strains, indicating that our mCherry tag interfered with the ability of NucS to cleave mutations. This result suggests that the five C-terminal amino acids of NucS may be necessary for functions of NucS other than binding, or that additional amino acids may be necessary for binding.
We cannot draw conclusions on the interaction between NucS and DnaN because our tagged NucS did not have standard function and may not have accurately reflected the interaction of wild type NucS with DnaN.
Conclusion
As mutations are a key cause of antimicrobial resistance in bacteria, learning more about proteins such as NucS which prevent mutations may open new paths to researching and addressing antimicrobial resistance.
This project constructed a plasmid to investigate the function of the replisome tag of NucS and found that separating it from the rest of the protein caused increased mutagenicity.
Further research may involve tagging NucS at its N-terminal and DnaN with GFP.