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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND PROTEOMIC ANALYSIS OF FOODBORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: Characterization and comparative analysis of a second thermonuclease in Staphylococcus aureus

Authors
item Hu, Yu -
item Meng, Jianghong -
item Shi, Chunlei -
item Hervin, Kirstin -
item Fratamico, Pina
item Shi, Xianming -

Submitted to: Microbiological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 19, 2012
Publication Date: March 30, 2013
Repository URL: http://handle.nal.usda.gov/10113/57174
Citation: Hu, Y., Meng, J., Shi, C., Hervin, K., Fratamico, P.M., Shi, X. 2013. Characterization and comparative analysis of a second thermonuclease in Staphylococcus aureus. Microbiological Research. 168(3):174-182.

Interpretive Summary: Staphylococcus aureus is a bacterium that causes food-borne illness. It is important to understand how the bacterium causes disease to develop better treatments for infection. In addition, methods to detect and identify S. aureus based on unique markers found in the pathogen are needed. S. aureus produces an enzyme known as a thermonuclease that hydrolyzes DNA and RNA in human cells, causing tissue destruction and spreading of the pathogen. In this study, various characteristics, such as amino acid sequence, structure, and nuclease activity of a novel thermonuclease were compared to those of a more well known enzyme with similar activity found in S. aureus. The two enzymes were both thermonucleases; however, a number of differences, including level of activity against DNA and RNA, structure, and heat stability were observed. Thus, these are clearly two distinct enzymes in S. aureus and are likely involved in the ability of this bacterium to cause human illness. The novel thermonuclease characterized in the current work could potentially be used as a target for the identification of S. aureus and for diagnosis and treatment for infections caused by this pathogen.

Technical Abstract: Staphylococcal nuclease (here termed as NUC1) is considered an important virulence factor and a unique marker widely used in detection of Staphylococcus aureus. A novel functional thermostable nuclease (here termed as NUC2) in S. aureus was characterized after recombinant expression in Escherichia coli. Sequence alignment and phylogenetic analysis revealed that NUC2 was a more conserved protein in the staphylococci group compared with NUC1. Recombinant NUC2 showed weaker nuclease activity in the zymogram test compared to NUC1, and it was able to degrade various types of nucleic acids similar to NUC1. The optimal reaction temperature and pH for NUC2 were 50 degrees C and pH 10, respectively. The enzymatic activity of NUC2 was stimulated in the presence of Ca2+ (0.05mM), Mg2+ (0.5mM), dithiothreitol, ß-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn2+ and Mn2+, and in the presence of EDTA or SDS. The weaker activity and lower thermostability of NUC2 compared with NUC1 were postulated to be related to sequence differences for the two enzymes and changes of the hydrophobic core residues in the predicted structure of NUC2. The comparative analysis of NUC2 in S. aureus distinguished it from NUC1, indicating that NUC2 is a new sugar non-specific thermonuclease in the genus Staphylococcus.

Last Modified: 7/31/2014
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