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Applied and Environmental Microbiology, January 2009, p. 72-77, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01647-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A{triangledown}

Shaw R. Gargis,1 Harry E. Heath,1 Lucie S. Heath,1 Paul A. LeBlanc,1 Robin S. Simmonds,2 Brian D. Abbott,3 Russell Timkovich,3 and Gary L. Sloan1*

Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama,1 Department of Microbiology, University of Otago, Dunedin, New Zealand,2 Department of Chemistry, The University of Alabama, Tuscaloosa, Alabama3

Received 17 July 2008/ Accepted 23 October 2008

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a D-alanyl-L-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.

* Corresponding author. Mailing address: Department of Biological Sciences, Box 870334, The University of Alabama, Tuscaloosa, AL 35487-0334. Phone: (205) 348-8444. Fax: (205) 348-5976. E-mail: gsloan{at}bama.ua.edu

{triangledown} Published ahead of print on 31 October 2008.

Applied and Environmental Microbiology, January 2009, p. 72-77, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01647-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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