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Applied and Environmental Microbiology, May 2005, p. 2442-2451, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2442-2451.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Phenotypic Characterization of Sphingomonas wittichii Strain RW1 by Peptide Mass Fingerprinting Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Rolf U. Halden,* David R. Colquhoun, and Eric S. Wisniewski

Center for Water and Health, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205

Received 11 September 2004/ Accepted 22 November 2004

Mass spectrometry is a potentially attractive means of monitoring the survival and efficacy of bioaugmentation agents, such as the dioxin-mineralizing bacterium Sphingomonas wittichii strain RW1. The biotransformation activity of RW1 phenotypes is determined primarily by the presence and concentration of the dioxin dioxygenase, an enzyme initiating the degradation of both dibenzo-p-dioxin and dibenzofuran (DF). We explored the possibility of identifying and characterizing putative cultures of RW1 by peptide mass fingerprinting (PMF) targeting this characteristic phenotypic biomarker. The proteome from cells of RW1—grown on various media in the presence and absence of DF—was partially purified, tryptically digested, and analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mascot online database queries allowed statistically significant identification of RW1 in disrupted, digested cells (P < 0.01 to 0.05) and in digested whole-cell extracts (P < 0.00001 to 0.05) containing hundreds of proteins, as determined by two-dimensional gel electrophoresis. Up to 14 peptide ions of the alpha subunit of the dioxin dioxygenase (43% protein coverage) were detected in individual samples. A minimum of 107 DF-grown cells was required to identify dioxin degradation-enabled phenotypes. The technique hinges on the detection of multiple characteristic peptides of a biomarker that can reveal at once the identity and phenotypic properties of the microbial host expressing the protein. The results demonstrate the power of PMF of minimally processed microbial cultures as a sensitive and specific technique for the positive identification and phenotypic characterization of certain microorganisms used in biotechnology and bioremediation.

* Corresponding author. Mailing address: JHU Center for Water and Health, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Room E6618, Baltimore, MD 21205. Phone: (410) 955-2609. Fax: (410) 955-9334. E-mail: rhalden{at}jhsph.edu.

Applied and Environmental Microbiology, May 2005, p. 2442-2451, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2442-2451.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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