Chapter 4
Microbial Characterization by Phospholipid Profiling Downloaded by UNIV MASSACHUSETTS AMHERST on July 29, 2012 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0541.ch004
Practice and Problems in Microbial Identification 1
Mark J. Cole and Christie G. Enke
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1
Drug Metabolism Department, Central Research Division, Pfizer, Inc., Groton, CT 06340 Department of Chemistry and Center for Microbial Ecology, Michigan State University, East Lansing, MI 48823
2
The variety and relative abundance of membrane phospholipids are widely thought to be unique for each microorganism. Tandem mass spectrometry has been shown capable of rapidly and sensitively characterizing the phospholipid content of crude microbial lipid extracts. This work confirms that the large data sets containing the masses and fatty acyl compositions for each phospholipid class obtainable by this technique include not only valuable taxonomic information but are also strongly influenced by environmental factors, including the growth conditions, growth medium, sample history, and the act of culturing itself. Due to the large size of these data sets and the limited amount of such data yet available, correlations between the various elements of the data sets and the factors of identity, history, activity, and function have not been obtained. Though clearly a powerful technique, the full utilization of phospholipid profiling by tandem mass spectrometry for both environment-independent taxonomy and studies on microbial function and activity awaits the development of suitable data analysis tools. A multitude of different biomolecules are needed by each microbial species to perform the functions required to fill, and survive in, its niche in nature. The natural selective forces imposed on different microorganisms have required microbes to adapt and optimize specific sets of biomolecules for survival in specific environments. A few examples of these biomolecules include the lipids needed for membrane function and integrity, the respiratory quinones needed for metabolism, and the polysaccharides needed for protection from the environment. The particular collection of these molecules incorporated in each microbial species is unique to that species. In fact, individual subsets of this complete collection of biomolecules are often unique to a particular species. Identification of these unique subsets can thus provide biomarkers for identification of microorganisms. The diversity of lipids in microorganisms signifies a diversity of functions. Lipids, either directly or indirectly, play a significant role in the specialized functions each species maintains to survive in its particular environmental habitat. Lipids are critical to many vital functions including storage, membrane structure and function, photosynthetic processes, and most energy-generating processes. The types and 0097-6156/94/0541-0036$07.50/0 © 1994 American Chemical Society In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
Downloaded by UNIV MASSACHUSETTS AMHERST on July 29, 2012 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0541.ch004
4. COLE & ENKE
Microbial Characterization by Phospholipid Profiling
distribution of lipids contained in microorganisms can be very species specific (7-5). However, not all of the cellular lipids are easily accessed. Phospholipids, which are responsible for the structure of the cell membrane, and thus, ultimately its function, are easily accessed through simple extraction procedures and provide a useful and abundant biomarker for microbial detection and identification. In addition, phospholipids are readily amenable to desorption-mass spectrometric techniques. This makes an attractive package for microbial identification: an abundance of specific information contained within the phospholipid content, coupled with the speed and sensitivity of mass spectrometry. Despite the above attractions, the use of phospholipids for microbial identification is daunting. Success depends on two factors: achieving high discriminating power and correlating the data with the identification. Mass spectrometry has been shown to provide the needed discriminating power (4,5), and tandem mass spectrometry has been particularly successful (6, 7). Only two uses of phospholipid data for the general identification of microorganisms have been reported (8,9), but neither of these have been applied to large data sets. Paradoxically, the qualities that make membrane phospholipids attractive for microbial identification - the abundance of information contained in the phospholipids and the functional uniqueness of the phospholipid membrane composition for each microorganism - also make analysis of these data difficult The difficulty arises in two areas: first, the growth conditions, nutritional status, and history of a microorganism can cause changes in its phospholipid profile as the microbe changes its membrane composition in response to its environmental requirements, and secondly, the amount of data generated from the phospholipids rapidly exceeds the capacities of the common data analysis methodologies in existence today. This research approaches the problem of microorganism identification through: 1) developing a general method for rapidly and sensitively characterizing the phospholipid content and structures in crude lipid extracts; 2) using this methodology to probe the extent to which the phospholipid data varies as microorganisms adjust to environmental stresses; 3) exploring the types of data obtained and the possibilities for new data analysis tools. Phospholipid Profiling The phospholipids with which this research is concerned are the glycerophospholipids (Structure 1). Glycerophospholipids consist of four primary functional groups: a glycerol-3-phosphate core on which two fatty acyls (R, R') have been esterified to the two free hydroxyl groups in the sn-1 and sn-2 positions, and a second alcohol (Y) has been esterified to the phosphate group in the sn-3 position. The exception to this basic structure is phosphatide acid, which only contains the phosphate group. The head group (Y) is the functional group that defines the specific class to which the phospholipid belongs, while the fatty acyls distinguish the individual phospholipid molecular species within each class. Examples of head groups from several phospholipid classes are shown in Figure 1. Crude lipid extracts are obtained using a modified Bligh-Dyer procedure (10). Phospholipid Dissociation Under Low-Energy C A D Conditions. When phospholipid ions undergo low-energy C A D , only a few different dissociation product ion masses are formed. However, the C A D product ions that are produced are the result of cleavages at specific points that are common to all classes of phospholipids. These cleavages occur around the phospholipid functional groups and thus provide significant structural information about the phospholipid. The lowenergy C A D fragmentation of phospholipids for both positive and negative ions is shown in Figure 2. In the positive ion mode, the major reaction occurring is cleavage of the phosphate/glycerol bond resulting in the loss of the polar head group as a
In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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MASS SPECTROMETRY FOR CHARACTERIZATION OF MICROORGANISMS
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Downloaded by UNIV MASSACHUSETTS AMHERST on July 29, 2012 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0541.ch004
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