METHODS AND PLATFORMS 



I have designed the methodological approach that I am taking specifically to avoid the 

 artifacts that may arise from bottle incubations. We are using 2-D gel electrophoresis to examine 

 proteins produced by cultured bacteria grown under specific physiological conditions. Our 

 purpose is to identify proteins that can be used as general biomarkers of inorganic N/P utilization 

 by bacteria. Appropriate proteins will be used for antibody production and these antibodies will 

 be applied to natural assemblages of bacteria to assess the physiological status of the bacteria. 



STRENGTHS AND LIMITATIONS OF PROPOSED RESEARCH 



A strength of this methodological approach is the ability to assess the physiological 

 condition of bacterial assemblages without prior incubation. Artifactual results due to bottle 

 incubation continues to be a major problem in microbial ecology. Another advantage of this 

 method is that it provides a means for subsequent identification of genes producing useful 

 biomarkers (via protein sequencing and gene isolation). This has been a powerful approach for 

 identifying genes associated with specific physiological abilities. This approach has not yet been 

 heavily exploited in marine research. This latter work (gene identification) is beyond the 

 immediate scope of this project but it will provide useful information to guide future research. 



Potential limitations of my approach involve the significant lead time for the identification 

 and isolation of proteins and the production and testing of antibodies. Another problem that 

 could arise is the lack of conservation among diverse bacterial taxa of the proteins associated 

 with inorganic nitrogen and phosphorus utilization. However, this conceptual approach has 

 proven fruitful for other projects using similar methodology. 



STATUS OF RESEARCH 



I have recently hired a postdoctoral investigator on this project. It took more time than 

 anticipated to identify a suitable candidate for this work, but the person I have hired is extremely 

 appropriate and well trained for this work. At this time we have obtained suitable bacterial 

 species for the culture work. We have tested these species in a variety of media designed to 

 specifically elicit (or repress) proteins associated with inorganic nitrogen and phosphorus uptake 

 and utilization. We are in the process now of performing and analyzing 2-D protein gels from 

 bacteria grown in batch cultures. We will expand this work in batch culture to include 

 continuous culture of these microorganisms within the next several weeks. Continuous culture 

 will permit us to grow bacteria under specific conditions of limitation in order to examine 

 changes in protein signatures with different physiological states. We have also recently acquired 

 and installed a Sparc 10 SUN workstation equipped with software for the analysis and 

 comparison of 2-D gels. Gel comparison is a key issue in distinguishing proteins that might be 



24 



