Appendix II 

 Molecular Approaches to Understanding Energetics 



Molecular approaches to understanding the inherent 

 limitations of metabolic processes require measurements of internal 

 fluxes. Molecular techniques for measuring fluxes are difficult to 

 apply in natural systems; however, some areas of research are 

 promising. 



Molecular Methods for Estimating Growth 



RNA/DNA ratios have been used (with mixed success) to estimate 

 bulk growth rates of microbes in situ . Can these measurements be 

 coupled to single-cell analysis by use of fluorescent probes for 

 DNA and RNA? These, in turn, can be detected and quantified by 

 flow cytometry or by quantitative epifluorescence microscopy. 



Fluorescently labelled antibodies to cell-division cycle (CDC) 

 proteins may indicate dormant vs. active stages. Can such 

 techniques be used to calculate growth rates, using algorithms 

 similar to those used in frequency of dividing cell (or frequency 

 of DNA replication) methods for growth-rate determinations 

 (Carpenter and Chang, 1988)? 



If these life-cycle stages are metabolic stages, i.e., dormant 

 vs. active, photoautotrophic vs. mixotrophic, they may be 

 characterized by different rates and/or by different pathways. If 

 there are different pathways, are they characterized by different 

 products that can be used as biomarkers to identify those life- 

 cycle stages in situ ? If these stages and their associated 

 pathways are also correlated with different rate functions, we may 

 be able to deduce rates by presence of the molecular biomarkers. 



Flow cytometry has been used to enumerate bacteria in aquatic 

 systems. The technique would be useful in looking for an organism 

 that comprises less than 1% of the population. The drawback to 

 flow cytometry is that it fragments consortia. However, if the 

 proper methods are found, particulate matter containing bacteria or 

 intact communities possibly could be sorted to obtain much the same 

 information as described above. 



Evidence from the response of starved E. coli (Matin et al . , 



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