Investigating the Impacts of Growth Rate on Nitrogen Isotope Values in E.coli

Abstract

Nitrogen (N) is an essential component for the growth of all living organisms, due to the key role it plays in the synthesis of amino acids, proteins, ATP, and nucleic acids. Amino acids are one of the main N-containing compounds in cells and are the primary vehicle for N assimilation in cells, making them useful for studying metabolism and N cycling within and between organisms. The distribution of 15N isotope values in amino acids is used to study trophic relationships between macro-organisms, or as tracers of organic matter processing in sediments. There is also potential for 15N of amino acids to illuminate N cycling within microbial communities, but so far little is known about the intracellular cycling and allocation of N in individual microbes. In preliminary experiments, it was observed that free amino acids in cells were more enriched in 15N than protein-bound amino acids. One hypothesis for why this phenomenon occurs is that there is an unidentified fractionating process associated with protein synthesis, resulting in a kinetic isotope effect where lighter isotopes are incorporated into protein. Because the rate that proteins are synthesized and recycled increases with growth rate (Lahtvee et al., 2014), we attempted to indirectly test this hypothesis by varying growth rates. In this study, we used Escherichia coli to investigate whether different growth rates, and in turn different rates of protein synthesis, impact the isotope values between the free amino acids and protein bound amino acids and the magnitude of isotope fractionation.

Growth rates were altered by changing the growth temperature of E.coli. Experiments were conducted at different temperatures, including 15°C, 22°C, and 37°C (E. coli optimum). Both free and protein bound amino acids samples were purified and analyzed for δ15N on a GC-IRMS. Bulk samples were freeze dried and run on an elemental analyzer. We found that average N isotopic compositions of individual amino acids differed between temperature treatments. Free amino acids from 37°C cultures were the most enriched in δ15N whereas those from 15°C cultures were the least enriched. This provides some evidence that growth rate may play a role in controlling the isotope values between free and protein-bound amino acids. There is potential evidence that the magnitude of fractionation is greater at 37°C than at 15°C. Additional work for the future could investigate the effect of growth rate on amino acid δ15N values in different microbial groups or uncover the specific mechanism during protein synthesis that causes the observed fractionation.