Abstract
Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g. mediated by small regulatory RNAs.
Highlights
Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation
Changes that occur in the central carbon metabolism at the onset of anaerobiosis are related to the need for alternative ways to maintain a proper intracellular redox balance because molecular oxygen, the terminal electron acceptor of the aerobic electron transport chain, is no longer available to remove reducing equivalents formed in catabolism
When grown in aerobic batch cultures with glucose as the energy source and in the absence of alternative electron acceptors, E. coli stops growing upon a switch to anaerobiosis for about 20 min and resumes growth at a slower pace [23]
Summary
Synthesis of L-Azhal—L-Azhal was synthesized from L-Boc-2,4diaminobutyric acid (Chem-Impex, Wood Dale, IL) as described previously [9]. Cells were split and transferred to either a fully anaerobic culture vessel (under nitrogen) or an aerobic culture vessel, both containing M9 minimal medium in which the methionine was replaced by 400 mg/liter azhal. Shifted fractions of secondary runs enriched in TCEP-induced reaction products from azhal-containing peptides were analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-tandem MS) as described in detail previously [9]. Non-shifted pooled fractions were collected, resuspended in 400 l of 0.1% TFA with the addition of 150 pmol of human [Glu1]fibrinopeptide B (Sigma-Aldrich) for internal calibration Of these samples, 5 l was injected and analyzed as described for the shifted fractions. Quantitation as described previously [9], whereas the iTRAQ reporter ions from peptides not containing azhal or methionine were used to quantify changes in protein levels. Proteins that changed more than 1.5-fold and for which the p value was adjusted for a false discovery rate of less 0.05 due to multiple testing [20] were considered to have a significantly altered expression level
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