The metabolic potential of Escherichia coli BL21 in defined and rich medium

Abstract

BackgroundThe proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis.ResultsThe presence of preformed biomass precursor molecules in rich media such as Luria Bertani supported rapid growth concomitant to acetate formation and apparently unbalanced abundances of central metabolic pathway enzymes, e.g. high levels of lower glycolytic pathway enzymes as well as pyruvate dehydrogenase, and low levels of TCA cycle and high levels of the acetate forming enzymes Pta and AckA. The proteome of cells growing exponentially in glucose-supplemented mineral salt medium was dominated by enzymes of amino acid synthesis pathways, contained more balanced abundances of central metabolic pathway enzymes, and a lower portion of ribosomal and other translational proteins. Entry into stationary phase led to a reconstruction of the bacterial proteome by increasing e.g. the portion of proteins required for scavenging rare nutrients and general cell protection. This proteomic reconstruction during entry into stationary phase was more noticeable in cells growing in rich medium as they have a greater reservoir of recyclable proteins from the translational machinery.ConclusionsThe proteomic comparison of cells growing exponentially in different media reflected the antagonistic and competitive regulation of central metabolic pathways through the global transcriptional regulators Cra, Crp, and ArcA. For example, the proteome of cells growing exponentially in rich medium was consistent with a dominating role of phosphorylated ArcA most likely a result from limitations in reoxidizing reduced quinones in the respiratory chain under these growth conditions. The proteomic alterations of exponentially growing cells into stationary phase cells were consistent with stringent-like and stationary phase responses and a dominating control through DksA-ppGpp and RpoS.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions

“…Gnd, TktA) have previously been found in lower amounts in cells growing under alkaline conditions [27]. We observed down-regulation of both transketolase A and B (TktA, TktB) involved in the nonoxidative branch of the pentose phosphate pathway in contrast to other studies where TktA and TktB have been suggested to be regulated in opposite ways, for instance the TktB gene is induced while TktA is repressed by RpoS [28].…”

“…Under alkaline conditions this is executed through active influx of protons and restrained outflux of protons from the cytosol. At aerobic conditions, E. coli produce NADH and FADH 2 through the TCA cycle, these reducing equivalents are oxidized in the respiratory chain, and the electrons generated from the reducing equivalents are subsequently transferred to cytochromes where O 2 is converted to H 2 O [27]. This process is coupled to the formation of a proton motive force (PMF) over the cytoplasmic membrane, which is utilized for ATP generation from ADP and Pi through the ATP synthase complex.…”

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

“…59 alkylAPs may enter cells by diffusion through porins 59 or by active nutrient transporters as in the cases of fosmidomycin, 60 fosfomycin, 61 and D-cycloserine. 62 Nutrient limitation itself may alter expression of transporters that aid cellular uptake for the alkylAP analogs 63 or lacks compounds found in rich medium that may otherwise serve to interfere with transport of the analogs (akin to the inhibited uptake of the antibiotic D-cycloserine by D-alanine through the D-alanine−glycine transport system). 62 Finally, some combination of effects altering cellular metabolism and alkylAP uptake could certainly be at play to cause this striking difference in alkylAP activity accompanying the change in growth environment.…”

RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

“…9a), a reduction in AceE levels might be expected, but we observed instead an increase in activity, transcript levels, and transcript half-life. Additionally, transcript levels of aceE have been shown to decrease significantly upon entry into stationary phase (51), and even if this reduction occurs in both the WT and the rnc deletion mutant, the observed increase in the half-life of aceEF-lpd (~5 min compared to ~3 min) could explain the observed increase in PDH activity in early stationary phase in the rnc deletion mutant. The effect of these processing events on transcripts encoding metabolic enzymes is complex because, although RNase III is known to be constitutively expressed (and autoregulates its own mRNA decay [29]), its activity has been shown to be affected by a protein inhibitor as well as by other, unknown factors (5).…”