We have designed an experimental/computational framework for studying complex phenotypes in bacteria.Our framework relies on whole-genome fitness profiling coupled with a module-level analysis to discover pathways that directly affect fitness.As a proof-of-principle, we studied ethanol tolerance in Escherichia coli and we identified key pathways that contribute to this phenotype.We then validated our findings through genetic manipulations, gene-expression profiling, metabolite-level measurements, and stable-isotope labeling.

Cellular metabolism converts available nutrients into usable energy and biomass precursors. The process is regulated to facilitate efficient nutrient use and metabolic homeostasis. Feedback inhibition of the first committed step of a pathway by its final product is a classical means of controlling biosynthesis1–4. In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate1,4,5. The physiological consequences of disrupting this regulation, however, have not been previously explored. Here we identify an alternative regulatory strategy that enables precise control of pyrimidine pathway end-product levels, even in the presence of dysregulated biosynthetic flux. The mechanism involves cooperative feedback regulation of the near-terminal pathway enzyme uridine monophosphate kinase6. Such feedback leads to build-up of the pathway intermediate uridine monophosphate, which is in turn degraded by a conserved phosphatase, here termed UmpH, with previously unknown physiological function7,8. Such directed overflow metabolism allows homeostasis of uridine triphosphate and cytidine triphosphate levels at the expense of uracil excretion and slower growth during energy limitation. Disruption of the directed overflow regulatory mechanism impairs growth in pyrimidine-rich environments. Thus, pyrimidine homeostasis involves dual regulatory strategies, with classical feedback inhibition enhancing metabolic efficiency and directed overflow metabolism ensuring end-product homeostasis.

A strain of Halomonas bacteria, GFAJ-1, has been reported to be able to use arsenate as a nutrient when phosphate is limiting, and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.Wolfe-Simon et al. isolated strain GFAJ-1 from the arsenic-rich sediments of California's Mono Lake by its ability to grow through multiple subculturings in artificial Mono Lake medium AML60 that lacked added phosphate but had high concentrations of arsenate (+As/ −P condition) (1). Because GFAJ-1 grew in −P medium only when arsenate was provided, and because significant amounts of arsenate were detected in subcellular fractions, growth was attributed to the use of arsenate in place of phosphate. However, the basal level of phosphate contaminating the −P medium was reported to be 3-4 M (1), which previous studies of low-phosphate microbial communities suggest is sufficient to support moderate growth (2). GFAJ-1 grew well on medium supplemented with ample phosphate but no arsenate (1500 M PO 4 , +P/-As condition), indicating that GFAJ-1 is not obligately arsenate-dependent. Wolfe-Simon et al.(1) further reported that arsenic was incorporated into the DNA backbone of GFAJ-1 in place of phosphorus, with an estimated 4% replacement of P by As based on the As:P ratio measured in agarose gel slices containing DNA samples. This † Corresponding author: redfield@zoology.ubc.ca. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript finding was surprising because arsenate is predicted to reduce rapidly to arsenite in physiological conditions (3, 4), and because arsenate esters in aqueous solution are known to be rapidly hydrolyzed (5). We have now tested this report by culturing GFAJ-1 cells supplied by the authors (1) and by analyzing highly purified DNA from phosphate-limited cells grown with and without arsenate.Wolfe-Simon et al. reported that GFAJ-1 cells grew very slowly in AML60 medium (doubling time ~12 hours), and that, when phosphate was not added to the medium, cells failed to grow unless arsenate (40 mM) was provided (1). However, although we obtained strain GFAJ-1 from these authors, in our hands GFAJ-1 was unable to grow at all in AML60 medium containing the specified trace elements and vitamins, even with 1500 M sodium phosphate added as specified in (1). We confirmed the strain's identity using RT-PCR and sequencing of 16S rRNA, using primers specified by Wolfe-Simon et al.(1); this gave a sequence identical to that reported for strain GFAJ-1. We then found that addition of small amounts of yeast extract, tryptone or individual amino acids to basal AML60 medium allowed growth, with do...

Due to the importance of microbes as model organisms, biotechnology tools, and contributors to mammalian and ecosystem metabolism, there has been longstanding interest in measuring their metabolite levels. Current metabolomic methods, involving mass spectrometry-based measurement of cell extracts, enable routine quantitation of most central metabolites. Metabolomics alone, however, is inadequate to understand cellular metabolic activity: Flux measurement and proteomic, genetic, and biochemical approaches with a metabolomics bent are all needed. Here we highlight examples where these integrated methods have contributed to discovery of metabolic pathways, regulatory interactions, and homeostasis mechanisms. We also indicate enduring challenges concerning unstable and low abundance compounds, subcellular compartmentalization, and quantitative amalgamation of different data types.Systems biology aims to explain physiological processes in terms of the concerted actions of numerous biochemicals. Microbial metabolism would seem to provide a promising arena for achieving this aim. Balanced growth in diverse environments a hallmark of microbial physiology is a metabolic capability. Microbes are relatively simple. Model microbes are also easy to grow and genetically tractable. The connections between metabolites, catalyzed by enzymatic transformations, are well mapped.Consistent with this promise, microbial metabolism has been among the fastest areas of systems biology to develop. A key driver in this regard has been the constraints-based computational approach of flux balance analysis (FBA). A remarkable finding has been that E. coli (although not most other microbes) often maximizes growth per unit of carbon source consumed: Fluxes are optimally efficient [1]. This raises key questions: What regulatory mechanisms lead to flux optimality? How are these mechanisms different in organisms that have different metabolic objectives? How do these mechanisms coordinate appropriate responses to changing nutrient availability?Metabolomics, defined broadly as the systems biology of metabolism, aims to address these questions. Metabolomics, defined more narrowly as comprehensive metabolite measurement, provides a critical tool for doing so. Figure 1 provides a schematic overview of microbial metabolomics and its integration with other experimental approaches. Metabolomics and genomics provide complementary tools for identifying metabolites and

Anapleurosis is the filling of the TCA cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite, phosphoenolpyruvate (PEP). Here we show that E. coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting build-up of PEP is used to quickly import glucose if it becomes re-available. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally on steady glucose. However, they fail to build-up PEP upon glucose removal, grow poorly on oscillating glucose, and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.