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Regulation underlying hierarchical and simultaneous utilization of carbon substrates by flux sensors in Escherichia coli.

ABSTRACT: Many microorganisms exhibit nutrient preferences, exemplified by the 'hierarchical' consumption of certain carbon substrates. Here, we systematically investigate under which physiological conditions hierarchical substrate utilization occurs and its mechanisms of implementation. We show utilization hierarchy of Escherichia coli to be ordered by the carbon-uptake flux rather than the identity of the substrates. A detailed study of glycerol uptake finds that it is fully suppressed if the uptake flux of another glycolytic substrate exceeds a threshold, which is set to the influx obtained when grown on glycerol alone. Below this threshold, limited glycerol uptake is 'supplemented' such that the total carbon uptake is maintained at the threshold. This behaviour results from total-flux feedback mediated by cAMP-Crp signalling but also requires inhibition by the regulator fructose 1,6-bisphosphate, which senses the upper-glycolytic flux and ensures that glycerol uptake defers to other glycolytic substrates but not to gluconeogenic ones. A quantitative model reproduces all of the observed utilization patterns, including those of key mutants. The proposed mechanism relies on the differential regulation of uptake enzymes and requires a specific operon organization. This organization is found to be conserved across related species for several uptake systems, suggesting the deployment of similar mechanisms for hierarchical substrate utilization by a spectrum of microorganisms.

SUBMITTER: Okano H

PROVIDER: S-EPMC6925339 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Regulation underlying hierarchical and simultaneous utilization of carbon substrates by flux sensors in Escherichia coli.

Okano Hiroyuki H Hermsen Rutger R Kochanowski Karl K Hwa Terence T

Nature microbiology 20191209 1

Many microorganisms exhibit nutrient preferences, exemplified by the 'hierarchical' consumption of certain carbon substrates. Here, we systematically investigate under which physiological conditions hierarchical substrate utilization occurs and its mechanisms of implementation. We show utilization hierarchy of Escherichia coli to be ordered by the carbon-uptake flux rather than the identity of the substrates. A detailed study of glycerol uptake finds that it is fully suppressed if the uptake flu ...[more]

PMID: 31819215

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Project description:As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.

Project description:Model topology is divided into two compartments, cell programming and performance testing. The cell programming compartment is split into history and pre-treatment. History ( History I or H1: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask after that. History II or H2: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask for 45 min. From this flask, 0.1O.D./ml transferred to rich medium and grown for 4 hours. From this,0.1O.D./ml transferred to fresh rich medium. History III or H3: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask for 45 min. From this flask, 0.1O.D./ml transferred to starvation medium and grown for 4 hours. From this,0.1O.D./ml transferred to fresh starvation medium. Pre-treatment (Pre-treatment 1(T1): 2.5g glucose/litre 5mM NH4Cl. Pre-treatment 2 (T2): 2.5g glucose/litre 0.25mM NH4Cl. Pre-treatment 3 (T3): - 0.25g glucose/litre 5mmM NH4Cl. Pre-treatment 4 (T4): 0.25g glucose/litre 0.25mM NH4Cl). Each pre-treatment given for 2.5 hours.The culture nomenclature indicates the adaptive path followed, for example, H1T1 indicates the culture has encountered history I (H1) and then transferred to pre-treatment 1 (T1).RNA was extracted for selected combinations. Performance testing : Performance testing describes the type of analysis done which is the growth pattern study onto three substrates, glucose, succinate and pyruvate. This performance testing revealed specific history-pretreatment combinations to be better suited for growth on certain substrate and some not suited for growth. The samples were harvested for RNA isolation at peak growth points and named worst_glucose, best_glucose,worst_succinate, best_succinate, worst_pyruvate and best_pyruvate according to the growth shown after testing all 12 history-pretreatment combinations. The differences in physiology were studied in details using microarray analysis of 13 samples including 3 history samples, 4 pre-treatment samples and 6 samples at performance testing level. RNA extraction was done using Qiagen RNeasy minikit (Germany). Standard Affymetrix protocol was followed for hybridization on Affymetrix E. coli Genome 2.0 Array.

Project description:BackgroundEnterohemorrhagic Escherichia coli (E. coli) are intestinal pathogenic bacteria that cause life-threatening disease in humans. Their cardinal virulence factor is Shiga toxin (Stx), which is encoded on lambdoid phages integrated in the chromosome. Stx phages can infect and lysogenize susceptible bacteria, thus either increasing the virulence of already pathogenic bacterial hosts or transforming commensal strains into potential pathogens. There is increasing evidence that Stx phage-encoded factors adaptively regulate bacterial host gene expression. Here, we investigated the effects of Stx phage carriage in E. coli K-12 strain MG1655. We compared the transcriptome and phenotype of naive MG1655 and two lysogens carrying closely related Stx2a phages: ?O104 from the exceptionally pathogenic 2011 E. coli O104:H4 outbreak strain and ?PA8 from an E. coli O157:H7 isolate.ResultsAnalysis of quantitative RNA sequencing results showed that, in comparison to naive MG1655, genes involved in mixed acid fermentation were upregulated, while genes encoding NADH dehydrogenase I, TCA cycle enzymes and proteins involved in the transport and assimilation of carbon sources were downregulated in MG1655::?O104 and MG1655::?PA8. The majority of the changes in gene expression were found associated with the corresponding phenotypes. Notably, the Stx2a phage lysogens displayed moderate to severe growth defects in minimal medium supplemented with single carbon sources, e.g. galactose, ribose, L-lactate. In addition, in phenotype microarray assays, the Stx2a phage lysogens were characterized by a significant decrease in the cell respiration with gluconeogenic substrates such as amino acids, nucleosides, carboxylic and dicarboxylic acids. In contrast, MG1655::?O104 and MG1655::?PA8 displayed enhanced respiration with several sugar components of the intestinal mucus, e.g. arabinose, fucose, N-acetyl-D-glucosamine. We also found that prophage-encoded factors distinct from CI and Cro were responsible for the carbon utilization phenotypes of the Stx2a phage lysogens.ConclusionsOur study reveals a profound impact of the Stx phage carriage on E. coli carbon source utilization. The Stx2a prophage appears to reprogram the carbon metabolism of its bacterial host by turning down aerobic metabolism in favour of mixed acid fermentation.

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Regulation underlying hierarchical and simultaneous utilization of carbon substrates by flux sensors in Escherichia coli.

Publications

Regulation underlying hierarchical and simultaneous utilization of carbon substrates by flux sensors in Escherichia coli.

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