Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

Abstract

Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13 C-labeling experiments in chemostat cultures of a wild-type strain, ⌬creB and ⌬arcA single mutants, and a ⌬creB ⌬arcA double mutant. Continuous cultures were conducted at D ‫؍‬ 0.1 h ؊1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the EmbdenMeyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ⌬arcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ⌬arcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.Transcriptional regulation comprises a complex network of global and specific regulators. Global regulators normally have pleiotropic effects on metabolism, since they control the transcription of several operons that belong to different functional groups. In Escherichia coli, seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate expression of about one-half of all genes (41). CreBC and ArcAB are two global sensing and regulation systems in E. coli which, along with some other regulatory systems, modulate the expression of genes involved in central metabolism and respiratory pathways allowing rapid adaptive responses to carbon source and oxygen availability in the culture environment.The cre (for carbon source responsive) locus comprises creABCD and was formerly known as the phoM locus (4). Whereas creA is a hypothetical open reading frame and creD, also known as cet, encodes an inner-membrane protein of unknown function, creB and creC encode a two-component system, i.e., a cytoplasmic regulator and a sensor kinase, respectively. After the discovery of CreBC, genes modulated by this system proved elusive. Based on sequence analysis and using bioinformatic tools Avison et al. (5) were able to define a cre tag sequence, to which CreB binds in vitro (8), in order to describe the...

YieJ (CbrC) Mediates CreBC-Dependent Colicin E2 Tolerance in Escherichia coli

“…In A. hydrophila, cepH expression is controlled by the BlrAB two-component system. The signal sensor BlrB is 65% identical to CreC (21), and the response regulator, BlrA, is 63% identical to CreB (1,21) and uses the same Cre tag motif to bind DNA and thus activate gene expression (6). Hence, cepH effectively has an upstream Cre tag and becomes part of the Cre regulon when cloned into E. coli (5,6).…”

CbrA Is a Flavin Adenine Dinucleotide Protein That Modifies the Escherichia coli Outer Membrane and Confers Specific Resistance to Colicin M

“…CreBC is a global two-component regulator of gene expression, with CreC as a sensor of unknown signals. CreBC-controlled transcription is initiated when cells are shifted from a rich medium to a poor medium, when cells are grown in minimal medium with fermentable glycolytic carbon sources, or when cells are grown aerobically with fermentation products as carbon sources (8,27).…”

Lactoferricin B Inhibits the Phosphorylation of the Two-Component System Response Regulators BasR and CreB

“…CreBC has been identified as an important metabolic regulator in a recent study (37). Although no direct relation between the CreBC of microbes and the metabolism within the gastrointestinal tract has been reported, previous studies have demonstrated that CreBC responds to the change of the carbon sources that are utilized by microbes (39).…”

“…Nikel et al (37) reported that the mutant strain of creB had a low glucose uptake rate and an abnormal biomass condition. Because CreB is a response regulator that is activated when bacteria are grown in minimal medium, we observed the antimicrobial capability of Lfcin B in minimal medium to determine whether Lfcin B may exhibit an enhanced effect on bacteria.…”