Study of Escherichia coli global regulator CreC in response to different oxygen avalability: its effect over LDH and ACK activity and organic acid formation.

GODOY, MANUEL S.; ZALDUENDO, MERCEDES; PETTINARI M. JULIA

Mar del Plata

Congreso; IX Congreso de la Sociedad Argentina de Microbiología General (SAMIGE 2013); 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Global regulators (GR) in E. coli work hierarchically to regulate a complex network of genes in response to enviromental conditions and resource availability. Most of them are well characterized (ArcA, Fnr, FadR, etc). Nevertheless, some GR are poorly known, such as CreBC, a two-component system involved in the regulation of carbohydrate catabolism. To investigate the effect of CreC in E. coli metabolism, we studied the relationship of some physiological pameters with oxygen availability in three strains: K1060 (wt) DC1060 (ΔcreC) and K1060C (creC const). Metabolite production and enzymatic activity of two important fermentation enzimes (LDH and ACK), were measured in three diferent oxygen availablity conditions, determined by the agitation speed and the the volume of the flask to media volume ratio as follows: hight aeration (HA) with 250rpm and 1/10 Vflask/Vmedia, low aeration (LA) 125 rpm and ½ Vflask/Vmedia, and anaerobiosis (AA) 4 rpm and 1/1 Vflask/Vmedia in sealed bottles. A mineral medium (M9) supplemented with glucose was used. As expected, no significant differences were observed between strains in HA, with the exception of acetate, the main acid secreted, and ethanol. The production of acetate was slightly lower in DC1060. In microaerobiosis, DC1060 produced more acetate and less lactate than K1060. DC1060 had 50% more ACK and 40% less LDH activities compared to the wild type strain, showing good correlation between enzyme activity and metabolite concentration. In AA, K1060 and K1060C had poor growth compared to DC1060. Acetate production decreased for all strains compared to LA, but it drecreased more in K1060 and K1060C. Once again, ACK activity was much (60%) higer in DC1060 than in K1060, but surprisingly, when aerobic respiration was supressed, LDH activiy and lactic acid production had the opposite trend from that observed in LA: they were higher for DC1060 than for K1060. To assess whether differences in growth and acid secretion profiles were mediated by CreB, all three mutants (∆creB, ∆creC and ∆creBC), and the wild type as the control, were grown in AA to compare their growth rates and organic acid profiles. No significative differences were seen among the mutant strains, neither on biomass formation nor in the metabolites produced, indicating that CreC acts mainly by means of the CreB response regulator in this condition. These results show that the effects of CreC over metabolism seem to be different depending on oxygen availability, and that this regulation in AA is most probably mediated by CreB (for which the ligand, according to literature, must be a metabolite present during fermentation). Our results suggest that the variations in metabolic distribution observed in this work in the different oxygen availablity conditions analyzed can be the product of the concerted action of several regulatory systems, that affects metabolic pathways at different levels. Further research will be dedicated to study these interactions.