K-093. The Legacy of HfrH: Mutations in the Two-Component System CreBC Modify the Phenotype of an Escherichia coli arcA Mutant

P. I. Nikel1,2, A. de Almeida1, M. J. Pettinari1, B. S. Mendez1;
1Facultad de Ciencias Exactas y Naturales, Univ. de Buenos Aires, Ciudad Autonoma de Buenos Aires, ARGENTINA, 2Instituto de Investigaciones Biotecnologicas, Univ. Nacional de San Martin, Buenos Aires, ARGENTINA.

Background: Escherichia coli can adapt quickly to changing environmental conditions by regulating gene expression through a complex transcription regulatory network. The two-component signal transduction systems ArcAB and CreBC respond to O2 supply and carbon source availability, respectively. Targets for CreBC regulation are not clear, but some genes are activated (ackA/pta, creD, talA) while at least malE is repressed. In previous work, we demonstrated that E. coli CT1061 (arcA::IS10-L, constructed by P1 transduction) is a suitable host for synthesis of reduced bioproducts. Our hypothesis was that CreBC could be also involved in its particular phenotype. Methods: The arc-cre region was PCR-amplified from strains K1060 (wild-type) and their derivatives CT1061 y CT1062 (ΔarcA), and sequenced. Cytochromes o and d content was measured (both regulated by ArcAB), as well as AckA activity (regulated by CreBC). Results: While CT1061 showed small colonies when grown in toluidine blue agar, CT1062 did not grow (as expected for arc null mutants). CT1062 remained dye-sensitive when complemented with the arcA::IS10-L allele from CT1061. Western blot analysis also confirmed that CT1061 is an arc null mutant. We thought that other mutations could be co-transduced along with the insertion in arcA. Cytochromes o and d content was similar for both arc strains, also suggesting that another gene could be responsible of the metabolic differences. By sequencing the region adjacent to arc in CT1061, we found a mutation in creC (causing a R77P-substitution, which confers a CreC constitutive phenotype). This mutation was also observed in strains ECL618 (donor for P1 transduction) and Hfr3000 (the ancestor strain from which ECL618 is derived), but was absent in K1060 and CT1062. AckA activity, measured in exponential cultures using glucose as the carbon source, was 10.7 ± 2.1 and 2.5 ± 0.7 U · mg protein-1 for CT1061 and K1060, respectively. AckA activity in CT1062 was similar to that of K1060, while ECL618 and Hfr3000 showed increased activities. Conclusion: Our results suggest that mutations in both arcA and creC contribute to the metabolic traits of E. coli CT1061.