The influence of a recombinant plasmid on the formation of escherichia coli biofilms

Abstract

The Gram-negative bacteria Escherichia coli is one of the most widely used organisms in biotechnology. Despite this fact studies using E. coli as a model organism on bacterial biofilm formation are rare compared to the wealth of information concerning other bacteria like for instance the Pseudomonas genus. E. coli plasmids are used as vectors for recombinant protein production and in order to ensure plasmid stability on the cell a resistance marker for an antibiotic is often included thus preventing the growth of cells that have lost the plasmid. Cells harbouring plasmids face two different challenges that impact on their metabolism: they have to deal with the metabolic burden of maintaining the plasmids at a certain copy number and they have to produce an agent that will confer resistance to the antibiotic. Plasmid maintenance requires the replication and proofreading of large segments of DNA which is consuming both in terms of the energy drain involved but also on the expenditure of metabolic precursors such as nucleotides. Regarding the antibiotic, the resistance agent is often an enzyme that will act upon the antibiotic reducing or eliminating its activity. In this case protein synthesis is involved and precursors such as amino acids and energy are consumed. On this work we have assessed the impact of using E. coli cells that have been transformed with a plasmid conferring resistance to kanamycin and grown under selective pressure with the behaviour of non-transformed cells growing on a glucose containing medium in the absence of selective pressure. We have evaluated the cell growth on the planktonic state, the glucose consumption as an indicator of the energy drain and we have characterised the formed biofilms regarding their weight and thickness. By analyzing the optical density of the planktonic cultures we observe that higher cell densities are obtained when the cells were not transformed with the plasmid. Although this may be an indication of the metabolic burden caused by plasmid maintenance and/or gene expression, we have also observed that the glucose consumption profiles are similar on both situations with higher glucose consumption for the transformed cells. By analysing the obtained biofilms we observe that thicker and heavier biofilms are obtained. We thus postulate that the metabolic load involved in maintaining the plasmid and/or the burden to express a resistance gene are major contributors to physiological stress that favours biofilms formation.