Recombinant Peptide Production Softens Escherichia coli Cells and Increases Their Size during C-Limited Fed-Batch Cultivation.

Stress-associated changes in the mechanical properties at the single-cell level of Escherichia coli (E. coli) cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. The cell growth, peptide content, and cell lysis were analysed, and changes in the mechanical properties were investigated using atomic force microscopy. Recombinant-tagged somatostatin-28 was expressed as soluble up to 197 ± 11 mg g-1. The length of both cultivated strains increased throughout the cultivation by up to 17.6%, with nearly constant diameters. The peptide-producing cells were significantly softer than the non-producers throughout the cultivation, and respective Young's moduli decreased by up to 57% over time. A minimum Young's modulus of 1.6 MPa was observed after 23 h of the fed-batch. Furthermore, an analysis of the viscoelastic properties revealed that peptide-producing BL21(DE3) appeared more fluid-like and softer than the non-producing reference. For the first time, we provide evidence that the physical properties (i.e., the mechanical properties) on the single-cell level are significantly influenced by the metabolic burden imposed by the recombinant peptide expression and C-limitation in bioreactors.

Power Laws Describe Bacterial Viscoelasticity.

Bacterial cells survive in a wide range of different environments and actively tune their mechanical properties for purposes of growth, movement, division, and nutrition. In Gram-negative bacteria, the cell envelope with its outer membrane and peptidoglycan are the main determinants of mechanical properties and are common targets for the use of antibiotics. The study of bacterial mechanical properties has shown promise in elucidating a structure-function relationship in bacteria, connecting, shape, mechanics, and biochemistry. In this work, we study frequency and time-dependent viscoelastic properties of E. coli cells by atomic force microscopy (AFM). We perform force cycles, oscillatory microrheology, stress relaxation, and creep experiments, and use power law rheology models to fit the experimental results. All data sets could be fitted with the models and provided power law exponents of 0.01 to 0.1 while showing moduli in the range of a few MPa. We provide evidence for the interchangeability of the properties derived from these four different measurement approaches.