Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli

Abstract

To get further information on bacterial surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless lacZ gene, was performed with an ompR234 adherent strain, and a simple screen was developed to assess changes in gene expression in biofilm cells versus planktonic cells. This screen revealed that major changes in the pattern of gene expression occur during biofilm development: the transcription of 38% of the genes was affected within biofilms. Different cell functions were more expressed in sessile bacteria: the OmpC porin, the high-affinity transport system of glycine betaine (encoded by the proU operon), the colanic acid exopolysaccharide (wca locus, formerly called cps), tripeptidase T (pepT), and the nickel high-affinity transport system (nikA). On the other hand, the syntheses of flagellin (fliC) and of a putative protein of 92 amino acids (f92) were both reduced in biofilms. Such a genetic reprogramming of gene expression in biofilms seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase.

FIG. 1

Kinetics of osmoregulated lacZ fusion expression in attached and free-living bacteria. Specific β-galactosidase activities of ompC-lacZ (A), proU-lacZ (B), fliC-lacZ (C), and wcaB-lacZ (D) fusions were measured in free-living (open squares) and sessile (filled squares) bacteria during biofilm development on petri dishes in M63-mannitol medium (A, B, and C) or on Plexiglas strips in M63-glucose medium (D) as described in Materials and Methods. Results are means ± standard deviations of 3 or 10 (∗) independent measurements.

FIG. 2

Electron micrographs of negatively stained bacteria of planktonic (A) or biofilm (B) cell suspensions of the motile PHL628 strain. The cells were grown in M63-mannitol medium, and biofilms were formed on plastic strips. The micrographs, corresponding to observations made after 24 h of culture, are representative of several microscopic observations performed at between 8 and 48 h of biofilm development.

FIG. 3

Comparison of down-regulated lacZ fusion expression (strain PHL825) in attached and free-living bacteria during biofilm development on petri dishes (A) and in shaken liquid cultures under different osmolarity conditions (B). (A) Biofilm cultures were performed on petri dishes in M63-mannitol medium, and specific β-galactosidase activities in free-living (open squares) and sessile (filled squares) bacteria were measured as described in Materials and Methods. Results are means ± standard deviations of three independent measurements. (B) Cells were grown at 30°C in low-osmolarity MOPS-glycerol medium supplemented with increasing NaCl concentrations of 0 to 0.3 M. Two independent measurements of specific β-galactosidase activities were performed on mid-exponential-growth-phase cells. Results are means ± standard deviations of two independent measurements.

FIG. 4

Kinetics of proU-lacZ (A) and wcaB-lacZ (B) fusion expression in attached and free-living bacteria of adherent ompR wild-type strains (PHL1064 and PHL1036, respectively) overproducing the CsgD specific activator of the curlin-encoding csgBA operon. Specific β-galactosidase activities were measured in free-living (open squares) and sessile (filled squares) bacteria during biofilm development on petri dishes in M63 medium supplemented with ampicillin and mannitol (A) or glucose (B), as described in Materials and Methods. Results are means ± standard deviations of three independent measurements.

FIG. 5

Comparison of nikA-lacZ expression in attached and free-living bacteria grown under standard conditions (A) or in anaerobiosis (B). Biofilm cultures were performed in M63-mannitol medium on petri dishes incubated in Generbox anaerobic jars (B) or not (A) for 23 and 45 h and specific β-galactosidase activities were measured in free-living (open bars) and sessile (hatched bars) bacteria as described in Materials and Methods. Results are means ± standard deviations of three independent measurements.

FIG. 6

Kinetics of up-regulated lacZ fusion expression (strain PHL665) in attached and free-living bacteria (A) and comparison of fusion expression under standard conditions and during anaerobiosis after 28 h of biofilm cultures (B). Biofilm cultures were performed in M63-mannitol medium on petri dishes, and specific β-galactosidase activities were measured in free-living bacteria (open squares and bars) and sessile bacteria (filled squares and hatched bars) as described in Materials and Methods. Results are means ± standard deviations of three independent measurements.

FIG. 7

Effects of cell density on expression of the pepT-lacZ fusion. (A) The PHL665 strain was grown in M63-mannitol medium in a shaker at 30°C. Samples were taken at intervals for determination of OD₆₀₀ (open circles) and specific β-galactosidase activity (filled circles). (B) Bacteria were grown in LB medium (open squares) or conditioned LB medium prepared as described in Materials and Methods from a stationary-phase culture of strain DH5α (filled squares). Growth curves in the two media were similar. This experiment is representative of three.