Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors

Abstract

Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development. Escherichia coli is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of E. coli by taking control of two biofilm-promoting factors, autotransported antigen 43 adhesin and the bscABZC cellulose operon. The modulation of the biofilm formation capacities of E. coli or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.

FIG. 1.

Construction of the RExTET and PcL transcriptional fusions. (A) RExTET fusions were constructed in three steps, as follows: (i) cloning of the tetR gene with its own constitutive promoter and terminator T1 downstream of the nptII gene encoding resistance to kanamycin and upstream of the t₀ terminator, on the pZE21-gfp plasmid; (ii) amplification of the 2,537-bp kmRExTET or the 2,555-bp kmRExTETrbs cassette by using primers annealing to the 3′ end of the t₀ terminator and just upstream from the 5′ end of the RBS sequence (1; kmRExTET; no RBS) or just upstream from the 5′ end of the gfp gene (2; kmRExTETrbs; RBS present in the cassette); (iii) insertion of the kmRExTET or kmRExTETrbs cassette by three-step PCR upstream of the RBS sequence or at the start codon of the target gene (lacZ, flu, or bcsA, as indicated), respectively. (B) Construction of constitutive PcL fusions by (i) amplification of the 1,222-bp kmPcL, 1,281-bp ampPcL, 1,254 kmPcLrbs, or 1,313-bp ampPcLrbs cassette by using primers annealing to the 3′ end of the t₀ terminator and just upstream from the 5′ end of the RBS sequence (1; kmPcL and ampPcL; no RBS) or just upstream from the 5′ end of the gfp gene (2; kmPcLrbs and ampPcLrbs; RBS present in the cassette) and (ii) insertion of the PcL or PcLrbs cassette by three-step PCR upstream of the RBS sequence or at the start codon of the target gene (lacZ or flu, as indicated), respectively.

FIG. 2.

Gene expression can be modulated by RExTET cassettes and can be taken over by PcL cassettes. β-Galactosidase activity measurements of RExTET and PcLlacZ fusions. Strains were grown up until stationary phase in M63B1glu medium at 37°C. Concentrations (nanograms per milliliter) of aTc, when added, are indicated below the bars. MG1655 grown in the presence of 1 mM IPTG was considered to demonstrate the wild-type expression of the lac operon. The experiments were performed in triplicate; error bars represent standard deviations of the means. All the results were qualitatively the same with cultures grown in LB medium (data not shown), with slight changes in the levels of enzymatic activity and aTc concentrations needed to reach the plateau. +, present; −, absent.

FIG. 3.

Modulation of Ag43 production by using PcL and RExTET cassettes. (A) RExTET and PcL cassettes can modulate Ag43 production. An amount of each culture equivalent to an OD₆₀₀ of 0.2 was loaded onto a sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis gel. Immunodetection was performed using a polyclonal rabbit antiserum raised against the α domain of Ag43. Concentrations (in nanograms per milliliter) of aTc, when added, are indicated above the lanes. (B) Overnight cultures of the different strains were grown at 37°C in LB medium without aTc or, for strain MG1655kmRExTETrbs-flu, with 50 ng of aTc/ml. After fixation and successive incubations with a 1:1,000 dilution of a primary polyclonal rabbit antiserum raised against the α domain of Ag43 and with a 1:300 dilution of a secondary polyclonal goat anti-rabbit serum coupled to Alexa488 along with DAPI, cells were observed with a 600× objective under oil immersion. A DAPI filter was used to reveal the bacterial nucleoid (in blue), and a green fluorescent protein filter was use to reveal the presence of Ag43 (in green) at the cell surface. (C) Closeup of a culture of MG1655kmRExTETrbs-flu in which the Ag43 production had been induced with 50 ng of aTc/ml. The cell surface localization of Ag43 is clearly demonstrated, as is the integrity of the bacterial membrane. The right panel shows the superimposition of the two preceding images.

FIG. 4.

Modulation of bacterial autoaggregation via controlled production of the Ag43 protein. The different strains were grown overnight in LB with increasing concentrations of aTc ranging from 1 to 50 ng/ml when indicated. After growth, cells were diluted to an OD₆₀₀ of 2.5 in a 3-ml volume and the autoaggregation of each strain over 24 h at room temperature was assessed by capturing images of the tubes at 24 h (A) and determining the OD₆₀₀ of the upper part of the standing culture tubes (white dashed line) at the indicated time (B). Aggregation tests were performed in triplicate; error bars represent the standard deviations of the means.

FIG. 5.

Modulation of biofilm formation via RExTET- and PcL-mediated control of Ag43 production. The ability of the different strains to form a biofilm in a microfermentor was tested in M63B1glu medium. aTc at 20 ng/ml was added to the medium where indicated. Images of the 30-h-old biofilms in microfermentors (A) were captured before the biofilms were resuspended and OD₆₀₀ measurements were taken (B). The histogram shows the ratios of the biofilm biomasses of the different strains to that of wild-type strain MG1655. Data are the averages of results from three independent experiments; error bars represent standard deviations of the means.

FIG. 6.

Modulation by the RExTETrbs cassette of cellulose production and biofilm formation by an E. coli natural isolate. The ability of the different 1094-derivative strains to produce cellulose was tested. (A) Calcofluor binding and fluorescence under UV light. Two-microliter volumes of overnight cultures were spotted onto LB-calcofluor (upper row) and LB-calcofluor-aTc (lower row) plates and incubated for 24 h at 37°C; the aTc concentration on the plate was 200 ng/ml. (B) Capacities for the formation of biofilms in microfermentors. Biofilms were grown for 26 h at 37°C in M63B1glu supplemented or not with 20 ng of aTc/ml. The levels of biofilm formation by the different strains are expressed as the ratios of the OD readings for the resuspended biofilms of the different strains to that for the 1094 biofilm in the absence of aTc. Data are averages of results from three independent experiments; error bars represent standard deviations of the means.