Dying for Good: Virus-Bacterium Biofilm Co-evolution Enhances Environmental Fitness

Abstract

Commonly used in biotechnology applications, filamentous M13 phage are non-lytic viruses that infect E. coli and other bacteria, with the potential to promote horizontal gene transfer in natural populations with synthetic biology implications for engineering community systems. Using the E. coli strain TG1, we have investigated how a selective pressure involving elevated levels of toxic chromate, mimicking that found in some superfund sites, alters population dynamics following infection with either wild-type M13 phage or an M13-phage encoding a chromate reductase (Gh-ChrR) capable of the reductive immobilization of chromate (ie, M13-phageGh-ChrR). In the absence of a selective pressure, M13-phage infection results in a reduction in bacterial growth rate; in comparison, in the presence of chromate there are substantial increases in both cellular killing and biomass formation following infection of E. coli strain TG1with M13-phageGh-ChrR that is dependent on chromate-reductase activity. These results are discussed in terms of community structures that facilitate lateral gene transfer of beneficial traits that enhance phage replication, infectivity, and stability against environmental change.

Keywords: bioremediation; chromate reduction; community stability; population dynamics; selective pressure; synthetic biology; temperate phage.

Figure 1

Functionally active chromate reductase is expressed on the surface of purified M13-phage_Gh-ChrR. (Panel A) Map of engineered pCDisplay-4 phagmid vector endcoding Gh-ChrR (green arrows) expressed as a fusion protein with Fos-Gh-ChrR and Jun-Gh-ChrR to facilitate dimerization following phage display. (Panel B) Kinietic reduction of chromate (monitored at 370 nm) by purified M13-phage_ChrR (red squares) in comparison to M13 helper phage (blue triangles). Notes: Measurements involved purified phage (5 × 10¹⁰ pfu/mL) in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM Cr₂O₄, and 0.1 mM NADH. Chromate reduction rates for M13-phage_Gh-ChrR (ΔOD_370nm = 0.006/min) are significantly increased in comparison to that observed for helper phage alone (ΔOD_370nm = 0.004/min) (P = 0.00015).

Figure 2

Phage-dependent lateral gene transfer of chromate reductase by M13-phage_Gh-ChrR results in enhanced biomass formation. Confocal images of live (Cyto9; green) or dead (PI; red) cells (top panels), rates of chromate reduction (middle panels), and measured biomass (bottom panels) for mature biofilms comprised of E. coli strain TG1 alone (left panels), in the presence of either phagmid Gh-ChrR or helper phage alone (center panels), or following infection (right panels). Chromate (5 mM) reduction was monitored by measuring the absorbance at 370 nm wavelength, and is normalized following correction for the absorbance of the LB medium. Total biomass was estimated by crystal violet staining and measured absorbance at 595 nm, and is normalized to that of TG1 alone (left panel). Note: Significant differences (*) in comparison to TG1 alone (P-value < 0.01) were based on a Student’s t-test (n = 3).