Engineered Phage Enables Efficient Control of Gene Expression upon Infection of the Host Cell

Abstract

Recently, we developed a spatial phage-assisted continuous evolution (SPACE) system. This system utilizes chemotaxis coupled with the growth of motile bacteria during their spatial range expansion in soft agar to provide fresh host cells for iterative phage infection and selection pressure for preserving evolved genes of interest carried by phage mutants. Controllable mutagenesis activated only in a subpopulation of the migrating cells is essential in this system to efficiently generate mutated progeny phages from which desired individuals are selected during the directed evolution process. But, the widely adopted small molecule-dependent inducible system could hardly fulfill this purpose because it always affects all cells homogeneously. In this study, we developed a phage infection-induced gene expression system using modified Escherichia coli (E. coli) phage shock protein operon or sigma factors from Bacillus subtilis. Results showed that this system enabled efficient control of gene expression upon phage infection with dynamic output ranges from small to large using combinations of different engineered phages and corresponding promoters. This system was incorporated into SPACE to function as a phage infection-induced mutagenesis module and successfully facilitated the evolution of T7 RNA polymerase, which generated diverse mutants with altered promoter recognition specificity. We expect that phage infection-induced gene expression system could be further extended to more applications involving partial induction in a portion of a population and targeted induction in specific strains among a mixed bacterial community, which provides an important complement to small molecule-dependent inducible systems.

Keywords: directed evolution; engineered phage; inducible gene expression; phage shock protein; sigma factor.

Figure 1

Application of the phage shock protein (psp) operon for construction of phage-inducible gene expression circuit. (A) Structure and working mechanism of psp operon in E. coli. Phage represents filamentous phage M13, of which the infection of E. coli cells activates the psp operon via a phage pIV-dependent signaling cascade. Psp, phage shock protein; UAS, upstream activating sequence; IHF BS, integration host factor binding site; TSS, transcription start site. (B) Design of a fluorescent gene expression system activated by phage infection using psp operon. The E. coli cells carry a reporter plasmid (RP), which harbors gfp under the control of the psp promoter. (C) Two versions of phages are used to activate the expression of gfp and the fluorescence intensity of their host cells across time after phage infection. Wild-type M13 was used in (1), and AP1-SPT7, which carries a T7 RNA polymerase gene in place of gIII and only produces infectious progeny phages in the presence of an accessory plasmid carrying gIII downstream of the T7 promoter, was used in (2). Native promoters are not annotated in the schematic diagram of the circuit design. The fluorescence intensity was normalized by the corresponding OD₆₀₀ value at each time point. The starting time point of the plots was set at 50 min after phage inoculation when the OD₆₀₀ values became significant enough to give stable normalized intensity values. Solid circle (●) represents the experimental group in which both E. coli FM15 cells and phages were added. Open circle (○) represents the control group in which only FM15 cells were added. The mean for two or three replicates is shown in the plot.

Figure 2

Modification of the psp operon for construction of phage-inducible gene expression circuit. (A) Construction of E. coli FM20 strain by deleting the native psp operon from FM15 genome with CRISPR-Cas system. FM20 was used as the bacterial host for the genetic circuit design using a modified psp operon. (B) Design of a fluorescent gene expression system activated by phage infection using the modified psp operon consisting only of the psp promoter without other psp genes and pspF carried by the activator phage. (C) Two versions of phages are used to activate the expression of gfp and the fluorescence intensity of their host cells across time after phage infection. AP2-SPT7F carries the T7 RNA polymerase gene followed by pspF in place of gIII. For further control of infectious progeny phage reproduction, gII and gV, two more phage genes were deleted, and pspF was inserted instead to construct another version of activator phage, AP3-SPF. Correspondingly, accessory plasmid pLAasc22 carrying gIII, gII, and gV downstream of the T7 promoter was constructed to enable the reproduction of AP3-SPF. Native promoters are not annotated in the schematic diagram of the circuit design. The fluorescence intensity was normalized by the corresponding OD₆₀₀ value at each time point. Solid circle (●) represents the experimental group in which both E. coli FM15 cells and phages were added. Open circle (○) represents the control group in which only FM15 cells were added. The mean for three replicates is shown in the plot.

Figure 3

Construction of phage-inducible gene expression circuit using heterologous sigma factors. (A) Schematic design of a fluorescent gene expression system in E. coli activated by phage infection using heterologous sigma factors and promoters from Bacillus. These sigma factors are expected to bind the host core RNA polymerase (RNAP) and form a holoenzyme to recognize/transcribe specifically from its cognate promoter to yield an orthogonal expression system. (B) Three versions of phages are used to activate the expression of gfp and the fluorescence intensity of their host cells across time after phage infection. AP4-SPSF and AP5-SPSB carry the T7 RNAP gene in place of phage gIII and genes of Bacillus sigma factors B and F, respectively, in place of phage gII-gV. These two phages both rely on accessory plasmid pLAasc22 carrying gIII, gII, and gV downstream of the T7 promoter to reproduce. AP6-SPJSF was derived from AP1-SPT7 by inserting the gene of sigma factor F controlled by a strong synthetic promoter J23100 (https://parts.igem.org/Part:BBa_J23100 (accessed on 26 November 2020)) downstream of the T7 RNAP gene. This activator can also use accessory plasmid pLAasc1 to reproduce. Native promoters are not annotated in the schematic diagram of the circuit design. The fluorescence intensity was normalized by the corresponding OD₆₀₀ value at each time point. Solid circle (●) represents the experimental group in which both E. coli FM15 cells and phages were added. Open circle (○) represents the control group in which only FM15 cells were added. The mean for three replicates is shown in the plot.

Figure 4

Spatial continuous directed evolution (SPACE) experiment using different versions of mutagenesis modules. (A) Two versions of activator phages carrying the T7 RNA polymerase (RNAP) gene as the target gene to be evolved and either the native phage gIV or Bacillus sigma factor gene of which the protein product can activate the expression of mutator genes from corresponding mutagenesis plasmids (pLM1/pLM2). (B) Upon phage infection, the mutagenesis process becomes active and leads to the production of various T7 RNAP mutants. Mutants with improved activity to recognize and transcribe from the target promoter 1C12 will lead to stronger infectious progeny phage propagation and will, in turn, produce a larger fan-shaped infection area with lower cell density on the bacterial lawn in the SPACE experiment. Evolved T7 RNAP mutant genes carried by sampled phages were sequenced, and the amino acid (AA) changes detected in these mutants are listed alongside the images of the SPACE agar plates from which the mutants were isolated. Photographs of a quarter of the semi-solid agar plate are shown in the Figure. Scale bar represents 1 cm.