FlgM as a secretion moiety for the development of an inducible type III secretion system

Abstract

Regulation and assembly of the flagellar type III secretion system is one of the most investigated and best understood regulational cascades in molecular biology. Depending on the host organism, flagellar morphogenesis requires the interplay of more than 50 genes. Direct secretion of heterologous proteins to the supernatant is appealing due to protection against cellular proteases and simplified downstream processing. As Escherichia coli currently remains the predominant host organism used for recombinant prokaryotic protein expression, the generation of a strain that exhibits inducible flagellar secretion would be highly desirable for biotechnological applications. Here, we report the first engineered Escherichia coli mutant strain featuring flagellar morphogenesis upon addition of an external inducer. Using FlgM as a sensor for direct secretion in combination with this novel strain may represent a potent tool for significant improvements in future engineering of an inducible type III secretion for heterologous proteins.

Figure 1. Transcription of the master operon flhDC.

(A) To verify the transcription of the plasmid-encoded master operon flhDC Reverse Transcriptase PCR Reaction of the isolated RNA was performed. It was assumed that within the overnight culture cells exhibiting flagellar assembly were found and therefore this culture was used as a positive control. Cells before addition of the inducer IPTG were used as reference compared to cells after induction of the plasmid-encoded flhDC. To evaluate genomic DNA contamination the isolated RNA samples were also subjected to subsequent PCR. Plasmid-encoded flhDC was used as a positive control for the PCR reaction. Fermentas 1 KB GeneRuler, ON overnight culture sample, -I whole cell sample without induction of plasmid-encoded flhDC, +I whole cell samples with induction of plasmid-encoded flhDC, RT Reverse Transcriptase Reaction, + plasmid-encoded flhDC; (B) Scheme of the plasmid-encoded master operon under the control of the lacUV5 promoter

Figure 2. Modification of the flhDC master operon.

The expression strain HMS174(DE3) was used for modifications of the bacterial genome. Upstream regulator elements of the master operon flhDC were deleted and replaced by the artificial araBAD promoter resulting in the HMS174(DE3)ΔinsAB araBAD-flhDC strain. Activator elements are depicted in green, whereas repressor elements are depicted in red , .

Figure 3. Comparison of the secretion competency via plasmid-encoded overexpression of FlgM.

(A) FlgM was secreted to the supernatant in the HMS174(DE3) strain. (B) In contrast, in the newly generated HMS174(DE3)ΔinsAB araBAD-flhDC no FlgM could be detected in the supernatant. (C) The ability to secrete FlgM was recovered in the HMS174(DE3)ΔinsAB araBAD-flhDC strain when plasmid-encoded flhDC was co-expressed. SDS-PAGE, M Fermentas PageRuler Prestained, -I whole cell sample without induction of recombinant protein expression, +I whole cell samples with induction of recombinant protein expression, C cytoplasmic fraction, Sn supernatant;

Figure 4. Inducible secretion of FlgM.

In the newly generated strain HMS174(DE3)LT7 the functional assembly of the hook basal body led to the secretion of plasmid-encoded FlgM into the supernatant. No secretion of FlgM occurred in the strain HMS174(DE3)LL. (A), HMS174(DE3)LL, pET30-lacUV5-flgM-6His (B), HMS174(DE3)LT7, pET30-lacUV5-flgM-6His (C) HMS174(DE3)LT7, pET30-lacUV5-flgM-SOD (D) HMS(174)LT7, pET30-lacUV5-flgM-GFPmut3.1-6H, M Fermentas PageRuler Prestained, -I whole cell sample without induction of recombinant protein expression, +I whole cell samples with induction of recombinant protein expression, C cytoplasmic fraction, Sn supernatant;

Figure 5. Quantitative Real Time PCR of generated HMS174(DE3) mutant strains.

(A) The generated mutant strains HMS174(DE3)ΔinsAB lacUV5-flhDC, HMS174(DE3)ΔinsAB lacUV5-flhDC, att7 lacUV5-flhDC, HMS174(DE3) ΔinsAB T7-flhDC and HMS174(DE3)LT7 were incubated for 1 h after induction with 1 mM IPTG at 37°C/225 rpm. Quantification of flhD mRNA levels was accomplished with the comparative ΔΔCt method using rpoD for normalization. Resulting data were analyzed via 2-tailed type 2 Student's t-test, ***p<0.001, n = number of replicates.

Figure 6. Analysis of the novel host strain HMS174(DE3)LT7.

(A) Scanning Electron Microscopy (SEM) of the HMS174(DE3)LT7 strain without addition of the inducer IPTG. (B) Scanning Electron Microscopy (SEM) of the HMS174(DE3)LT7 strain with addition of the inducer IPTG. Samples were prepared after 90 min cultivation. (C) Cell culture homogeneity. The homogeneity regarding fliC promoter activity of three parallel HMS174(DE3)LT7 strain cultivations containing the plasmid p5′UTR-fliC20-GFPmut-Flag-Linker-StrepII-3′UTR with and without addition of the inducer IPTG were analyzed via Fluorescence Activated Cell Sorting (FACS). After incubation for 2 h at 220 rpm at 37 °C all samples were normalized to OD 600: 1.0 and the percentage of GFP expressing cells indicating flagellar assembly was determined. (D) FACS analysis of GFP expressing cells. A sample of the HMS174(DE3)LT7 strain cultivation containing the plasmid p5′UTR-fliC20-GFPmut-Flag-Linker-StrepII-3′UTR (from Figure 6C) with and without addition of the inducer IPTG.