Construction of strains with constitutively expressed FlhDC

A series of strains were constructed where the native class I promoter, flhDp, was replaced with a synthetic constitutive promoter of different transcriptional strength (the “Pro” promoters) (31). The native RBS for FlhD was also replaced with either the T7 RBS or a mutant RBS (“mut4”) with the sequence TTTAAGAATTGCATATACAT (mutated bases in bold). To construct these strains, plasmids consisting of a synthetic Pro promoter, a T7 or mut4 RBS, and FlhDC coding sequence were constructed. Subsequently, a linear fragment consisting of the Pro promoter, T7 or mut4 RBS, and FlhD, with overhangs homologous to the 5′-end of the flhDp was amplified from the plasmid template. This linear fragment was chromosomally inserted via red recombination. See the “Construction of strains with constitutively expressed FlhDC” section in the Supplementary Materials for more details.

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