| Summary: | Bistable expression of the <i>Salmonella enterica</i> pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1<sup>OFF</sup> Flag<sup>OFF</sup>, SPI-1<sup>OFF</sup> Flag<sup>ON</sup>, SPI-1<sup>ON</sup> Flag<sup>OFF</sup>, and SPI-1<sup>ON</sup> Flag<sup>ON</sup>. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that Flag<sup>OFF</sup> cells might contribute to optimal invasion as previously proposed for SPI-1<sup>OFF</sup> cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1<sup>ON</sup> cells. As a consequence, growth resumption in SPI-1<sup>ON</sup> Flag<sup>ON</sup> cells requires switching to both SPI-1<sup>OFF</sup> and Flag<sup>OFF</sup> states.
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