FIG. 8.

csrA activates motility and represses glycogen biosynthesis in EPEC. (A) For motility assays, cultures grown overnight were diluted in LB broth or in LB broth supplemented with the appropriate antibiotic and allowed to grow to an OD of 1.0. One microliter of the culture was stabbed onto 0.3% 0.5× DMEM agar plates and incubated for 30 h at 37°C. Motility assays were performed in triplicate, and the diameter of the bacterial halo was measured as an indicator of bacterial motility. The unpaired Student t test was used to assay for the statistical significance of differences between the csrA mutant and EPEC or the csrA lacZ csrA⁺ strain or between EPEC(pBR322) and EPEC(pCRA16). A P value of <0.01 was considered statistically significant. ** denotes a P value of <0.001, and * denotes a P value of <0.01. (B) Real-time qRT-PCR for flhD (master regulator of flagella) was performed as described above. flhD transcript levels were substantially reduced in the csrA mutant and restored when the mutant was complemented with csrA in monocopy. ** denotes a P value of <0.001, whereas * denotes a P value of <0.005. (C) To measure glycogen biosynthesis, bacterial cultures were grown to an OD₆₀₀ of ∼1.0, streaked onto Kornberg plates, and incubated at 37°C overnight, after which the plates were exposed to iodine vapor. Glycogen levels were elevated in the csrA mutant compared to those in the wild type (EPEC) or the complemented strain (csrA lacZ csrA⁺), as evident from increased black-brown color formation in the mutant. csrA repressed glycogen biosynthesis in a dose-dependent manner because multicopy expression of csrA in the wild-type background [EPEC(pCRA16)] did not lead to a color change, in contrast to the brown color observed for the empty-vector-containing strain [EPEC(pBR322)], which contains a single copy of csrA.