FIG 5.

Fis regulatory elements identified in the regulatory regions of effector-encoding genes. (A) The regulatory regions of the effectors found to be repressed by the Fis1 and Fis3 regulators are presented. The nucleotides representing the putative Fis consensus are in red and underlined, the transcription start sites are in bold and underlined, the −10 and −35 promoter elements are in blue, the CpxR (sidM and cegC4) and PmrA (legA9, ceg20 and sdbB) consensus sequences are in purple, and the nucleotides that were mutated are marked by asterisks. The effector designations are indicated on the left. (B and C) Mutations constructed in the putative Fis regulatory elements resulted in elevated levels of expression at exponential phase. (B) The expression levels of effector (indicated below the bars) wild-type lacZ fusions (white bars) and lacZ fusions of the same effector containing a mutation in a putative Fis binding site (gray bars) were examined at the exponential phase in the L. pneumophila wild-type strain. The mutations constructed are marked by asterisks in panel A. (C) In three genes (sidC, cegC4, and legC8) two individual mutations in two Fis sites were generated (M1 and M2) as well as a double mutation in both sites together (M1 + 2). For sidC and cegC4 the upstream Fis site was named M1, and the downstream Fis site was named M2. For legC8 the downstream Fis site was named M1, and the upstream Fis site was named M2. β-Galactosidase activity was measured as described in Materials and Methods. Data (expressed in Miller units [M.U.]) are the averages ± standard deviations (error bars) of the results of at least three different experiments. The effector-encoding genes were divided according to their levels of expression.