T-POP array identifies EcnR and PefI-SrgD as novel regulators of flagellar gene expression

Abstract

The T-POP transposon was employed in a general screen for tetracycline (Tet)-induced chromosomal loci that exhibited Tet-activated or Tet-repressed expression of a fliC-lac transcriptional fusion. Insertions that activated flagellar transcription were located in flagellar genes. T-POP insertions that exhibited Tet-dependent fliC-lac inhibition were isolated upstream of the ecnR, fimZ, pefI-srgD, rcsB, and ydiV genes and in the flagellar gene flgA, which is located upstream of the anti-sigma(28) factor gene flgM. When expressed from the chromosomal P(araBAD) promoter, EcnR, FimZ, PefI-SrgD, and RcsB inhibited the transcription of the flagellar class 1 flhDC operon. YdiV, which is weakly homologous to EAL domain proteins involved in cyclic-di-GMP regulation, appears to act at a step after class 1 transcription. By using a series of deletions of the regulatory genes to try to disrupt each pathway, these regulators were found to act largely independently of one another. These results identify EcnR and PefI-SrgD as additional components of the complex regulatory network controlling flagellar expression.

FIG. 1.

T-POP insertions at six different loci inhibited flagellar transcription when induced with Tet. Each T-POP insertion is represented by a triangle containing an arrow. The arrow indicates the direction in which transcripts initiated from the tetA promoter proceed into the adjacent chromosomal DNA. The smaller, solid triangles represent polar MudJ insertions which either disrupt the inhibition of flagellar transcription or have no effect on inhibition by the T-POP insertions. As depicted in panel F, the only isolated insertion in feoB consisted of 6 kb of E. coli F plasmid DNA (wavy line) flanked by T-POP insertions.

FIG. 2.

Motility of T-POP insertion strains with or without induction by autoclaved chlortetracycline (A) or P_ara constructs with or without arabinose induction (B). For the colonies poked into motility plates containing chlortetracycline or arabinose, the expression of negative regulators had already been induced by growth on LB-Tet or LB-Ara plates. Pictures were taken after 6 h. Strains are indicated by the affected gene. wt, wild type.

FIG. 3.

Motility and class 3 flagellar transcription of strains with deletions of the negative regulators. Motility was determined by measuring the diameters in motility plates after 6 h. Each reported diameter is an average for six replicates and is normalized with respect to the diameter for the wild type (wt). A representative photo of each strain is shown beneath the bar graph. Class 3 transcription from the motA::MudJ transcriptional reporter was quantified through β-galactosidase assays.

FIG. 4.

Epistatic analysis of negative regulators of motility. A deletion of each regulator was introduced into strains in which one of the five negative regulators could be induced. Colonies picked from LB-Ara plates were poked into motility plates containing arabinose using toothpicks. Pictures were taken after 6 h. wt, wild type.

FIG. 5.

Flagellar transcription and motility in strains expressing the negative regulators from the arabinose promoter. For both panels, the activities of MudJ transcriptional fusions to the flhC class 1 promoter, the fliL class 2 promoter, and the motA class 3 promoter were quantified through the β-galactosidase assay. Transcription activities and swimming-area diameters were normalized with respect to the wild-type levels. In the strains analyzed in panel A, the flhDC operon was expressed from its wild-type promoter. In those analyzed in panel B, the flhDC operon was expressed from a T-POP induced with Tet. The key in panel A also applies to panel B.

FIG. 6.

Locations of spontaneous mutations in the flhDC promoter region that suppress the inhibition of motility by EcnR and PefI-SrgD overexpression. Transcription start sites (P1, P2, P3, P4, and P6) as determined by Yanagihara et al. (61) are marked with bent arrows, and −10 hexamers are underlined. The sequence that matches the consensus for the RcsAB box is identified by a wavy line (17, 36). The start codon of flhD is boxed. One double mutation was isolated and is indicated by a line connecting the two changes (G−211A and G−213A). The A−198G mutation was found in 6 of the 10 flhDC promoter mutants that suppressed PefI-SrgD inhibition and 20 of the 26 flhDC promoter mutants that suppressed EcnR inhibition.

FIG. 7.

Effect of the flagellar system on the transcription of the negative regulator genes. The fusion of lac to each negative regulator gene was used to quantify changes in transcription. The flhDC operon was expressed from its wild-type promoter or from the tetA promoter in the T-POP. When under the control of the T-POP, the flhDC operon is not expressed without the inducer chlortetracycline (chlortet.).

FIG. 8.

Diagram of interactions for the five negative regulators identified in this study. While arrows show interactions between the regulators and the flagellar system, it is not known whether the majority of the interactions are direct or are mediated by regulators other than the five that were studied. A binding site in the class 1 promoter has been identified only for RcsB (57). It is also not clear how RcsB helps EcnR to inhibit class 1 transcription, and this interaction is labeled with a question mark. Since part of RcsB's inhibition of motility occurs after class 1 transcription, EcnR may not inhibit flagellar transcription by directly activating RcsB.