FlbT couples flagellum assembly to gene expression in Caulobacter crescentus

Abstract

The biogenesis of the polar flagellum of Caulobacter crescentus is regulated by the cell cycle as well as by a trans-acting regulatory hierarchy that functions to couple flagellum assembly to gene expression. The assembly of early flagellar structures (MS ring, switch, and flagellum-specific secretory system) is required for the transcription of class III genes, which encode the remainder of the basal body and the external hook structure. Similarly, the assembly of class III gene-encoded structures is required for the expression of the class IV flagellins, which are incorporated into the flagellar filament. Here, we demonstrate that mutations in flbT, a flagellar gene of unknown function, can restore flagellin protein synthesis and the expression of fljK::lacZ (25-kDa flagellin) protein fusions in class III flagellar mutants. These results suggest that FlbT functions to negatively regulate flagellin expression in the absence of flagellum assembly. Deletion analysis shows that sequences within the 5' untranslated region of the fljK transcript are sufficient for FlbT regulation. To determine the mechanism of FlbT-mediated regulation, we assayed the stability of fljK mRNA. The half-life (t(1/2)) of fljK mRNA in wild-type cells was approximately 11 min and was reduced to less than 1.5 min in a flgE (hook) mutant. A flgE flbT double mutant exhibited an mRNA t(1/2) of greater than 30 min. This suggests that the primary effect of FlbT regulation is an increased turnover of flagellin mRNA. The increased t(1/2) of fljK mRNA in a flbT mutant has consequences for the temporal expression of fljK. In contrast to the case for wild-type cells, fljK::lacZ protein fusions in the mutant are expressed almost continuously throughout the C. crescentus cell cycle, suggesting that coupling of flagellin gene expression to assembly has a critical influence on regulating cell cycle expression.

FIG. 1

The C. crescentus flagellar regulatory hierarchy (reviewed in references , , and 80). A schematic of the flagellar regulatory hierarchy is shown. The hypothetical structures are depicted in the order in which they are assembled. Below each intermediate structure is indicated the class of flagellar genes that encode it. Flagellar assembly is coupled to gene expression at two distinct levels of the regulatory hierarchy. A cell cycle cue activates the response regulator CtrA, which in turn activates the transcription of the class II subset of flagellar genes. These genes encode regulatory proteins such as FlbD and FlbE, as well as the MS ring, the flagellar switch, and components of the flagellum-specific class III secretory apparatus. The expression and activation of FlbD and FlbE are required for the transcription of class III genes, which encode components of the basal body and hook structure. In addition, in the absence of assembly of the MS ring-switch secretory complex, the transcription of class III genes is negatively regulated by the bfa gene product. The proper assembly of class III genes is, in turn, necessary for the expression of the class IV genes, which encode flagellins. In this report, we describe how FlbT negatively regulates the expression of flagellin in the absence of assembly of the basal body-hook complex. OM, outer membrane; IM, inner membrane.

FIG. 2

Posttranscriptional regulation of the 25-kDa flagellin gene, fljK. Expression of flagellin protein or fljK-lacZ fusions in either wild-type or hook mutant cells was assayed by immunoprecipitation. Cells were grown in M2 medium to mid-log phase, and an aliquot of cells was removed and labeled with Tran³⁵S-label for 5 min. Labeled protein was immunoprecipitated with either antiflagellin (lanes 1 and 2) or anti-β-galactosidase (lanes 3 to 6) antibody and separated by SDS-PAGE as described in Materials and Methods. Lane 1, NA1000; lane 2, SC511; lane 3, NA1000 containing a fljK::lacZ protein fusion; lane 4, SC511 containing a fljK::lacZ protein fusion; lane 5, NA1000 containing a fljK-lacZ transcription fusion; lane 6, SC511 containing a fljK-lacZ transcription fusion. The apparent molecular masses of the immunoprecipitated proteins are indicated with arrows.

FIG. 3

A mutation in flbT restores flagellin expression in flagellar mutants. Proteins from whole-cell extracts from mutant strains were separated by SDS-PAGE and subjected to immunoblotting as described in Materials and Methods. Mutations in a single flagellar gene were introduced into the flbT mutant strain SC276 by generalized transduction. The mobilities of flagellins derived from these strains are compared to that of purified flagellin protein. W.T., wild type.

FIG. 4

Schematic diagram of deletion derivatives of fljK::lacZ translation fusions. Site-directed mutagenesis was used to construct different deletions of fljK. The sequence encoding amino acids 1 to 23 was fused in frame to lacZ to create fljK::lacZ. fljK1::lacZ corresponds to a deletion of amino acids 15 to 23 fused in frame to lacZ. The sequence encoding amino acids 2 to 23 was deleted, resulting in an in-frame fusion of the ATG from fljK to lacZ to create fljK2::lacZ. fljK3::lacZ is a deletion of the upstream leader, leaving only the ribosomal binding site intact, fused in frame to lacZ. IHF, integration host factor.

FIG. 5

Effect of deletions on the expression of fljK::lacZ translation fusions in wild-type and mutant strains. Wild-type and deleted fljK::lacZ translation fusions were introduced into the mutant strains indicated on the x axis. Values on the y axis represent β-galactosidase activity, in units (48), assayed in triplicate from three different mid-logarithmic-phase cultures. NA1000 is a synchronizable derivative of wild-type C. crescentus CB15. AE8006 contains a Tn5-VB32 insertion in flgE (hook). JG551 contains a Tn5-VB32 insertion in flgE and a flbT650 mutation from strain SC276. SC276 contains the flbT650 allele. (A) Mean β-galactosidase activity generated from pfljK::lacZ, which contains the entire 5′ untranslated region of fljK and the first 23 codons fused in frame to lacZ. The mean β-galactosidase activities were 2,772 U for NA1000, 246 U for AE8006, 9,253 U for JG551, and 5,955 U for SC276. (B) β-Galactosidase activity generated from pfljK1::lacZ, which contains the entire 5′ untranslated region of fljK and the first 14 codons fused in frame to lacZ. The mean β-galactosidase activities were 1,328 U for NA1000, 503 U for AE8006, 14,926 U for JG551, and 3,407 U for SC276. (C) β-Galactosidase activity generated from pfljK2::lacZ, which contains the entire 5′ untranslated region of fljK and the first codon fused in frame to lacZ. The mean β-galactosidase activities were 2,426 U for NA1000, 373 U for AE8006, 12,279 U for JG551, and 9,135 U for SC276. (D) β-Galactosidase activity (note difference in scale) generated from pfljK3::lacZ, in which the entire 5′ untranslated region of fljK except the ribosome binding site was deleted, fused in frame to lacZ. The mean β-galactosidase activities were 685 U for NA1000, 132 U for AE8006, 479 U for JG551, and 1,791 U for SC276. See Fig. 4 for a schematic representation of these fusions.

FIG. 6

Effect of flagellar mutations on fljK::lacZ mRNA stability. C. crescentus NA1000 cells were grown to mid-logarithmic phase in M2 medium. At 0 min, 200 μg of rifampin per ml was added to inhibit transcription. At 3, 6, 9, 12, and 15 min, an aliquot was removed, RNA was isolated from each sample, and primer extension was performed with a ³²P-labeled oligonucleotide that hybridized to the coding sequence of lacZ. The primer extension products were subjected to electrophoresis in a denaturing polyacrylamide gel. (A) C. crescentus NA1000 cells. The mRNA half-life is approximately 11 min. (B) Strain AE8006 (flgE::Tn5-VB32). The mRNA half-life is less than 1.5 min. (C) Strain SC276 (flbT650). The mRNA half-life is greater than 30 min. (D) Strain JG551 (flgE::Tn5-VB32 flbT650). The mRNA half-life is greater than 30 min.

FIG. 7

Effect of a flbT mutation on the temporal expression of a fljK::lacZ translation fusion. The temporal expression of β-galactosidase was assayed with either C. crescentus NA1000 or SC276 cells containing a fljK::lacZ translation reporter fusion. Isolated swarmer cells were suspended in fresh M2 medium and were permitted to progress through the cell cycle. At various times during the cell cycle (0, 30, 60, 90, 120, 150, and 180 min), an aliquot was removed and proteins were labeled with Tran³⁵S-label for 10 min. Labeled protein was immunoprecipitated with a monoclonal anti-β-galactosidase antibody and subjected to SDS-PAGE as described in Materials and Methods. The gel was dried and exposed to X-ray film. (Top) fljK::lacZ expression in wild-type strain NA1000. The drawing above the fluorogram shows the cell types present at each time point, as determined by light microscopy. Labeled β-galactosidase is indicated by an arrow. (Bottom) fljK::lacZ expression in a flbT mutant strain, SC276. A low-molecular-mass (approximately 20-kDa) proteolytically generated fragment of β-galactosidase is also indicated by an arrow.