Role of Integration Host Factor in the Transcriptional Activation of Flagellar Gene Expression in Caulobacter crescentus

Cell division in Caulobacter crescentus results in the generation of two dissimilar daughter cells, a sessile stalked cell and a motile swarmer cell. The synthesis of the single polarly localized flagellum occurs in a temporally and spatially regulated fashion over the course of each cell cycle (14, 17, 27). Over 50 C. crescentus genes are required for construction of the flagellum, with the majority organized within a trans-acting regulatory hierarchy of four classes (I to IV). The class I-encoded transcription factor CtrA activates the cell cycle-regulated expression of class II genes, encoding the cytoplasmic membrane-embedded MS ring, the flagellar switch, and the secretory apparatus of the flagellum. The expression and successful assembly of a class II-encoded structure is required for the transcriptional activation of class III genes, which encode the rod transversing the cytoplasmic membrane and the periplasm, the outer membrane rings, and the extracellular hook structure (37, 48, 65). The assembly of the hook structure is, in turn, required for translation of the flagellin subunits (class IV) that comprise the filament. The temporal regulation of class III and IV flagellar gene expression is influenced by both cell cycle events and the progression of flagellar assembly. These late flagellar genes share a conserved promoter sequence that includes a consensus binding site for RNA polymerase containing the σ54 subunit (8, 21, 28, 38, 39, 45, 47) and one or more ftr enhancer elements recognized by the class II-encoded transcription factor FlbD (5, 6, 18, 29, 46, 62-64). The activation of FlbD occurs in response to the successful assembly of early class II-encoded flagellar components. FlbD contains an amino-terminal receiver domain, conserved among two-component regulatory systems, that is phosphorylated in the predivisional stage of the cell cycle (63). The activity of FlbD peaks in the predivisional cell type and becomes restricted to the swarmer compartment of the predivisional cell once a cell division constriction has formed (20, 63). FlbD-mediated transcriptional activation has been shown to be regulated by flagellar assembly through the activity of the trans-acting factor FliX (42-44). FliX-regulated coordination of early flagellar assembly with cell division allows FlbD-dependent, swarmer compartment-specific expression of class III and IV flagellar genes in the late predivisional cell. The C. crescentus class III and IV flagellar promoters also contain a conserved binding site for integration host factor (IHF) located between the promoter sequence and the upstream FlbD binding site (18, 19, 38). IHF is a small (approximately 20 kDa in Escherichia coli), heterodimeric, sequence-specific DNA binding protein that was first identified in E. coli as having a critical role in integration and excision of the prophage form of bacteriophage λ (33). During bacteriophage λ integration, IHF binding induces a bend in the DNA (22, 30, 52, 53, 60) of greater than 160° (53). This DNA bending activity was shown to be required for Int-catalyzed recombination between the attB and attP sites on the bacterial and lambda chromosomes in vitro (22, 30, 53). Subsequently, IHF was found to play an important role in diverse cellular processes, such as the initiation of DNA replication, certain recombination events, and the regulation of gene expression (1, 11, 12, 23-25, 34, 59). When present in σ54 promoters, the IHF-induced bend is thought to bring RNA polymerase bound to the promoter and transcriptional activators bound to the relatively distant enhancer sequences into close proximity with each other, thus increasing the probability of interaction and transcriptional activation (10, 18, 19, 26, 54). Experiments with the Pseudomonas putida Pu promoter have shown that IHF can also function to recruit RNA polymerase holoenzyme to promoter sequences (7, 35). IHF has been demonstrated to be required for the transcription of the σ54 nifH promoter (26) and C. crescentus class III and IV flagellar promoters in in vitro experiments (5). Furthermore, mutations in the IHF binding site of either of the class III flagellar gene promoters fliK (formerly flbG) or flgK (formerly flaN) indicate that IHF is required for maximal levels of transcription in vivo (18, 19). Interestingly, a mutagenized fliK promoter that was shown in vitro to no longer be bound by IHF was still capable of driving FlbD-dependent reporter gene expression, though to a much lesser extent than the wild-type promoter (18, 19). Thus, the higher-order DNA structure created by IHF binding is not strictly required for fliK transcription. Although these previous experiments suggest a role for IHF in fine-tuning the temporal and spatial regulation of class III and IV flagellar gene expression, it is not known what influence IHF has on motility and swarmer cell differentiation in C. crescentus. Here we investigate the role of IHF activity on the regulation of transcription of late flagellar genes in C. crescentus by determining the effects of deleting ihfB (himD) on growth, cell morphology, motility, and the transcription of flagellar promoters. Strains containing a deletion of ihfB exhibited a motility defect and a reduction in the level of transcriptional activation of several class III/IV flagellar genes. Interestingly, cells lacking IHF displayed a wild-type temporal pattern of class III, fliK-lacZ reporter gene expression, although the promoter activity was diminished. Absence of IHF was found to most significantly affect expression and accumulation of the class IV flagellin, FljL.