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Journal of Bacteriology, February 2005, p. 949-960, Vol. 187, No. 3
0021-9193/05/$08.00+0     doi:10.1128/JB.187.3.949-960.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Rachel E. Muir1,{dagger} and James W. Gober1*

Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California1

Received 14 September 2004/ Accepted 21 October 2004


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ABSTRACT
 
In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the {sigma}54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the ß-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The {Delta}ihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a wild-type cell cycle-regulated pattern of expression in the absence of IHF. Thus, IHF is required for maximal transcription of several late flagellar genes. The {Delta}ihfB strain was found to express significantly reduced amounts of the class IV flagellin, FljL, as a consequence of reduced transcriptional activity. Our results indicate that the motility defect exhibited by the {Delta}ihfB strain is most likely attributable to its failure to accumulate the class IV-encoded 27-kDa flagellin subunit, FljL.


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INTRODUCTION
 
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 {sigma}54 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 {lambda} (33). During bacteriophage {lambda} 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 {sigma}54 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 {sigma}54 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.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this work are listed below. C. crescentus strains were LS107 (wild-type strain), syn-1000 bla-6 (58), JG1182, syn-1000 bla-6 flbD198::Tn5 (44), and JG1216, syn-1000 bla-6 ihfB1 (this work). E. coli strains were DH5{alpha}, endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 and S17-1, Rp4-2 Tc::Mu Km::Tn7 (57). Plasmids used were the following: pREM85, 747-bp PCR-generated SalI (natural site)-PstI (introduced site) fragment encompassing ihfB and endogenous ihfB promoter in pBBR1MCS-1 (31); pREM86, 547-bp SalI-PstI ihfB fragment lacking an internal 200-bp AvaII fragment originating from pREM85 in pNPTS139; and transcriptional reporters pfliF/lacZ/290 (62), pfliL/lacZ/290 (41), pfliX/lacZ/290 (44), pflgF/lacZ/290 (38), pflgI/lacZ/290 (42), pflgK/lacZ/290 (18), pfliK/lacZ/290 (18), and pfljL/lacZ/290 (36). In order to overexpress His-tagged IHF-B, the entire ihfB open reading frame was subcloned in frame downstream of the His6-encoding sequence in pET21b (Novagen). The overexpressed His-tagged IHF-B was purified by nickel affinity chromatography under denaturing conditions (4). DNA manipulations were performed essentially as described elsewhere (4). Plasmids were introduced into C. crescentus by bacterial conjugation. C. crescentus strains were grown at 31°C in peptone-yeast extract medium (PYE) (50) or M2 minimal medium (28) either alone or supplemented with one or more of the following: chloramphenicol (2.5 µg ml–1), kanamycin (25 µg ml–1), or tetracycline (2.0 µg ml–1). PYE motility plates contained 0.3% agar. The E. coli strains were grown at 37°C in Luria-Bertani medium (4) either alone or supplemented with one or more of the following: chloramphenicol (30 µg ml–1), kanamycin (50 µg ml–1), or tetracycline (12.5 µg ml–1).

Construction of the {Delta}ihfB strain JG1216. The ihfB1 deletion strain was generated utilizing the sacB selection method (55). Plasmid pREM86 was conjugated into C. crescentus LS107, and transformants were selected on PYE agar containing kanamycin (25 µg ml–1) and naladixic acid (20 µg ml–1). Transformants were grown overnight in PYE without selection and plated onto PYE agar containing 2.5% sucrose. A total of 192 sucrose-resistant colonies were isolated and tested for kanamycin sensitivity and assayed for motility in PYE-0.3% agar. Of the 192 colonies chosen, only one was both kanamycin sensitive and displayed a motility defect, seen as a motile swarm much smaller in diameter than any produced from the other 191 colonies assayed. Deletion of the 200-bp AvaII fragment encompasses the ribosome binding site and start of translation through the first 52 encoded residues of the 96 codons which comprise the ihfB open reading frame. The presence of this deletion in the chromosome of JG1216 was confirmed by PCR. Note that 30 different kanamycin-sensitive, sucrose-resistant isolates that exhibited wild-type motility did not contain a deletion in ihfB as determined by PCR (data not shown). Complementation tests were performed by transducing either the wild-type allele into the {Delta}ihfB strain or transducing the deleted ihfB allele into wild-type cells. In order to accomplish this, the gene directly adjacent to ihfB, mscL (CC3585), was amplified by PCR and subcloned into plasmid pNPST139, which cannot replicate in C. crescentus. The mscL-containing plasmid was then mated into either LS107 (wild-type) or JG1216 ({Delta}ihfB) cells, and integrants were selected by kanamycin resistance. Transducing lysates of bacteriophage {phi}CR30 were then prepared from each strain (13). Transduction recipients were selected for resistance to kanamycin and scored for motility defects. The effect of the ihfB deletion on the accumulation of IHF {alpha}- and ß-subunits was demonstrated by immunoblot analysis, using anti-IHF rabbit antiserum provided by S. Goodman or antisera raised against purified C. crescentus IHF-B.

Flagellar gene expression. The synchronizable LS107 and JG1216 C. crescentus strains either alone or harboring the fliK-lacZ reporter fusion were used in cell cycle gene expression experiments performed as described previously (38). The expression of all flagellar gene transcriptional reporters in unsynchronized cultures was determined in triplicate (on three independent cultures) by quantitative measurements of ß-galactosidase activity as previously described (37). Immunoblots were prepared as described previously (61) and were analyzed using antiflagellin (C. crescentus), anti-IHF-B (C. crescentus), anti-IHF (E. coli), anti-MreB (C. crescentus) (16), and anti-HU (C. crescentus) (J. C. England and J. W. Grober, unpublished data) antisera.


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RESULTS AND DISCUSSION
 
Cell cycle expression pattern of IHF. We first determined whether the cellular concentration of IHF changed during the course of the cell cycle, since this could conceivably influence the temporal regulation of class III/IV flagellar gene transcription. Microarray experiments have indicated that the transcription of ihfA (himA), encoding the IHF {alpha}-subunit, is under cell cycle control (34). Using antiserum generated against E. coli IHF, we observed two cross-reacting bands in an immunoblot of cell extracts that had been shown by previous experiments to correspond to C. crescentus IHF {alpha}- and ß-subunits (Fig. 1) (18, 19). We found that levels of both polypeptides increased gradually over the course of the cell cycle, with peak cellular concentrations occurring in predivisional cells (Fig. 1). This result is consistent with previous experiments showing that IHF was more concentrated in isolated predivisional cells than in isolated swarmer or stalked cells (18). It is interesting, however, that this temporal expression pattern does not parallel the cell cycle microarray data showing that himA (ihfA) mRNA levels peak in the swarmer cell type (33). These contrasting findings may indicate that an additional posttranscriptional mechanism is responsible for the cell cycle regulation of IHF levels.



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  		FIG. 1. Cell cycle expression of C. crescentus IHF. (A) Pure populations of wild-type swarmer cells were isolated (15) and allowed to progress synchronously through the cell cycle in PYE medium. At the time points indicated, cells from a 1-ml portion of culture (optical density at 600 nm, 0.35) were isolated by centrifugation. The resulting cell pellets were suspended in SDS sample buffer and boiled for 5 min, and an equal amount of each sample was subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analyses. A schematic of the cell types present at each time point during the cell cycle, as determined by light microscopy, is shown above the immunoblots. A sample of an E. coli cell extract was electrophoresed with the C. crescentus cell cycle for comparison. As previously demonstrated (17, 18), the E. coli anti-IHF antibodies cross-react with the two C. crescentus polypeptides that are similar in molecular mass to the E. coli IHF {alpha}-subunit (10.5 kDa) and ß-subunit (9.5 kDa). The C. crescentus IHF {alpha}- and ß-subunits (indicated as IHF-A and IHF-B, respectively) gradually increased in intensity over the course of the cell cycle. Note that the C. crescentus IHF ß-subunit migrates slower than the {alpha}-subunit (see below). The anti-MreB and antiflagellin antisera served as two different cell type controls. (B) Graph depicting quantitation of IHF levels from the immunoblot shown in panel A during the course of the C. crescentus cell cycle. The x axis represents relative levels, with 100% representing the peak cell cycle level for each indicated subunit.

An IHF deficiency results in a motility defect. In order to assess the role of IHF in the transcriptional regulation of late flagellar genes, we deleted a gene encoding one of the subunits of the IHF heterodimer. We chose to disrupt the single gene locus encoding the ß-subunit, ihfB (himD) (CC3586), reasoning that a disruption in the himA locus could potentially affect expression of a downstream conserved hypothetical gene (CC1371) that together with himA (ihfA) comprises a two-gene operon. A plasmid containing ihfB was digested with AvaII and religated, generating a 200-bp internal deletion (Fig. 2A). The deleted gene was then integrated in the C. crescentus genome by homologous recombination. These cells were then grown in liquid medium and selected for excision of the integrated plasmid by plating onto sucrose-containing medium, followed by screening for kanamycin sensitivity (see Materials and Methods) (55). Previous experiments have demonstrated the presence of IHF binding sites upstream of at least 12 flagellar genes. Thus, we performed a secondary screen for a motility defect by inoculating the sucrose-resistant, kanamycin-sensitive colonies into semisolid motility agar. When grown in motility agar, strains in which motility defects are evident produce a halo of growth or swarm that is noticeably smaller in diameter than that produced by the wild-type strain. Surprisingly, the frequency of sucrose-resistant, kanamycin-sensitive colonies that also exhibited an unequivocal motility defect was quite low (1 out of 192 screened). The cells from this single isolate were confirmed to have a deletion in ihfB when tested by PCR (data not shown), indicating a low frequency of allelic exchange via homologous recombination. This may have been a consequence of the small amount of homologous DNA on the plasmid containing the deleted ihfB gene (see Materials and Methods). The motility of the {Delta}ihfB strain (JG1216) was compared to that of wild-type and nonmotile flbD::Tn5 cells by growth in motility agar (Fig. 2C). The {Delta}ihfB strain appeared motile, with a swarm size approximately 50 to 60% that of wild-type cells (Fig. 2C), although liquid cultures contained significantly less motile cells than wild-type cultures when observed by phase-contrast microscopy (data not shown). We then performed a complementation test by introducing a multicopy plasmid containing the ihfB promoter and open reading frame (pREM85) into the {Delta}ihfB strain. This plasmid was able to restore a wild-type proportion of motile cells to the mutant strain as judged by light microscopy (data not shown), suggesting that the motility defect displayed by the {Delta}ihfB strain was a consequence of the absence of a functional ihfB gene. Both wild-type and {Delta}ihfB cells possessing pREM85 produced a smaller-sized swarm than wild-type cells without plasmid, which may be attributable to a slower growth rate of the plasmid-bearing cells (data not shown). The diameter of the swarm produced by both wild-type and {Delta}ihfB cells containing pREM85 was identical, indicating that expression of ihfB from pREM85 probably complements the motility defect exhibited by the {Delta}ihfB strain (Fig. 2D).



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  		FIG. 2. The {Delta}ihfB strain displays a motility defect and expresses reduced levels of the 27-kDa flagellin. (A) Schematic showing the region of the genome adjacent to ihfB and the introduced deletion in ihfB within strain JG1216. The open reading frame downstream of ihfB, mscL (CC3565), is predicted to encode a polypeptide with similarity to the large subunit of mechano-sensitive channels. (B) Schematic showing the pNPTS139 plasmid containing mscL integrated into either wild-type (JG1220) or {Delta}ihfB (JG1221) cells. Integration of this plasmid via single-crossover, homologous recombination results in the presence of a duplicated mscL locus. Complementation tests were performed by transducing the kanamycin resistance marker from JG1220 or JG1221 into either wild-type or {Delta}ihfB cells (see below). (C) C. crescentus wild-type (LS107), flbD::Tn5 (SC1032), and {Delta}ihfB (JG1216) cells were inoculated into motility agar and grown for 4 days. The nonmotile flbD::Tn5 strain lacks the concentric ring of swarming cells displayed by the wild-type and {Delta}ihfB strains. The {Delta}ihfB strain displays a slight motility defect, demonstrated here as a motile swarm smaller in diameter (approximately 40%) than that generated by the wild-type strain. (D) The {Delta}ihfB strain harboring ihfB on a multicopy plasmid, pREM85, generates a motile swarm of cells comparable in diameter to that generated by the wild-type strain harboring the same plasmid. (E) The integrated mscL::npts139 plasmid from either JG1221 ({Delta}ihfB) or JG1220 (wild-type ihfB) or that was transduced into the wild-type strain (wild type x JG1221) or the {Delta}ihfB strain (JG1216 x JG1220), respectively. The kanamycin-resistant transductants were then scored for a motility defect by inoculation into semisolid motility agar (see Results). Examples of strains that acquired a motility defect, in the case of wild-type recipient (wild-type x JG1221), or a wild-type motility phenotype, in the case of {Delta}ihfB recipient (JG1216 x JG1220), are shown.

Since the swarm size of {Delta}ihfB cells possessing pREM85 was significantly smaller than that exhibited by wild-type cells without plasmid, we performed an additional complementation test by transduction. We constructed strains in which a plasmid containing the adjacent mscL gene and a kanamycin resistance marker was integrated into either the wild-type or deleted ihfB gene (see Materials and Methods) (Fig. 2B). The transduction frequency of the kanamycin resistance marker into either wild-type or JG1216 cells was similar (approximately 10–5 per PFU) whether the phage were derived from the wild-type strain or JG1216, indicating that acquisition of the deleted ihfB allele was not especially deleterious or required a secondary mutation. In order to determine whether the ihfB deletion was responsible for the observed motility defect in the original strain, we also assessed the frequency of motility defects in each transduction experiment. Transducing phage derived from wild-type cells plated onto JG1216 resulted in a high frequency of recipients (94%) that had normal motility, thus complementing the motility defect in JG1216 (Fig. 2E). In a complementary experiment, we found that transducing phage prepared from JG1216 conferred a motility defect (Fig. 2E) to wild-type recipients at a similar frequency (95%). Thus, the integrated mscL-containing plasmid is 94 to 95% linked to ihfB by transduction. Importantly, this result shows that the low frequency of motility-deficient, {Delta}ihfB cells recovered in the original screen is not a consequence of the required acquisition of second-site, compensatory mutations and that the observed motility defect is probably attributable to the deletion in ihfB.

The cellular levels of the 27-kDa flagellin are reduced in an {Delta}ihfB mutant strain. Next we performed immunoblot analysis to determine whether the {Delta}ihfB mutant strain was deficient in IHF. Initially, we used antiserum raised against the E. coli protein (Fig. 3A). As shown above (Fig. 1), this antiserum reacts with two low-molecular-weight polypeptides present in extracts of wild-type C. crescentus cells that presumably correspond to the {alpha}- and ß-subunits of IHF. This antiserum reacted weakly with the polypeptide of apparent higher molecular weight (Fig. 3A, lane 1) and showed an absence of this slower-migrating species in the {Delta}ihfB cells (Fig. 3A, lane 3). Since the slower-migrating band disappeared in this mutant strain, we propose that this polypeptide corresponds to the ß-subunit of C. crescentus IHF. This is in contrast to the behavior of IHF from E. coli, in which the slower-migrating species corresponds to the higher-molecular-weight IHF {alpha}-subunit. In support of this idea, the introduction of an intact copy of ihfB on a plasmid (pREM85) restored, and increased, the level of expression of the slower-migrating polypeptide (Fig. 3A, lane 4). Since the C. crescentus IHF ß-subunit and 9.6-kDa HU proteins share approximately 31% identity and the antiserum we employed in these experiments cross-reacts with E. coli HU (Fig. 1), we performed a control to determine whether the polypeptides observed in these immunoblots corresponded to HU. In order to accomplish this, we employed C. crescentus anti-HU antibody (11) in immunoblots of cell extracts prepared from each of these strains (Fig. 3A). The HU antiserum showed two distinct polypeptides, one that was of similar apparent molecular weight to the faster-migrating band observed using anti-IHF antiserum (Fig. 3A). These polypeptides were of a molecular weight distinct from that of the slower-migrating polypeptide detected with the anti-IHF antibody. Furthermore, the levels of the HU subunits were unaffected in the {Delta}ihfB strain, suggesting that the slower-migrating band is IHF.



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  		FIG. 3. The {Delta}ihfB strain expresses reduced levels of the 27-kDa flagellin. (A) Crude cell extracts of LS107 (wild type) (lanes 1 and 6), SC1032 (flbD::Tn5) (lanes 2 and 7), JG1216 ({Delta}ihfB) (lanes 3 and 8), JG1216 containing pREM85 (lanes 4 and 9), and LS107 containing pREM85 (lanes 5 and 10) were prepared and subjected to immunoblot analyses with antiflagellin, E. coli anti-IHF (lanes 1 to 5), anti-HU (lanes 6 to 10), and anti-MreB (loading control) antisera. The IHF {alpha}-subunit and ß-subunit electrophoresed as two distinct bands, with the slower-migrating band (indicated as IHF) absent in extract prepared from the {Delta}ihfB strain. The {Delta}ihfB strain contains reduced amounts of the 27-kDa flagellin while still possessing wild-type levels of the 25- and 29-kDa flagellins. Expression of ihfB from the multicopy plasmid construct pREM85 restores wild-type levels of the 27-kDa flagellin to the {Delta}ihfB strain. (B) Cell extracts of LS107 (wild type) (lane 2), JG1216 ({Delta}ihfB) (lane 3), JG1216 containing pREM85 (lane 4), and LS107 containing pREM85 (lane 5) were prepared and subjected to immunoblot analysis with C. crescentus anti-IHF-B antibody. Purified His6-IHF-B was loaded in lane 1. The slower-migrating band is absent in the {Delta}ihfB strain.

In order to confirm that the slower-migrating polypeptide absent in the {Delta}ihfB strain was in fact the ß-subunit of C. crescentus IHF, we raised antibodies to purified His-tagged IHF-B from C. crescentus. This antiserum reacted strongly with the purified His-tagged IHF-B (Fig. 3B, lane 1), as well as with two polypeptides present in extracts from wild-type cells of approximate predicted molecular masses corresponding to either subunit of IHF (Fig. 3B, lane 2). The slower-migrating species was once again absent in extracts prepared from the {Delta}ihfB strain (Fig. 3B, lane 3) and was restored by the introduction of ihfB on plasmid pREM85 (Fig. 3B, lane 4). This result indicates that the {Delta}ihfB strain is deficient in the synthesis of the ihfB gene product and that this subunit of IHF migrates slower under these electrophoretic conditions than the {alpha}-subunit. We speculate that the slower migration of this polypeptide may be attributable to either differences in the degree of its association with sodium dodecyl sulfate (SDS) and/or its isoelectric point.

Since the {Delta}ihfB mutant strain exhibited a motility defect, we next determined if the {Delta}ihfB mutation affected the cellular levels of flagellin (Fig. 3A). Previous mutational analysis of flagellar promoters predicted that the IHF-deficient strain may lack significant class III flagellar gene expression, thus resulting in a partially assembled basal body-hook complex (18, 19, 38). In the absence of a completed class III-encoded hook-basal body structure, the mRNAs of class IV genes, encoding the flagellin proteins of the filament, are not translated (2, 3, 36, 37). Immunoblot analysis of cell extracts prepared from the wild-type, flbD::Tn5 (the class III/IV transcriptional activator) and {Delta}ihfB strains revealed that the {Delta}ihfB strain expressed wild-type levels of the most abundant filament protein, the 25-kDa flagellins, that are encoded by the class IV fljKMNO genes (Fig. 3A). Interestingly, the 27-kDa flagellin, encoded by fljL, was present in significantly reduced amounts in the {Delta}ihfB strain, resembling that produced in the flbD::Tn5 strain. Expression of ihfB from pREM85 completely restored production of FljL to the {Delta}ihfB strain. In fact, the amount of the 25- and 27-kDa flagellins was found to be slightly higher in both the wild-type and {Delta}ihfB strains harboring pREM85 than in the wild-type strain alone (Fig. 3A).

Cells possessing a {Delta}ihfB mutation exhibit a minor cell division defect. Class II flagellar mutants exhibit a cell division defect characterized by the presence of an abundant frequency of elongated cells when grown in PYE medium (41, 66). Likewise, such mutants typically grow at a reduced rate compared to either wild-type or late flagellar assembly mutant strains when cultured in liquid medium (41). In contrast, class III and IV flagellar mutants are morphologically indistinguishable from wild-type cells when observed under the light microscope (41). We compared the cell morphology (Fig. 4A to C) and growth rate (Fig. 4D) of the {Delta}ihfB strain to those of the wild-type and the flbD::Tn5 cells grown in PYE medium. The {Delta}ihfB mutant strain exhibited a relatively minor cell division defect, with the accumulation of some slightly elongated and filamentous cells (approximately 10%) in late logarithmic phase (Fig. 4B). This is in contrast to a flbD::Tn5 strain (Fig. 4C) and other class II flagellar mutants, which exhibit an abundance of filamentous cells as the culture approaches stationary phase. Although the {Delta}ihfB cells were less motile and possessed a minor cell division defect, they grew at a rate almost identical to that of wild-type cells, approximately 1.5-fold faster than the growth rate observed for the flbD::Tn5 cells (Fig. 3D). Interestingly, recent experiments have shown that the C. crescentus origin of DNA replication contains IHF binding sites (56). It is hypothesized that IHF binding to these sites enhances the initiation of replication. The growth rate and cell morphology data presented here indicate that there is probably no gross cell cycle defect in this strain lacking IHF. We have also found that there is no increase in the frequency of anucleate cells in the {Delta}ihfB strain (data not shown).



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  		FIG. 4. Loss of IHF alters cell morphology but does not affect the growth rate of C. crescentus cells. Shown are phase-contrast micrographs of the wild-type (A), ihfB1 ({Delta}ihfB) (B), and flbD::Tn5 (C) strains grown in liquid PYE to an optical density at 600 nm of 0.5 and stained with the lipophilic fluorescent dye FM4-64. C. crescentus class II flagellar mutants exhibited a characteristic cell division defect, seen as an accumulation of filamentous cells during the mid- to late-log phase of growth. Cultures of {Delta}ihfB cells contained some elongated and filamentous cells (indicated by arrows) (B); however, they do not resemble the cell division phenotype typical of class II flagellar mutants, such as flbD::Tn5 (C). (D) Comparison of growth rates of the wild-type, {Delta}ihfB, and flbD::Tn5 strains grown in liquid PYE medium. The class II flagellar mutant, flbD::Tn5, grows 1.5-fold slower than either the wild-type or {Delta}ihfB strain. While the {Delta}ihfB strain displays a cell division defect mildly similar to that exhibited by the flbD::Tn5 strain (B), it grows at a wild-type rate.

Effect of {Delta}ihfB on flagellar gene expression. A role for IHF in the temporal and spatial expression of late flagellar genes in C. crescentus had been previously suggested by assaying the expression of flagellar reporter gene fusions that possessed mutated IHF binding sites (18, 19). These initial studies focused on the transcription of the divergently transcribed class III flagellar genes, flgK and fliK (formerly flaN and flbG, respectively). Both flgK and fliK require FlbD and {sigma}54-containing RNA polymerase for transcription, as well as an intact IHF binding site, in order to achieve maximal expression levels (18, 19). The intergenic regulatory region shared by flgK and fliK is unique in C. crescentus in that it is the only flagellar promoter region that contains two divergently transcribed class III genes (14, 17) (Fig. 5). It is organized such that IHF binding and bending the DNA, in conjunction with FlbD binding, could create a higher-order nucleosome-like structure (18). The remaining class III flagellar genes are divergently transcribed from class II flagellar genes (or class II flagellar gene operons) that require {sigma}73-containing RNA polymerase and CtrA for expression (14, 17, 27) (Fig. 5). For example, the class III flagellar gene flgF, encoding the proximal rod, is divergently transcribed from the class II gene fliL (Fig. 5) (67). Likewise, the class III, flgI operon, which also contains putative IHF and FlbD binding sites, is divergently transcribed from the class II fliX gene (Fig. 5). As with the fliK and flgK promoters, mutations in the IHF binding site of the flgF promoter have indicated that IHF is required for maximal transcription (38). In contrast, the class IV flagellin genes all exist as solitary transcription units (Fig. 5). We hypothesized that the higher-order structure induced upon IHF binding may have varied effects on the transcriptional activity of these differently organized divergent promoters. For example, does IHF binding to a class III promoter influence the CtrA-activated transcription of the divergent class II promoter? We investigated the role of IHF in transcriptional activation of late flagellar genes, including any effects on expression of the divergently transcribed early class II flagellar genes. In order to accomplish this, we assayed the expression of flagellar promoter-lacZ transcription fusions in the {Delta}ihfB mutant strain. The promoters in this experiment consisted of the divergent class II fliL-lacZ and class III flgF-lacZ fusions and the divergent class II fliX-lacZ and class III flgI-lacZ fusions (Fig. 5). ß-Galactosidase activity generated from these fusions was compared with that measured in wild-type and flbD::Tn5 cells, as well as in {Delta}ihfB cells containing the complementing plasmid, pREM85. We also measured the expression of the two divergent class III promoters, using flgK- and fliK-lacZ reporter fusions, as well as the class IV, fljL-lacZ fusion. A schematic of the promoters used in these assays and a summary of the results is depicted in Fig. 5.



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  		FIG. 5. C. crescentus class II, III, and IV flagellar promoter organization. The schematic depicts the locations of cis-acting regulatory elements relative to the transcription initiation site of the flagellar promoters used in this study. The consensus sequences corresponding to the {sigma}54 and {sigma}73 binding sites, –12 and –24 (gray circles) and –10 and –35 (black circles), respectively, are shown. A checkered box illustrates the consensus sequence for the IHF binding site, a striped box shows the consensus CtrA binding site, and a dotted box denotes the enhancer-like ftr (flagellar transcription regulator) site recognized by the transcription factor FlbD. To the left is a summary of the data presented in the figure. +++, expression level equal to wild-type levels; +, expression level 20% of wild type. Note, for the class II-class III-divergent promoters shown, reporter gene expression measured in the {Delta}ihfB strain was marginally lower for the class II promoters and was slightly higher for the class III promoters than those measured in the wild-type strain.

As a control, we tested the expression of the class II fliF-lacZ fusion, which contains a binding site for the transcriptional activator, CtrA, but does not possess an IHF binding site (Fig. 5). Expression of fliF-lacZ in the {Delta}ihfB strain was no different than that observed for the wild-type strain and, as previously reported, was found to be fivefold higher in the flbD::Tn5 strain (Fig. 6A). Thus, a deletion in IHF probably does not have an adverse affect on CtrA-activated gene expression. In the {Delta}ihfB strain, the divergently transcribed class II flagellar reporters, fliL-lacZ and fliX-lacZ, were expressed at essentially wild-type levels, being reduced only 5% compared to expression in the wild-type strain (Fig. 6A). The activity of both of these increased approximately twofold in the flbD::Tn5 strain relative to wild-type, similar to what has been previously reported for these class II promoters (35). Interestingly, promoter activity of reporter gene fusions of the two genes divergently transcribed from fliL and fliX, the class III flgF and flgI genes (Fig. 6B), was slightly increased in the {Delta}ihfB strain (ca. 10 to 20% increase) compared to that in wild-type cells (Fig. 6B). This was an unanticipated result, because flgF-lacZ reporter expression, which is governed solely by the class III promoter elements, had been previously shown to decrease when IHF binding sites were mutated (38). These results also differ from the example of the divergently transcribed {sigma}54-dependent prpBCDE operon of Salmonella enterica serovar Typhimurium (49). Like these two C. crescentus flagellar promoters, the S. enterica prpBCDE promoter shares a regulatory region with the divergently transcribed {sigma}73-governed prpR gene. In contrast to the results presented here, transcription of the prpBCDE promoter is reduced 15-fold in the absence of IHF (49).



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  		FIG. 6. Effect of IHF on expression of flagellar genes. (A) Activity of the indicated class II flagellar gene-lacZ transcriptional reporter fusions was assayed in strains containing the {Delta}ihfB (JG1216) allele (white bars) as well as in wild-type cells (LS107) (gray bars), a strain containing flbD::Tn5 (SC1032) (dark gray bars), JG1216 containing ihfB on a multicopy plasmid (pREM85) (hatched bars), and wild-type cells containing pREM85 (black bars). Levels of reporter gene expression are shown in units of ß-galactosidase activity. The expression of the flagellar gene transcriptional reporters in unsynchronized cultures was determined in triplicate (on three independent cultures) by quantitative measurements of ß-galactosidase activity as previously described (37). The shown values represent the mean activity. The error bars represent the standard deviation. (B) Effect of IHF on expression of class III and IV flagellar genes. The activity of the indicated flagellar gene-lacZ transcriptional reporter fusions was assayed in strains containing the {Delta}ihfB (JG1216) allele (white bars) as well as in wild-type cells (LS107) (gray bars), a straincontaining flbD::Tn5 (SC1032) (dark gray bars), JG1216 containing ihfB on a multicopy plasmid (pREM85) (hatched bars), and wild-type cells containing pREM85 (black bars). Levels of reporter gene expression are shown in units of ß-galactosidase activity. The expression of the flagellar gene transcriptional reporters in unsynchronized cultures was determined in triplicate (in three independent cultures) by quantitative measurements of ß-galactosidase activity as previously described (37). The shown values represent the mean activity. The error bars represent the standard deviations. The deletion in ihfB results in a significant decrease in flgK (hook-associated protein), fliK (hook operon), and fljL (27-kDa flagellin) promoter activity.

The activity of the class III divergently transcribed promoters flgK and fliK was significantly decreased in the absence of IHF (Fig. 6B). Although this effect was not as severe as that observed in the flbD::Tn5 strain (approximately 10% compared to wild-type cells), the flgK-lacZ and fliK-lacZ expression levels assayed in the {Delta}ihfB strain were found to be 60 and 48% of wild-type, respectively (Fig. 6B). Introduction of the complementing plasmid containing ihfB restored wild-type reporter gene expression to the {Delta}ihfB mutant (Fig. 6B). Previous experiments using fliK and flgK reporter gene fusions with mutated IHF binding sites showed an approximate 35 to 50% reduction in ß-galactosidase expression (18, 19). Thus, these results are consistent with a previously proposed model for IHF in the transcriptional activation of {sigma}54 promoters, in which IHF-mediated looping of the DNA intervening between the promoter and enhancer sequences facilitates interaction between RNA polymerase and the FlbD transcriptional activator but is not absolutely required for transcriptional activation.

Since {Delta}ihfB cells lack significant amounts of the 27-kDa flagellin (FljL) (Fig. 3), we next investigated the influence IHF has on transcription of the class IV fljL gene (40) (Fig. 6B). At least two possible mechanisms might account for the decreased concentration of the 27-kDa flagellin in the {Delta}ihfB strain. First, the observation that {Delta}ihfB cells have a decrease in fliK and flgK expression may indicate an absence of a completed class III structure in a significant population of {Delta}ihfB mutant cells which, in turn, inhibits the translation of the fljL-encoded message (2, 36, 37). A second possibility for the decreased amount of FljL in the {Delta}ihfB mutant is that transcription of fljL is reduced in the absence of IHF. Consistent with this latter case, fljL-lacZ expression was the most severely affected of the flagellar genes we assayed in the IHF mutant strain (Fig. 6B). The fljL-lacZ reporter gene expression levels dropped to 20% of wild-type in IHF-deficient cells (Fig. 6B). Introduction of the ihfB-complementing plasmid restored a significant level of fljL-lacZ expression to the {Delta}ihfB cells (Fig. 6B). These data suggest that the observed defect in fljL transcription in the {Delta}ihfB strain contributes to the loss of the 27-kDa flagellin and the subsequent motility defect exhibited by the {Delta}ihfB mutant. Consistent with this conclusion, the motility defect exhibited by the {Delta}ihfB strain is similar to that observed when fljL is deleted (40).

Effect of IHF on cell cycle expression of late flagellar genes. In C. crescentus, coordination of the progression of flagellar assembly and cell division ensures the correct timing of expression of late flagellar genes (14, 17). A delay in class III flagellar gene transcription would yield an incomplete flagellar structure by the time of cell division, resulting in a loss of motility. One plausible possibility was that the impaired motility phenotype exhibited by the {Delta}ihfB strain could be attributed to just such a delay in late flagellar gene expression. In order to address this idea, we determined whether IHF influences the temporal expression of late flagellar genes by assaying the cell cycle pattern of fliK-lacZ expression in the {Delta}ihfB strain and compared it to that observed for wild-type cells (Fig. 7). The {Delta}ihfB strain was found to possess an almost identical cell cycle-regulated pattern of fliK-lacZ reporter gene expression as that observed for the wild-type strain, with maximal promoter activity in the predivisional cell stage (Fig. 7). Thus, it appears that a delay in late flagellar gene expression cannot account for either the decrease in class III fliK transcription or impaired motility.



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  		FIG. 7. Cell cycle expression of late flagellar genes is unaffected by the loss of IHF. Pure populations of wild-type and {Delta}ihfB swarmer cells containing a class III, fliK-lacZ transcriptional reporter fusion were isolated and allowed to progress synchronously through the cell cycle in M2 minimal medium. At the indicated time points a portion of the culture was pulse-labeled with 35STrans-label for 5 min followed by an ice-cold methionine chase. The labeled reporter fusion was immunoprecipitated using anti-ß-galactosidase antibody from portions of each time point sample containing equal amounts of labeled protein and separated by SDS-PAGE. Cells from a 1-ml portion of each culture were also isolated by centrifugation at the time points indicated. The resulting cell pellets were processed and subjected to immunoblot analyses. The anti-MreB (loading control) immunoblot is shown directly below the corresponding phosphorimage depicting the fliK-lacZ cell cycle expression pattern observed for the wild-type and {Delta}ihfB strains. A schematic of the cell types present at each time point during the cell cycle, as determined by light microscopy, is shown above the phosphorimages of the dried electrophoresed gels. The {Delta}ihfB strain exhibited an almost identical cell cycle pattern of fliK-lacZ reporter gene expression as the wild-type strain, with peak expression occurring in the predivisional cell.

Conclusions. The data presented here show that IHF is required for achieving maximal expression of a subset of the cell cycle-regulated, FlbD-dependent class III and IV flagellar genes and, consequently, for normal flagellar assembly and motility. These findings are analogous to those reported for an ihfA mutant of P. putida that exhibited reduced expression of the {sigma}54-dependent xyl operon and consequently was unable to degrade benzyl alcohol (9). We propose that the motility defect in the C. crescentus ihfB mutant strain is attributable to markedly reduced levels of the 27-kDa flagellin (FljL). This is more than likely the result of reduced transcription of fljL caused directly by the absence of IHF, and not the result of a flagellar assembly defect, since previous experiments have shown that flagellar assembly represses fljL translation but not transcription.

One surprising finding in C. crescentus was that an absence of IHF had varied effects on the different flagellar class III and IV promoters. We speculate that this may be attributed to the differences in the overall organization of each of the late flagellar promoter regions. Experiments in E. coli that examined IHF function in regulating bacteriophage Mu transcription were among the first to indicate that the ability of IHF to organize regulatory DNA sequences into stable supercoiled structures is important in influencing or controlling gene expression (24). The negative supercoils, possibly generated during transcription of the class II genes at the class II-class III divergent promoter regions, could function in place of IHF to enhance interaction of ftr-bound FlbD and {sigma}54-containing RNA polymerase at the class III regulatory region and, thus, promote transcriptional initiation. In fact, IHF function has been demonstrated to be dispensable for the {sigma}54-dependent expression of the NR1-regulated glnHp2 promoter when negative supercoils are present in the expression templates and NR1-phosphate is supplied to the transcription reaction mixture (10).

We propose that the reduced expression levels observed for the class III divergently transcribed flgK and fliK flagellar genes in the {Delta}ihfB strain may be attributed to the multiple ftr sites present within this promoter region (Fig. 5). FlbD belongs to a class of transcriptional activators that is known to oligomerize at the enhancer DNA sequences (26, 51). Oligomerization of these transcriptional activators is thought to greatly enhance their function in stimulating the isomerization of RNA polymerase-bound closed complex to an open complex (32, 51). FlbD binding at one of the two ftr elements positioned within each half of the class III divergent promoter region may aid in the recruitment of additional molecules of FlbD to the regulatory region shared by the flgK and fliK flagellar genes. IHF-induced DNA bending may serve to enhance the interaction of FlbD molecules bound to these distantly spaced enhancer sequences. Interestingly, the expression of the transcription of fljL was markedly more sensitive than other class III or IV promoters to an absence of IHF. It is thought that the major role of IHF in the transcriptional activation of these promoters is to deliver FlbD bound at the enhancer sequences to RNA polymerase bound to the promoter region. Previous experiments using nif promoters have indicated that an IHF-induced bend is required when RNA polymerase binds weakly to promoter DNA sequences (26, 54). Therefore, a plausible mechanism to account for the stringent requirement for IHF in fljL transcriptional activation could be a reflection of weak binding of RNA polymerase and/or FlbD to the fljL promoter region.


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ACKNOWLEDGMENTS
 
We are grateful to S. Goodman for the generous gift of E. coli anti-IHF antibody. We thank members of our laboratory for critical reading of the manuscript.

This work was supported by Public Health Service grant GM48417 from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095-1569. Phone: (310) 206-9449. Fax: (310) 206-5213. gober{at}chem.ucla.edu. Back

{dagger} Present address: Department of Genetics, Stanford University, Stanford, CA 94305. Back


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REFERENCES
 
    1
  1. Abril, M. A., M. Buck, and J. L. Ramos. 1991. Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein. J. Biol. Chem. 266:15832-15838.[Abstract/Free Full Text]
    2. 2
  2. Anderson, D. K., and A. Newton. 1997. Posttranscriptional regulation of Caulobacter flagellin genes by a late flagellum assembly checkpoint. J. Bacteriol. 179:2281-2288.[Abstract/Free Full Text]
    3. 3
  3. Anderson, P. E., and J. W. Gober. 2000. FlbT, the posttranscriptional regulator of flagellin synthesis in Caulobacter crescentus, interacts with the 5' UTR of flagellin mRNA. Mol. Microbiol. 38:41-52.[CrossRef][Medline]
    4. 4
  4. Ausubel, F. M., R. Brent, R. E. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley and Sons, Inc., New York, N.Y.
    5. 5
  5. Benson, A. K., G. Ramakrishnan, N. Ohta, J. Feng, A. J. Ninfa, and A. Newton. 1994. The Caulobacter crescentus FlbD protein acts at ftr sequence elements both to activate and to repress transcription of cell cycle-regulated flagellar genes. Proc. Natl. Acad. Sci. USA 91:2369-2373.
    6. 6
  6. Benson, A. K., J. Wu, and A. Newton. 1994. The role of FlbD in regulation of flagellar gene transcription in Caulobacter crescentus. Res. Microbiol. 145:420-430.[Medline]
    7. 7
  7. Bertoni, G., N. Fujita, A. Ishihama, and V. de Lorenzo. 1998. Active recruitment of {sigma}54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and {alpha}CTD. EMBO J. 117:5120-5128.[CrossRef]
    8. 8
  8. Brun, Y. V., and L. Shapiro. 1992. A temporally controlled sigma-factor is required for polar morphogenesis and normal cell division in Caulobacter. Genes Dev. 6:2395-2408.[Abstract/Free Full Text]
    9. 9
  9. Calb, R., A. Davidovitch, S. Koby, H. Giladi, D. Goldenberg, H. Margalit, A. Holtel, K. Timmis, J. M. Sanchez-Romero, V. de Lorenzo, and A. B. Oppenheim. 1996. Structure and function of the Pseudomonas putida integration host factor. J. Bacteriol. 178:6319-6326.[Abstract/Free Full Text]
    10. 10
  10. Carmona, M., and B. Magasanik. 1996. Activation of transcription at {sigma}54-dependent promoters on linear templates requires intrinsic or induced bending of the DNA. J. Mol. Biol. 261:348-356.[CrossRef][Medline]
    11. 11
  11. de Lorenzo, V., M. Herrero, M. Metzke, and K. N. Timmis. 1991. An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J. 10:1159-1167.[Medline]
    12. 12
  12. Drlica, K., and J. Rouviere-Yaniv. 1987. Histone-like proteins of bacteria. Microbiol. Rev. 51:301-319.[Free Full Text]
    13. 13
  13. Ely, B., and R. C. Johnson. 1977. Generalized transduction in Caulobacter crescentus. Genetics 87:391-399.[Abstract/Free Full Text]
    14. 14
  14. England, J. C., and J. W. Gober. 2001. Cell cycle control of cell morphogenesis in Caulobacter. Curr. Opin. Microbiol. 4:674-680.[CrossRef][Medline]
    15. 15
  15. Evinger, M., and N. Agabian. 1977. Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J. Bacteriol. 132:294-301.[Abstract/Free Full Text]
    16. 16
  16. Figge, R. M., A. V. Divakaruni, and J. W. Gober. 2004. MreB, the cell shape-determining bacterial actin homolog, coordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 51:1321-1332.[CrossRef][Medline]
    17. 17
  17. Gober, J. W., and J. C. England. 2000. Regulation of flagellum biosynthesis and motility in Caulobacter, p. 319-339. In Y. V. Brun and L. J. Shimkets (ed.), Prokaryotic development. ASM Press, Washington, D.C.
    18. 18
  18. Gober, J. W., and L. Shapiro. 1992. A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements. Mol. Biol. Cell 3:913-926.[Abstract]
    19. 19
  19. Gober, J. W., and L. Shapiro. 1990. Integration host factor is required for the activation of developmentally regulated genes in Caulobacter. Genes Dev. 4:1494-1504.[Abstract/Free Full Text]
    20. 20
  20. Gober, J. W., R. Champer, S. Reuter, and L. Shapiro. 1991. Expression of positional information during cell differentiation in Caulobacter. Cell 64:381-391.[CrossRef][Medline]
    21. 21
  21. Gober, J. W., H. Xu, A. K. Dingwall, and L. Shapiro. 1991. Identification of cis and trans-elements involved in the timed control of a Caulobacter flagellar gene. J. Mol. Biol. 217:247-257.[CrossRef][Medline]
    22. 22
  22. Goodman, S. D., and H. A. Nash. 1989. Functional replacement of a protein-induced bend in a DNA recombination site. Nature 341:251-254.[CrossRef][Medline]
    23. 23
  23. Goosen, N., and P. van de Putte. 1995. The regulation of transcription initiation by integration host factor. Mol. Microbiol. 16:1-7.[CrossRef][Medline]
    24. 24
  24. Higgins, N. P., D. A. Collier, M. W. Kilpatrick, and H. M. Krause. 1989. Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription in phage Mu. J. Biol. Chem. 264:3035-3042.[Abstract/Free Full Text]
    25. 25
  25. Holtel, A., D. Goldenberg, H. Giladi, A. B. Oppenheim, and K. N. Timmis. 1995. Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid. J. Bacteriol. 177:3312-3315.[Abstract/Free Full Text]
    26. 26
  26. Hoover, T. R., E. Santero, S. Porter, and S. Kustu. 1990. The integration host factor (IHF) stimulates interaction of RNA polymerase with NifA, the transcriptional activator for nitrogen fixation operons. Cell 63:11-21.[CrossRef][Medline]
    27. 27
  27. Hung, D., H. McAdams, and L. Shapiro. 2000. Regulation of the Caulobacter cell cycle, p. 361-378. In Y. V. Brun and L. J. Shimkets (ed.), Prokaryotic development. ASM Press, Washington, D.C.
    28. 28
  28. Johnson, R. C. and B. Ely. 1977. Isolation of spontaneously derived mutants of C. crescentus. Genetics 86:25-32.[Abstract/Free Full Text]
    29. 29
  29. Khambaty, F. M., and B. Ely. 1992. Molecular genetics of the flgI region and its role in flagellum biosynthesis in Caulobacter crescentus. J. Bacteriol. 174:4101-4109.[Abstract/Free Full Text]
    30. 30
  30. Kosturko, L. D., E. Daub, and H. Murialdo. 1989. The interaction of E. coli integration host factor and l cos DNA: multiple complex formation and protein induced bending. Nucleic Acids Res. 17:317-334.[Abstract/Free Full Text]
    31. 31
  31. Kovach, M. E., R. W. Phillips, P. H. Elzer, R. M. Roop, and K. M. Peterson. 1994. pBBR1MCS: a broad host range cloning vector. BioTechniques 16:800-802.[Medline]
    32. 32
  32. Kustu, S., E. Santero, J. Keener, D. Popham, and D. S. Weiss. 1989. Expression of {sigma}54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol. Rev. 53:367-376.[Free Full Text]
    33. 33
  33. Landy, A. 1989. Dynamic, structural and regulatory aspects of {lambda} site specific recombination. Annu. Rev. Biochem. 38:913-949.[CrossRef]
    34. 34
  34. Laub, M. T., H. M. McAdams, T. Feldblyum, C. M. Fraser, and L. Shapiro. 2000. Global analysis of the genetic network controlling a bacterial cell cycle. Science 290:2144-2148.[Abstract/Free Full Text]
    35. 35
  35. Macchi, R., L. Montesissa, K. Murakami, A. Ishihama, V. De Lorenzo, and G. Bertoni. 2003. Recruitment of {sigma}54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element. J. Biol. Chem. 278:27695-276702.[Abstract/Free Full Text]
    36. 36
  36. Mangan, E. K., J. Malakooti, A. Caballero, P. E. Anderson, B. Ely, and J. W. Gober. 1999. FlbT couples flagellum assembly to gene expression in Caulobacter crescentus. J. Bacteriol. 181:6160-6170.[Abstract/Free Full Text]
    37. 37
  37. Mangan, E. K., M. Bartamian, and J. W. Gober. 1995. A mutation that uncouples flagellum assembly from transcription alters the temporal pattern of flagellar gene expression in Caulobacter crescentus. J. Bacteriol. 177:3176-3184.[Abstract/Free Full Text]
    38. 38
  38. Marques, M. V., and J. W. Gober. 1995. Activation of a temporally regulated Caulobacter promoter by upstream and downstream sequence elements. Mol. Microbiol. 16:279-289.[CrossRef][Medline]
    39. 39
  39. Minnich, S. A., and A. Newton. 1987. Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 84:1142-1146.[Abstract/Free Full Text]
    40. 40
  40. Minnich, S. A., N. Ohta, N. Taylor, and A. Newton. 1988. Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement. J. Bacteriol. 170:3953-3960.[Abstract/Free Full Text]
    41. 41
  41. Muir, R. E., and J. W. Gober. 2001. Regulation of late flagellar gene transcription and cell division by flagellum assembly in Caulobacter crescentus. Mol. Microbiol. 41:117-130.[CrossRef][Medline]
    42. 42
  42. Muir, R. E., and J. W. Gober. 2002. Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus. Mol. Microbiol. 43:597-616.[CrossRef][Medline]
    43. 43
  43. Muir, R. E., and J. W. Gober. Regulation of FlbD activity by flagellum assembly is accomplished through direct interaction with the trans-acting factor, FliX. Mol. Microbiol. 54:715-730.
    44. 44
  44. Muir, R. E., T. M. O'Brien, and J. W. Gober. 2001. The Caulobacter crescentus flagellar gene, fliX, encodes a novel trans-acting factor that couples flagellar assembly to transcription. Mol. Microbiol. 39:1623-1637.[CrossRef][Medline]
    45. 45
  45. Mullin, D. A., and A. Newton. 1989. Ntr-like promoters and upstream regulatory sequence ftr are required for transcription of a developmentally regulated Caulobacter crescentus flagellar gene. J. Bacteriol. 171:3218-3227.[Abstract/Free Full Text]
    46. 46
  46. Mullin, D. A., and A. Newton. 1993. A sigma 54 promoter and downstream sequence elements ftr2 and ftr3 are required for regulated expression of divergent transcription units flaN and flbG in Caulobacter crescentus. J. Bacteriol. 175:2067-2076.[Abstract/Free Full Text]
    47. 47
  47. Mullin, D., S. Minnich, L. S. Chen, and A. Newton. 1987. A set of positively regulated flagellar gene promoters in Caulobacter crescentus with sequence homology to the nif gene promoters of Klebsiella pneumoniae. J. Mol. Biol. 195:939-943.[CrossRef][Medline]
    48. 48
  48. Newton, A., N. Ohta, G. Ramakrishnan, D. Mullin, and G. Raymond. 1989. Genetic switching in the flagellar gene hierarchy of Caulobacter requires negative as well as positive regulation of transcription. Proc. Natl. Acad. Sci. USA 86:6651-6655.[Abstract/Free Full Text]
    49. 49
  49. Palacios, S., and J. C. Escalante-Semerena. 2000. prpR, ntrA, and ihf are required for expression of the prpBCDE operon, encoding enzymes that catabolize propionate in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 182:905-910.[Abstract/Free Full Text]
    50. 50
  50. Poindexter, J. S. 1964. Biological properties and classification of Caulobacter group. Bacteriol. Rev. 28:231-295.[Free Full Text]
    51. 51
  51. Porter, S. C., A. K. North, A. B. Wedel, and S. Kustu. 1993. Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation. Genes Dev. 7:2258-2273.[Abstract/Free Full Text]
    52. 52
  52. Rice, P. A., S. Yang, K. Mizuuchi, and H. A. Nash. 1996. Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell 87:1295-1306.[CrossRef][Medline]
    53. 53
  53. Robertson, C. A., and H. A. Nash. 1988. Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor. J. Biol. Chem. 263:3554-3557.[Abstract/Free Full Text]
    54. 54
  54. Santero, E., T. R. Hoover, A. K. North, D. K. Berger, S. C. Porter, and S. Kustu. 1992. Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter. J. Mol. Biol. 227:602-620.[CrossRef][Medline]
    55. 55
  55. Schweizer, H. P. 1992. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtillis sacB marker. Mol. Microbiol. 6:1195-11204.[Medline]
    56. 56
  56. Siam, R., A. K. Brassinga, and G. T. Marczynski. 2003. A dual binding site for integration host factor and the response regulator CtrA inside the Caulobacter crescentus replication origin. J. Bacteriol. 185:5563-5572.[Abstract/Free Full Text]
    57. 57
  57. Simon, R., U. Piefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Biotechnology 1:784-790.[CrossRef]
    58. 58
  58. Stephens, C., C. Mohr, C. Boyd, J. Maddock, J. Gober, and L. Shapiro. 1997. Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system. J. Bacteriol. 179:5355-5365.[Abstract/Free Full Text]
    59. 59
  59. Stenzel, T. T., C. S. Dow, and L. Vitkovic. 1987. The integration host factor of E. coli binds to bent DNA at the origin of replication of the plasmid pSC101. Cell 49:709-717.[CrossRef][Medline]
    60. 60
  60. Thompson, J. F., and A. Landy. 1988. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 16:9687-9705.[Abstract/Free Full Text]
    61. 61
  61. Towbin, H., T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.[Abstract/Free Full Text]
    62. 62
  62. Ussery, D., T. S. Larsen, K. T. Wilkes, C. Friis, P. Worning, A. Krough, and S. Brunak. 2001. Genome organisation and chromatin structure in Escherichia coli. Biochimie 83:201-212.[Medline]
    63. 63
  63. Wingrove, J. A., and J. W. Gober. 1994. A {sigma}54 transcriptional activator also functions as a pole-specific repressor in Caulobacter. Genes Dev. 8:1839-1852.[Abstract/Free Full Text]
    64. 64
  64. Wingrove, J. A., E. K. Mangan, and J. W. Gober. 1993. Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter. Genes Dev. 7:1979-1992.[Abstract/Free Full Text]
    65. 65
  65. Wu, J., A. K. Benson, and A. Newton. 1995. Global regulation of a {sigma}54-dependent flagellar gene family in Caulobacter crescentus by the transcriptional activator FlbD. J. Bacteriol. 177:3241-3250.[Abstract/Free Full Text]
    66. 66
  66. Xu, H., A. Dingwall, and L. Shapiro. 1989. Negative transcriptional regulation in the Caulobacter flagellar hierarchy. Proc. Natl. Acad. Sci. USA 86:6656-6660.[Abstract/Free Full Text]
    67. 67
  67. Yu, J., and L. Shapiro. 1992. Early Caulobacter crescentus genes fliL and fliM are required for flagellar gene expression and normal cell division. J. Bacteriol. 174:3327-3338.[Abstract/Free Full Text]

Journal of Bacteriology, February 2005, p. 949-960, Vol. 187, No. 3
0021-9193/05/$08.00+0     doi:10.1128/JB.187.3.949-960.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Shaheen, S. M., Ouimet, M.-C., Marczynski, G. T. (2009). Comparative analysis of Caulobacter chromosome replication origins. Microbiology 155: 1215-1225 [Abstract] [Full Text]  

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