Cell cycle control of cell morphogenesis in Caulobacter.

In Caulobacter crescentus, morphogenic events, such as cytokinesis, the establishment of asymmetry and the biogenesis of polar structures, are precisely regulated during the cell cycle by internal cues, such as cell division and the initiation of DNA replication. Recent studies have revealed that the converse is also true. That is, differentiation events impose regulatory controls on other differentiation events, as well as on progression of the cell cycle. Thus, there are pathways that sense the assembly of structures or the localization of complexes and then transduce this information to subsequent biogenesis or cell cycle events. In this review, we examine the interplay between flagellar assembly and the C. crescentus cell cycle.

The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus.

In Caulobacter crescentus, the genes encoding the chromosome partitioning proteins, ParA and ParB, are essential. Depletion of ParB resulted in smooth filamentous cells in which DNA replication continued. Immunofluorescence microscopy revealed that the formation of FtsZ rings at the mid-cell, the earliest molecular event in the initiation of bacterial cell division, was blocked in cells lacking ParB. ParB binds sequences near the C. crescentus origin of replication. Cell cycle experiments show that the formation of bipolarly localized ParB foci, and presumably localization of the origin of replication to the cell poles, preceded the formation of FtsZ rings at the mid-cell by 20 min. These results suggest that one major function of ParA and ParB may be to regulate the initiation of cytokinesis in C. crescentus.

Regulation of late flagellar gene transcription and cell division by flagellum assembly in Caulobacter crescentus.

Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a trans-acting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a trans-acting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.

The Caulobacter crescentus flagellar gene, fliX, encodes a novel trans-acting factor that couples flagellar assembly to transcription.

The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a trans-acting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a trans-acting factor. Furthermore, extragenic motile suppressors which arise in DeltafliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. beta-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.

Temporal regulation of genes encoding the flagellar proximal rod in Caulobacter crescentus.

The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. The flgBC-fliE genes were identified as a result of the C. crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with sigma(70) is responsible for regulating transcription of other early flagellar genes in C. crescentus. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and flgB deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus.

FlbT, the post-transcriptional regulator of flagellin synthesis in Caulobacter crescentus, interacts with the 5' untranslated region of flagellin mRNA.

Flagellar gene expression' in Caulobacter crescentus is regulated by a complex trans-acting hierarchy, in which the assembly of early structural proteins is required for the expression of later structural proteins. The flagellins that comprise the filament are regulated at both the transcriptional and the post-transcriptional levels. Post-transcriptional regulation is sensitive to the assembly of the flagellar basal body and hook structures. In mutant strains lacking these structures, flagellin genes are transcribed, but not translated. Mutations in the flagellar regulatory gene, flbT, restore flagellin translation in the absence of flagellar assembly. In this report, we investigate the mechanism of FlbT-mediated post-transcriptional regulation. We show that FlbT is associated with the 5' untranslated region (UTR) of fljK (25 kDa flagellin) mRNA and that this association requires a predicted loop structure in the transcript. Mutations within this loop abolished FlbT association and resulted in increased mRNA stability, indicating that FlbT promotes the degradation of flagellin mRNA by associating with the 5' UTR. We also assayed the effects on gene expression using mutant transcripts fused to lacZ. Interestingly, the mutant transcript that failed to associate with FlbT in vitro was still repressed in mutants defective in flagellum assembly, suggesting that other factors in addition to FlbT couple assembly to translation.

FlbT couples flagellum assembly to gene expression in Caulobacter crescentus.

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.

Protein localization during the Caulobacter crescentus cell cycle.

New research on bacterial cells has demonstrated that they have a dynamic and complex subcellular organization. Work in Caulobacter crescentus shows that essential and nonessential proteins localize to discrete positions in the cell as a function of cell-cycle progression. The flagellum and chemotaxis receptor are asymmetrically localized to a single pole in the predivisional cell by coordinated proteolysis and transcriptional regulation. Cell type- and compartment-specific localization of the CtrA global transcriptional regulator is essential for proper cell-cycle progression, and subcellular localization of key chromosome partitioning proteins is correlated with proper nucleoid segregation. Given this structural complexity, we are driven to ask how localization is achieved, and to what end.

A gene coding for a putative sigma 54 activator is developmentally regulated in Caulobacter crescentus.

In Caulobacter crescentus, the alternative sigma factor sigma54 plays an important role in the expression of late flagellar genes. Sigma54-dependent genes are temporally and spatially controlled, being expressed only in the swarmer pole of the predivisional cell. The only sigma54 activator described so far is the FlbD protein, which is involved in activation of the class III and IV flagellar genes and repression of the fliF promoter. To identify new roles for sigma54 in the metabolism and differentiation of C. crescentus, we cloned and characterized a gene encoding a putative sigma54 activator, named tacA. The deduced amino acid sequence from tacA has high similarity to the proteins from the NtrC family of transcriptional activators, including the aspartate residues that are phosphorylated by histidine kinases in other activators. The promoter region of the tacA gene contains a conserved sequence element present in the promoters of class II flagellar genes, and tacA shows a temporal pattern of expression similar to the patterns of these genes. We constructed an insertional mutant that is disrupted in tacA (strain SP2016), and an analysis of this strain showed that it has all polar structures, such as pili, stalk, and flagellum, and displays a motile phenotype, indicating that tacA is not involved in the flagellar biogenesis pathway. However, this strain has a high percentage of filamentous cells and shows a clear-plaque phenotype when infected with phage phiCb5. These results suggest that the TacA protein could mediate the effect of sigma54 on a different pathway in C. crescentus.

Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus.

In the bacterium C. crescentus, the cellular homologs of plasmid partitioning proteins, ParA and ParB, localize to both poles of the predivisional cell following the completion of DNA replication. ParB binds to DNA sequences adjacent to the origin of replication suggesting that this region of the genome is tethered to the poles of the cell at a specific time in the cell cycle. Increasing the cellular levels of ParA and ParB disrupts polar localization and results in defects in both cell division and chromosome partitioning. These results suggest that ParA and ParB are involved in partitioning newly replicated chromosomes to the poles of the predivisional cell and may function as components of a bacterial mitotic apparatus.

Identification of an asymmetrically localized sensor histidine kinase responsible for temporally and spatially regulated transcription.

Caulobacter crescentus undergoes asymmetric cell division, resulting in a stalked cell and a motile swarmer cell. The genes encoding external components of the flagellum are expressed in the swarmer compartment of the predivisional cell through the localized activation of the transcription factor FlbD. The mechanisms responsible for the temporal and spatial activation of FlbD were determined through identification of FlbE, a histidine kinase required for FlbD activity. FlbE is asymmetrically distributed in the predivisional cell. It is located at the pole of the stalked compartment and at the site of cell division in the swarmer compartment. These findings suggest that FlbE and FlbD are activated in response to a morphological change in the cell resulting from cell division events.

Temporal and spatial regulation of fliP, an early flagellar gene of Caulobacter crescentus that is required for motility and normal cell division.

In Caulobacter crescentus, the genes encoding a single polar flagellum are expressed under cell cycle control. In this report, we describe the characterization of two early class II flagellar genes contained in the orfX-fliP locus. Strains containing mutations in this locus exhibit a filamentous growth phenotype and fail to express class III and IV flagellar genes. A complementing DNA fragment was sequenced and found to contain two potential open reading frames. The first, orfX, is predicted to encode a 105-amino-acid polypeptide that is similar to MopB, a protein which is required for both motility and virulence in Erwinia carotovora. The deduced amino acid sequence of the second open reading frame, fliP, is 264 amino acids in length and shows significant sequence identity with the FliP protein of Escherichia coli as well as virulence proteins of several plant and mammalian pathogens. The FliP homolog in pathogenic organisms has been implicated in the secretion of virulence factors, suggesting that the export of virulence proteins and some flagellar proteins share a common mechanism. The 5' end of orfX-fliP mRNA was determined and revealed an upstream promoter sequence that shares few conserved features with that of other early Caulobacter flagellar genes, suggesting that transcription of orfX-fliP may require a different complement of trans-acting factors. In C. crescentus, orfX-fliP is transcribed under cell cycle control, with a peak of transcriptional activity in the middle portion of the cell cycle. Later in the cell cycle, orfX-fliP expression occurs in both poles of the predivisional cell. Protein fusions to a lacZ reporter gene indicate that FliP is specifically targeted to the swarmer compartment of the predivisional cell.

A mutation that uncouples flagellum assembly from transcription alters the temporal pattern of flagellar gene expression in Caulobacter crescentus.

The transcription of flagellar genes in Caulobacter crescentus is regulated by cell cycle events that culminate in the synthesis of a new flagellum once every cell division. Early flagellar gene products regulate the expression of late flagellar genes at two distinct stages of the flagellar trans-acting hierarchy. Here we investigate the coupling of early flagellar biogenesis with middle and late flagellar gene expression. We have isolated mutants (bfa) that do not require early class II flagellar gene products for the transcription of middle or late flagellar genes. bfa mutant strains are apparently defective in a negative regulatory pathway that couples early flagellar biogenesis to late flagellar gene expression. The bfa regulatory pathway functions solely at the level of transcription. Although flagellin promoters are transcribed in class II/bfa double mutants, there is no detectable flagellin protein on immunoblots prepared from mutant cell extracts. This finding suggests that early flagellar biogenesis is coupled to gene expression by two distinct mechanisms: one that negatively regulates transcription, mediated by bfa, and another that functions posttranscriptionally. To determine whether bfa affects the temporal pattern of late flagellar gene expression, cell cycle experiments were performed in bfa mutant strains. In a bfa mutant strain, flagellin expression fails to shut off at its normal time in the cell division cycle. This experimental result indicates that bfa may function as a regulator of flagellar gene transcription late in the cell cycle, after early flagellar structures have been assembled.

Regulation of the Caulobacter crescentus dnaKJ operon.

The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.

Activation of a temporally regulated Caulobacter promoter by upstream and downstream sequence elements.

The flagellar genes of Caulobacter crescentus are expressed under cell-cycle control. Expression is regulated by both flagellar assembly cues and cell-cycle events. In this paper we define the sequences required for the expression of the flgF operon, a new class of sigma 54 flagellar promoter. This promoter type is expressed in the middle portion of the cell cycle and regulates the expression of basal-body genes. DNase I footprinting and mutagenesis demonstrates that an integration host factor (IHF)-binding site is required for maximal levels of transcription of the flgF promoter. In addition to containing a conventional upstream enhancer element (RE-1), this promoter is unusual in that it also requires sequences (element RE-2) immediately downstream of the transcriptional start site for maximal levels of gene expression. Cell-cycle experiments indicate that RE-1 and RE-2 contribute equally to the regulation of temporal transcription. The presence of two intact elements in the promoter results in a fourfold increase in promoter activity compared with a promoter containing only one intact element, suggesting that these two elements may function synergistically to activate transcription.

Regulation of cellular differentiation in Caulobacter crescentus.

In Caulobacter crescentus, asymmetry is generated in the predivisional cell, resulting in the formation of two distinct cell types upon cell division: a motile swarmer cell and a sessile stalked cell. These progeny cell types differ in their relative programs of gene expression and DNA replication. In progeny swarmer cells, DNA replication is silenced for a defined period, but stalked cells reinitiate chromosomal DNA replication immediately following cell division. The establishment of these differential programs of DNA replication may be due to the polar localization of DNA replication proteins, differences in chromosome higher-order structure, or pole-specific transcription. The best-understood aspect of Caulobacter development is biogenesis of the polar flagellum. The genes encoding the flagellum are expressed under cell cycle control predominantly in the predivisional cell type. Transcription of flagellar genes is regulated by a trans-acting hierarchy that responds to both flagellar assembly and cell cycle cues. As the flagellar genes are expressed, their products are targeted to the swarmer pole of the predivisional cell, where assembly occurs. Specific protein targeting and compartmentalized transcription are two mechanisms that contribute to the positioning of flagellar gene products at the swarmer pole of the predivisional cell.

A sigma 54 transcriptional activator also functions as a pole-specific repressor in Caulobacter.

The differential localization of proteins in the Caulobacter predivisional cell leads to the formation of two distinct progeny cells: a motile swarmer cell and a sessile stalked cell. Pole-specific transcription in the predivisional cell is one mechanism responsible for protein localization. Here we show that the sigma 54 transcriptional activator FlbD, which activates swarmer pole-specific transcription of a subset of late flagellar genes, is also capable of functioning as a pole-specific repressor of the early flagellar fliF operon. DNase I footprinting and methylation interference assays indicate that FlbD binds to regions of the fliF promoter at regions that would be likely to interfere with the binding of RNA polymerase. A mutation that abolishes FlbD binding results in up to a fourfold increase in fliF promoter expression. This mutation alters both the spatial and temporal pattern of fliF expression resulting in the inappropriate expression of the fliF operon in the swarmer pole of the predivisional cell. These results demonstrate that FlbD represses early flagellar gene expression in the swarmer pole of the Caulobacter predivisional cell. This is the first instance in which a protein specifically involved in pole-specific repression has been identified in Caulobacter. The restriction of FlbD activity to the swarmer pole accomplishes two regulatory missions by simultaneously activating late flagellar gene expression and repressing early flagellar genes.

Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter.

Polar localization of proteins in the Caulobacter predivisional cell results in the formation of two distinct progeny cells, a motile swarmer cell and a sessile stalked cell. The transcription of several flagellar promoters is localized to the swarmer pole of the predivisional cell. We present evidence that the product of the flbD gene is the transcriptional activator of these promoters. We show that FlbD is distributed in all cell types and in both poles of the predivisional cell. We also demonstrate that FlbD can be phosphorylated, and that a FlbD kinase activity is under cell cycle control. Cells expressing a FlbD mutant that should activate transcription in the absence of phosphorylation, exhibited an alteration in the temporal pattern of flagellin transcription. Furthermore, predivisional cells expressing the mutant FlbD failed to polarly localize flagellin synthesis. We propose that the phosphorylation of FlbD is restricted to the swarmer compartment of the predivisional cell, and serves as the control point for regulating the spatial transcription of flagellar promoters.

A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

Compartmentalized transcription and the establishment of cell type during sporulation in Bacillus subtilis.

An early step in sporulation of the bacterium Bacillus subtilis, is the formation of two compartments in the developing sporangium: the mother cell and the forespore. These compartments differ in their programs of gene expression and developmental fate. The establishment of cell type within this simple developmental program, is accomplished by the compartmentalization of sigma subunits of RNA polymerase. The localization of these sigma factors results in compartment-specific gene expression. Recent experiments have elucidated some of the early steps in the establishment of cell type. After septum formation, the activity of the sigma factor, sigma F, is confined to the forespore compartment. This, in turn, results in the localized expression of another developmental sigma factor, sigma G. The forespore localization of these two sigma factors, establishes the forespore line of gene expression. sigma F and sigma G also regulate mother cell events. sigma F activity in the forespore regulates the proteolytic processing of sigma E within the mother cell compartment. The localization sigma E activity leads to mother cell expression of another sigma factor, pro-sigma K. The proteolytic processing of pro-sigma K to mature sigma K is controlled by the forespore sigma factor, sigma G. Mature sigma K then directs the transcription of mother cell specific genes. Therefore, the initial localization of sigma F activity to the forespore compartment, orchestrates the establishment of cell type in both forespore and mother cell compartments.