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Positional information during Caulobacter cell differentiation
Positional information during Caulobacter cell differentiation James w. Gober, M.R.K. Alley and Lucy Shapiro Stanford University School of Medicine, Stanford, California, USA The formation of two distinct daughter cells upon division of the bacterium Caulobacter crescentus is the result of asymmetry in the predivisional cell, in part due to localization of both flagellar and chemotaxis proteins to the swarmer cell pole. Recent evidence suggests that both localized transcription and protein targeting directed by specific amino acid sequence are involved in the localization. Current Opinion in Genetics and Development 1991, 1:324-329
Introduction The expression of positional information is a fundamental aspect of pattern formation during animal development (reviewed in [1]). The cell division cycle of the bacterium Caulobacter crescentus is an elegant system for understanding how positional information is generated within a single cell. Caulobacter undergoes a simple developmental program, which, upon cell division, results in the formation of two distinct daughter cells (for more extensive reviews, see [2-4]. The two cell types, a swarmer cell and a stalked cell are a result of asymmetry generated before cell division in the predivisional cell type. The localization of gene products within the predivisional cell is an important component of the polarity that accompanies the Caulobacterdevelopmental program. The most studied aspect of Caulobacter development is the temporallycontrolled biogenesis of the polar flagellum and the chemotaxis machinery. As these genes are expressed, their products are targeted to the incipient swarmer pole of the predivisional cell, where the flagellum is assembled. This review focuses on two mechanisms Caulobacter employs to position flagellar and chemotaxis gene products to the swarmer cell pole; the localized expression of mRNA and the positioning of proteins.
The cell cycle and the generation of polarity The use of Caulobacter as an organism to study cell cycle events is particularly attractive because of the ease with which synchronized populations can be obtained. Pure populations of swarmer cells can be separated from other cell types by centrifugation through colloidal silica [5]. Isolated swarmer cells resuspended in fresh growth medium progress synchronously through the cell division cycle (Fig. 1).
The swarmer cells are unable to initiate chromosomal DNA replication for about one-fourth of the cell division cycle. Then, in response to an unknown internal cue, the flagellum is ejected, a stalk begins to grow at the pole once occupied by the flagellum, and DNA replication is initiated. As replication progresses, the genes encoding the flagellum are expressed in a fixed sequence. Although the precise nature of the cellular event that triggers the expression of flagellar genes is unknown, it is likely to be linked to DNA replication. DNA replication appears to be necessary for the expression of some flageflar genes [6]. The flagellar gene products are specifically localized to the swarmer pole of the predivisional cell, where the flagellum is assembled. This asymmetry generated in the predivisional cell results in the formation of the two distinct daughter cells upon cell division. The progeny swarmer cell inherits the polar flagellum and chemotaxis apparatus, and is unable to initiate DNA replication. The progeny stalked cell does not possess flageflar or chemotaxis components, but is able to initiate DNA replication and immediately re-enters the cell cycle. Therefore, asymmetry in the regulation of DNA replication is also generated at the time of cell division. Differences in nucleoid structure reflect yet another level in the asymmetry expressed in the predivisional cell. The swarmer cell inherits a chromosome which differs from the stalked cell chromosome in its physical properties. Specifically, nucleoids isolated from swarmer cells sediment more rapidly in a sucrose gradient than nucleiods isolated from stalked cells [3,5,7,8]. As the swarmer cell enters the transition to the stalked cell type the sedimentation coefficient of the chromosome decreases to that originally seen in the progeny stalked cell. These differences in nucleoid sedimentation coefficient may reflect differences in the superhelical density of the chromosomes and/or may result from the asymmetric distribution of bacterial histone-like proteins. Chromosome structural differences may dictate whether a chromosome
Abbreviations IHF~integration host factor; MCP--methyl-accepting chemotaxis protein; Tsr~E. coli MCP.
324
(~) Current Biology Ltd ISSN 0959-437X
Positional information during Caulobacter cell differentiation Gober, Alley and Shapiro 325
Flagellar
Stalk
ejected
gellar
Swarmer cell Motile
Chemotactically competent
MCP
Methyltransferase Methylesterase 25,27,29kD flagellins Hook protein
92kD Lon homolog 37kD RNAP subunit 70kD DnaK
Predivisional
cell
Stalk cell DNA replication initiation
is competent to initiate DNA replication. Currently, experiments are underway to define the nature of the differences in nucleoid structure during the cell cycle and the relationship they have to the global programming of gene expression and DNA replication in the two progeny cell types.
Temporal and spatial expression of the flagellar gene products Genetic studies have revealed that approximately 50 genes are required for motility and chemotaxis in Caulobacter [9]. The structure of the Caulobacter flagelum is similar to that of other bacteria. The flagellum consists of three major components, a basal body (rotor) that traverses both the inner and outer membranes, a flexible hook (universal joint) and a rigid flagellar filament (propeler). As is the case for Salmonella O*phimurium and E.scberichia coli [10], the Caulobacter basal body structure is the first to be assembled, followed by the external structures, the hook and the filament [11]. A cascade of events governs the temporal sequence of flagelar gene expression. It is currendy thought that a cell cycle event switches on the expression of flagellar regulatory genes. The regulatory genes, in turn, act in a positive fashion to activate the expression of the flagellar
Fig. 1. Diagram of the Caulobacter crescentus cell cycle showing the asymmetric distribution of proteins upon cell division. The rings within the cells represent the replicating chromosome and stages of DNA replication.
structural genes. As in E. coli [12] and S. typbimurium [13], Caulobacter flagellar gene expression is controlled by a transacting regulatory hierachy [14-19]. The hierachy imposes control at the transcriptional level, so that the temporal order of transcription of the flageflar structural genes approximates their order of assembly in the flageflar structure [14-23]. For example, the basal body L-ring gene, fibN, is transcribed before the hook structural gene, flaK [24], which is expressed before the flagellin genes flgL and flgK [20]. It is not known whether this sequential order of expression of flageflar genes is required for proper assembly of the flagellum. The assembly process does, however, influence the transcription of several flagella genes. For example, mutations in flaK result in increased transcription initiating from its promoter and a marked decrease in the transcription of flagellin genes flgK and flgL [16]. The flagellar regulatory network is somehow able to transduce information regarding the progress of flagellar assembly to the transciption machinery. This is supported by the fact that some of the genes that affect transcription of the basal body, hook and flagellin genes encode structural proteins; for example, the flageflar switch protein encoded by r i M (J Yu and L Shapiro, unpublished data). Recent work regarding the mechanisms of transcriptional activation of flagelar genes has focused on flbN, flaK
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Molecular genetics of simple developmental systems
flgL and flgK All of these genes possess cr54 promoters [24-26] and therefore require a cr54-containing RNA polymerase holoenzyme for transcription. At approximately 100 bp upstream of the transcription start site the promoters all contain binding sites for transcriptional activator proteins. The hook and flagellin gene promoters possess the same upstream sequence, known as the fir [24,25,27]. A different upstream sequence occurs in the f/bN promoter and is a binding site for a 70 kD protein known as RF-2 [26]. When the RF-2 binding site is altered by site-directed mutagenesis the characteristic cell cycle increase in flbN transcription does not occur, indicating that RF-2 has a role in temporal activation of flbN transcription [26]. RF-2 binding activity itself is not under cell cycle control (JW Gober, unpublished data), suggesting that other factors may modify RF-2 to activate transcription. The hook operon (flaKis the fourth gene in the operon) [20], flgL and flgKall possess similar promoter architecture [24,25,28-]. These promoters contain a conserved fir sequence located approximately 100bp from the transcriptional start [24,25,27] as well as 30-40 bp of ATrich DNA that lies between the fir and the o54 promoter [28"]. Mutagenesis of the fir sequence in the hook promoter abolished transcription in vivo [27]. Two proteins which bind to theflr have been identified; a 95 kD protein (RF-1) and 55kD protein [29"]. Both binding activities are expressed under cel cycle control and occur at maximal levels when the hook promoter is expressed, suggesting that the cell cycle modulation of the level of these binding activities plays a role in the transcriptional activation of the hook promoter [29"]. Another protein that may activate the transcription of the hook promoter is the product of the flbD gene. Strains harboring mutations in flbD do not transcribe the hook promoter. The predicted sequence of f/bD, is similar to the NtrC class of transcriptional activators [30"]. When expressed in E. coli FIbD can activate transcription of the E. coliglnA promoter and the hook promoter [30"]. Delaying the expression of theflaOflbD operon results in a delay of hook operon expression in the cell cycle [31"]. This supports the idea that FIbD has a critical role in activation of hook transcription. It is still unclear whether the delay of hook expression is directly due to a lack of FIbD protein or is a consequence of the absence of a properly assembled flagellum. Strains which contain flbD mutations still express RF-1 and 55 kD binding activity [29"]. It is possible that FIbD works in concert with these proteins to activate transcription of the hook promoter. The AT-rich DNA sequence in the hook, flgL andflgKpromoters is a binding site for integration host factor (IHF) [28.]. IHF is a small, basic DNA-binding protein that was originally identified as a host requirement for lambda integration (reviewed in [32]). Mutagenesis of the IHFbinding site in the hook promoter results in five to ninefold lower transcription when assayed in vivo [28-]. Likewise, I-IF is required for the in vitro transcription of the c54 nail-/promoter of Klebsiella pneumoniae [33"]. In the lambda system, I-IF facilitates integrase-mediated recombination by introducing a bend in the DNA which
permits integrase to interact simultaneously with two distantly spaced DNA sequences [32]. I-IF probably participates in a similar long-distance interaction in these cr54 promoters. It has been proposed that the bending in the DNA caused by IHF brings proteins bound at the upstream activator sequence into close proximity to RNA polymerase bound at the promoter, thereby increasing their interactions which then enhance transcriptional activation [33"]. In addition, the level of IHF is under cell cycle control (.JW Gober, unpublished data). Like RF-1 and the 55 kD binding activity, IHF is present maximally in the predivisional cell. T h e f l r [27] and IHF-binding site [28"] also occur in the flaNQ operon, another flagellar gene with a cr54 promoter [25]. In this case, however, the sites are located downstream of the transcription start [27]. Like other promoters, the IHF-binding site is located between the fir elements and the promoter (between + 24 and + 57bp) [28.]. This promoter possesses two f l r elements at + 110 and +130 [27]. Both downstream fir elements [27] (JW Gober, L Shapiro, unpublished data) and the IHF-binding site (JW Gober, L Shapiro, unpublished data) are required for maximal levels of transcription. This finding demonstrates that the fir sequences participate in transcriptional activation whether they are located upstream or downstream of the promoter. The hook and llagellin genes not only share similar regulatory sequences in their promoters but also share a common mechanism by which their products are l o calized to the swarmer pole of the predivisional cell. Several years ago, Milhausen and Agabian [34] demonstrated that the mRNA of the flgK flagellin gene segregated specifically to progeny swarmer ceils. Such segregation occurred even when the predivisional cells were treated just prior to cell division, with rifampicin. This suggests that the flgK mRNA is not transcribed in the newly formed swarmer cells, but is rather localized to the incipient swarmer pole of the predivisional cell and then segregated to the progeny swarmer cell [34]. Recently, we have used transcriptional fusions to show that in addition toflgKmRNA, the mRNAs of the hook operon [35 °'] and the flaNQ operon (JW Gober, unpublished data) are also localized to the swarmer pole of the predivisional cell. Several possible mechanisms could account for flagellar mRNA localization. The mRNA of these flagellar genes could be specifically targeted or sequestered to the swarmer pole of the predivisional cell. This would be similar to the mechanism by which bicoid mRNA is localized during Drosophila embryogenesis [36]. Alternatively, transcription of these genes could occur specifically in the swarmer pole of the predivisional cell. We reasoned that for the first mechanism to be operating, a specific mRNA sequence would be required to target the mRNA. We therefore tested whether the reporter gene product could be localized when its synthesis was driven by a transcriptional fusion that contained no naturally occuring hook mRNA [35"]. This fusion contained 112bp of hook promoter DNA and could drive the localized expression of the reporter gene product [35"]. This result rules out localization mechanisms that require mRNAspecific sequences for targeting or differential turnover,
Positional information during Cau/obacter cell differentiation Gober, Alley and Shapiro 327 but allows for the possibility that transcription is localized to the chromosome at the swarmer pole of the predivisional cell. If this is the case, then the Caulobacter predivisional cell must possess two chromosomes that are programmed for different functions. We are currently studying the mechanism bywhich differential, global programming of chromosomal DNA could be accomplished. In the specific case of localized flagellar gene transcription, we are exploring three possible mechanisms. Because all the locally transcribed promoters possess RF-1 and IHF binding sites, one of these transcription factors may be specifically targeted to the swarmer pole of the predivisional cell. Alternatively, a critical transcription factor may only be active in the swarmer cell pole. Activation of the factor could be the result of a local event or structure that is unique to the swarmer cell pole. This unique local event may be the assembling flagellum. Interestingly, the flagellar genes that are locally transcribed (hook, flaNQ and flgK), require proper flagellar assembly for transcription. Finally, it is possible that the transcription factors are able to discriminate between the two chromosomes. The differences in swarmer and stalked cell nucleoid structure may play a role in this process (reviewed in [3]). It is possible that histone-like proteins compete for binding with specific transcription factors in a manner analogous to nucleosome-mediated repression of transcription in eukaryotic chromatin.
Mechanism of protein localization The specific positioning of proteins is another mechanism through which polarity is established in the Caulobacter predivisional cell. The heat shock proteins, DnaK and Lon, as well as the heat shock sigma factor segregate to the stalk cell upon division of the predivisional cell [37], whereas polar pili and phage ~CbK receptors [38,39], the llagellins, the methyl-accepting chemotaxis protein (MCP) and the chemotaxis methyltransferase and methylesterase [21,40,41] are all targeted to the swarmer pole of the predivisional cell (Fig. 1). To define the protein sequences involved in localization, we have recently carried out extensive deletion analysis on one of these genes, that encoding a MCP. The partially deleted MCP genes were fused to either 13-1actamase or 13-galactosidase reporter genes [42] and the intracellular localization of the resulting proteins was detected by immuno-gold electron microscopy and immuno-fluorescence microscopy. Full-length fusions of the MCP to either 13-1actamase or 13-galactosidase segregated to the swarmer cell upon cell division ( M R Alley and L Shapiro, unpublished data), and immuno-electron microscopy revealed that the proteins were localized to the incipient swarmer cell pole of the predivisional cell (J Maddock and L Shapiro, unpublished data). When a region within the carboxy-terminus of the MCP gene was removed, still leaving the transmembrane portion of the protein, the fusion proteins were distributed randomly within the inner membrane of the cell, indicating that membrane localization is not the sole determinant for polar localization. The amino acid sequence that contributes to the polar target-
ing is highly conserved in bacterial MCPs. Proteins with deletions within this region of homology were no longer segregated to the swarmer cell nor were they localized to the swarmer pole of the predivisional cell. This suggests that the highly conserved sequences located in the signaling domain of the MCP, are required for both protein localization and other functions. It has been demonstrated that the E. coli MCP, Tsr, is transcribed and synthesized in Caulobacter in a cell-cycle-dependent fashion, co-incident with the native MCP [43]. As predicted from the conserved 'localization' site, the E. coliTsr is also specifically segregated to the Caulobacter swarmer cell (MRK Alley, unpublished data). The results suggest that the targeting of the MCPs to the swarmer pole is at least in part determined by a conserved stretch of amino acids. Currently, the precise mechanism by which localization is achieved is unknown. The localization of the Caulobacter MCP to a discrete portion of the cell membrane raises several questions regarding the mechanism of protein targeting in bacteria. Is the MCP protein localized to a distinct polar cellular structure or compartment, akin to the periseptal annulus described in E. coli [44]? Does the E. coli MCP localize to the pole in the E. coli cell? More generally it is also uncertain whether the localization of MCP is unique or whether other proteins may be subject to subcellular localization. For example, cell division proteins, especially those involved in determining the precise location of the division site, may also be localized to a discrete part of the cell by a similar mechanism.
Conclusions The Caulobacter cell cycle, with its unique developmental program provides a tractable system to explore the problem of site-specific localization. Recent results suggest that both localized transcription and protein targeting direct localization. Similar mechanisms may be involved in other systems, where proteins are localized to a discrete position in a cell.
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OHTAN, CHEN LS, MULL~ DA, NEWTONA: Timing of Flagellar Gene Expression in the Caulobacter Cell Cycle is Determined by a Transcriptional Cascade of Positive Regulatory Genes. J Bacteriol 1991, 173:1514-1522. Demonstrates that a delay in expression of a flagellar regulatory operon results in a temporal delay of hook and flaN expression. 32.
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329
Introduction The expression of positional information is a fundamental aspect of pattern formation during animal development (reviewed in [1]). The cell division cycle of the bacterium Caulobacter crescentus is an elegant system for understanding how positional information is generated within a single cell. Caulobacter undergoes a simple developmental program, which, upon cell division, results in the formation of two distinct daughter cells (for more extensive reviews, see [2-4]. The two cell types, a swarmer cell and a stalked cell are a result of asymmetry generated before cell division in the predivisional cell type. The localization of gene products within the predivisional cell is an important component of the polarity that accompanies the Caulobacterdevelopmental program. The most studied aspect of Caulobacter development is the temporallycontrolled biogenesis of the polar flagellum and the chemotaxis machinery. As these genes are expressed, their products are targeted to the incipient swarmer pole of the predivisional cell, where the flagellum is assembled. This review focuses on two mechanisms Caulobacter employs to position flagellar and chemotaxis gene products to the swarmer cell pole; the localized expression of mRNA and the positioning of proteins.
The cell cycle and the generation of polarity The use of Caulobacter as an organism to study cell cycle events is particularly attractive because of the ease with which synchronized populations can be obtained. Pure populations of swarmer cells can be separated from other cell types by centrifugation through colloidal silica [5]. Isolated swarmer cells resuspended in fresh growth medium progress synchronously through the cell division cycle (Fig. 1).
The swarmer cells are unable to initiate chromosomal DNA replication for about one-fourth of the cell division cycle. Then, in response to an unknown internal cue, the flagellum is ejected, a stalk begins to grow at the pole once occupied by the flagellum, and DNA replication is initiated. As replication progresses, the genes encoding the flagellum are expressed in a fixed sequence. Although the precise nature of the cellular event that triggers the expression of flagellar genes is unknown, it is likely to be linked to DNA replication. DNA replication appears to be necessary for the expression of some flageflar genes [6]. The flagellar gene products are specifically localized to the swarmer pole of the predivisional cell, where the flagellum is assembled. This asymmetry generated in the predivisional cell results in the formation of the two distinct daughter cells upon cell division. The progeny swarmer cell inherits the polar flagellum and chemotaxis apparatus, and is unable to initiate DNA replication. The progeny stalked cell does not possess flageflar or chemotaxis components, but is able to initiate DNA replication and immediately re-enters the cell cycle. Therefore, asymmetry in the regulation of DNA replication is also generated at the time of cell division. Differences in nucleoid structure reflect yet another level in the asymmetry expressed in the predivisional cell. The swarmer cell inherits a chromosome which differs from the stalked cell chromosome in its physical properties. Specifically, nucleoids isolated from swarmer cells sediment more rapidly in a sucrose gradient than nucleiods isolated from stalked cells [3,5,7,8]. As the swarmer cell enters the transition to the stalked cell type the sedimentation coefficient of the chromosome decreases to that originally seen in the progeny stalked cell. These differences in nucleoid sedimentation coefficient may reflect differences in the superhelical density of the chromosomes and/or may result from the asymmetric distribution of bacterial histone-like proteins. Chromosome structural differences may dictate whether a chromosome
Abbreviations IHF~integration host factor; MCP--methyl-accepting chemotaxis protein; Tsr~E. coli MCP.
324
(~) Current Biology Ltd ISSN 0959-437X
Positional information during Caulobacter cell differentiation Gober, Alley and Shapiro 325
Flagellar
Stalk
ejected
gellar
Swarmer cell Motile
Chemotactically competent
MCP
Methyltransferase Methylesterase 25,27,29kD flagellins Hook protein
92kD Lon homolog 37kD RNAP subunit 70kD DnaK
Predivisional
cell
Stalk cell DNA replication initiation
is competent to initiate DNA replication. Currently, experiments are underway to define the nature of the differences in nucleoid structure during the cell cycle and the relationship they have to the global programming of gene expression and DNA replication in the two progeny cell types.
Temporal and spatial expression of the flagellar gene products Genetic studies have revealed that approximately 50 genes are required for motility and chemotaxis in Caulobacter [9]. The structure of the Caulobacter flagelum is similar to that of other bacteria. The flagellum consists of three major components, a basal body (rotor) that traverses both the inner and outer membranes, a flexible hook (universal joint) and a rigid flagellar filament (propeler). As is the case for Salmonella O*phimurium and E.scberichia coli [10], the Caulobacter basal body structure is the first to be assembled, followed by the external structures, the hook and the filament [11]. A cascade of events governs the temporal sequence of flagelar gene expression. It is currendy thought that a cell cycle event switches on the expression of flagellar regulatory genes. The regulatory genes, in turn, act in a positive fashion to activate the expression of the flagellar
Fig. 1. Diagram of the Caulobacter crescentus cell cycle showing the asymmetric distribution of proteins upon cell division. The rings within the cells represent the replicating chromosome and stages of DNA replication.
structural genes. As in E. coli [12] and S. typbimurium [13], Caulobacter flagellar gene expression is controlled by a transacting regulatory hierachy [14-19]. The hierachy imposes control at the transcriptional level, so that the temporal order of transcription of the flageflar structural genes approximates their order of assembly in the flageflar structure [14-23]. For example, the basal body L-ring gene, fibN, is transcribed before the hook structural gene, flaK [24], which is expressed before the flagellin genes flgL and flgK [20]. It is not known whether this sequential order of expression of flageflar genes is required for proper assembly of the flagellum. The assembly process does, however, influence the transcription of several flagella genes. For example, mutations in flaK result in increased transcription initiating from its promoter and a marked decrease in the transcription of flagellin genes flgK and flgL [16]. The flagellar regulatory network is somehow able to transduce information regarding the progress of flagellar assembly to the transciption machinery. This is supported by the fact that some of the genes that affect transcription of the basal body, hook and flagellin genes encode structural proteins; for example, the flageflar switch protein encoded by r i M (J Yu and L Shapiro, unpublished data). Recent work regarding the mechanisms of transcriptional activation of flagelar genes has focused on flbN, flaK
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Molecular genetics of simple developmental systems
flgL and flgK All of these genes possess cr54 promoters [24-26] and therefore require a cr54-containing RNA polymerase holoenzyme for transcription. At approximately 100 bp upstream of the transcription start site the promoters all contain binding sites for transcriptional activator proteins. The hook and flagellin gene promoters possess the same upstream sequence, known as the fir [24,25,27]. A different upstream sequence occurs in the f/bN promoter and is a binding site for a 70 kD protein known as RF-2 [26]. When the RF-2 binding site is altered by site-directed mutagenesis the characteristic cell cycle increase in flbN transcription does not occur, indicating that RF-2 has a role in temporal activation of flbN transcription [26]. RF-2 binding activity itself is not under cell cycle control (JW Gober, unpublished data), suggesting that other factors may modify RF-2 to activate transcription. The hook operon (flaKis the fourth gene in the operon) [20], flgL and flgKall possess similar promoter architecture [24,25,28-]. These promoters contain a conserved fir sequence located approximately 100bp from the transcriptional start [24,25,27] as well as 30-40 bp of ATrich DNA that lies between the fir and the o54 promoter [28"]. Mutagenesis of the fir sequence in the hook promoter abolished transcription in vivo [27]. Two proteins which bind to theflr have been identified; a 95 kD protein (RF-1) and 55kD protein [29"]. Both binding activities are expressed under cel cycle control and occur at maximal levels when the hook promoter is expressed, suggesting that the cell cycle modulation of the level of these binding activities plays a role in the transcriptional activation of the hook promoter [29"]. Another protein that may activate the transcription of the hook promoter is the product of the flbD gene. Strains harboring mutations in flbD do not transcribe the hook promoter. The predicted sequence of f/bD, is similar to the NtrC class of transcriptional activators [30"]. When expressed in E. coli FIbD can activate transcription of the E. coliglnA promoter and the hook promoter [30"]. Delaying the expression of theflaOflbD operon results in a delay of hook operon expression in the cell cycle [31"]. This supports the idea that FIbD has a critical role in activation of hook transcription. It is still unclear whether the delay of hook expression is directly due to a lack of FIbD protein or is a consequence of the absence of a properly assembled flagellum. Strains which contain flbD mutations still express RF-1 and 55 kD binding activity [29"]. It is possible that FIbD works in concert with these proteins to activate transcription of the hook promoter. The AT-rich DNA sequence in the hook, flgL andflgKpromoters is a binding site for integration host factor (IHF) [28.]. IHF is a small, basic DNA-binding protein that was originally identified as a host requirement for lambda integration (reviewed in [32]). Mutagenesis of the IHFbinding site in the hook promoter results in five to ninefold lower transcription when assayed in vivo [28-]. Likewise, I-IF is required for the in vitro transcription of the c54 nail-/promoter of Klebsiella pneumoniae [33"]. In the lambda system, I-IF facilitates integrase-mediated recombination by introducing a bend in the DNA which
permits integrase to interact simultaneously with two distantly spaced DNA sequences [32]. I-IF probably participates in a similar long-distance interaction in these cr54 promoters. It has been proposed that the bending in the DNA caused by IHF brings proteins bound at the upstream activator sequence into close proximity to RNA polymerase bound at the promoter, thereby increasing their interactions which then enhance transcriptional activation [33"]. In addition, the level of IHF is under cell cycle control (.JW Gober, unpublished data). Like RF-1 and the 55 kD binding activity, IHF is present maximally in the predivisional cell. T h e f l r [27] and IHF-binding site [28"] also occur in the flaNQ operon, another flagellar gene with a cr54 promoter [25]. In this case, however, the sites are located downstream of the transcription start [27]. Like other promoters, the IHF-binding site is located between the fir elements and the promoter (between + 24 and + 57bp) [28.]. This promoter possesses two f l r elements at + 110 and +130 [27]. Both downstream fir elements [27] (JW Gober, L Shapiro, unpublished data) and the IHF-binding site (JW Gober, L Shapiro, unpublished data) are required for maximal levels of transcription. This finding demonstrates that the fir sequences participate in transcriptional activation whether they are located upstream or downstream of the promoter. The hook and llagellin genes not only share similar regulatory sequences in their promoters but also share a common mechanism by which their products are l o calized to the swarmer pole of the predivisional cell. Several years ago, Milhausen and Agabian [34] demonstrated that the mRNA of the flgK flagellin gene segregated specifically to progeny swarmer ceils. Such segregation occurred even when the predivisional cells were treated just prior to cell division, with rifampicin. This suggests that the flgK mRNA is not transcribed in the newly formed swarmer cells, but is rather localized to the incipient swarmer pole of the predivisional cell and then segregated to the progeny swarmer cell [34]. Recently, we have used transcriptional fusions to show that in addition toflgKmRNA, the mRNAs of the hook operon [35 °'] and the flaNQ operon (JW Gober, unpublished data) are also localized to the swarmer pole of the predivisional cell. Several possible mechanisms could account for flagellar mRNA localization. The mRNA of these flagellar genes could be specifically targeted or sequestered to the swarmer pole of the predivisional cell. This would be similar to the mechanism by which bicoid mRNA is localized during Drosophila embryogenesis [36]. Alternatively, transcription of these genes could occur specifically in the swarmer pole of the predivisional cell. We reasoned that for the first mechanism to be operating, a specific mRNA sequence would be required to target the mRNA. We therefore tested whether the reporter gene product could be localized when its synthesis was driven by a transcriptional fusion that contained no naturally occuring hook mRNA [35"]. This fusion contained 112bp of hook promoter DNA and could drive the localized expression of the reporter gene product [35"]. This result rules out localization mechanisms that require mRNAspecific sequences for targeting or differential turnover,
Positional information during Cau/obacter cell differentiation Gober, Alley and Shapiro 327 but allows for the possibility that transcription is localized to the chromosome at the swarmer pole of the predivisional cell. If this is the case, then the Caulobacter predivisional cell must possess two chromosomes that are programmed for different functions. We are currently studying the mechanism bywhich differential, global programming of chromosomal DNA could be accomplished. In the specific case of localized flagellar gene transcription, we are exploring three possible mechanisms. Because all the locally transcribed promoters possess RF-1 and IHF binding sites, one of these transcription factors may be specifically targeted to the swarmer pole of the predivisional cell. Alternatively, a critical transcription factor may only be active in the swarmer cell pole. Activation of the factor could be the result of a local event or structure that is unique to the swarmer cell pole. This unique local event may be the assembling flagellum. Interestingly, the flagellar genes that are locally transcribed (hook, flaNQ and flgK), require proper flagellar assembly for transcription. Finally, it is possible that the transcription factors are able to discriminate between the two chromosomes. The differences in swarmer and stalked cell nucleoid structure may play a role in this process (reviewed in [3]). It is possible that histone-like proteins compete for binding with specific transcription factors in a manner analogous to nucleosome-mediated repression of transcription in eukaryotic chromatin.
Mechanism of protein localization The specific positioning of proteins is another mechanism through which polarity is established in the Caulobacter predivisional cell. The heat shock proteins, DnaK and Lon, as well as the heat shock sigma factor segregate to the stalk cell upon division of the predivisional cell [37], whereas polar pili and phage ~CbK receptors [38,39], the llagellins, the methyl-accepting chemotaxis protein (MCP) and the chemotaxis methyltransferase and methylesterase [21,40,41] are all targeted to the swarmer pole of the predivisional cell (Fig. 1). To define the protein sequences involved in localization, we have recently carried out extensive deletion analysis on one of these genes, that encoding a MCP. The partially deleted MCP genes were fused to either 13-1actamase or 13-galactosidase reporter genes [42] and the intracellular localization of the resulting proteins was detected by immuno-gold electron microscopy and immuno-fluorescence microscopy. Full-length fusions of the MCP to either 13-1actamase or 13-galactosidase segregated to the swarmer cell upon cell division ( M R Alley and L Shapiro, unpublished data), and immuno-electron microscopy revealed that the proteins were localized to the incipient swarmer cell pole of the predivisional cell (J Maddock and L Shapiro, unpublished data). When a region within the carboxy-terminus of the MCP gene was removed, still leaving the transmembrane portion of the protein, the fusion proteins were distributed randomly within the inner membrane of the cell, indicating that membrane localization is not the sole determinant for polar localization. The amino acid sequence that contributes to the polar target-
ing is highly conserved in bacterial MCPs. Proteins with deletions within this region of homology were no longer segregated to the swarmer cell nor were they localized to the swarmer pole of the predivisional cell. This suggests that the highly conserved sequences located in the signaling domain of the MCP, are required for both protein localization and other functions. It has been demonstrated that the E. coli MCP, Tsr, is transcribed and synthesized in Caulobacter in a cell-cycle-dependent fashion, co-incident with the native MCP [43]. As predicted from the conserved 'localization' site, the E. coliTsr is also specifically segregated to the Caulobacter swarmer cell (MRK Alley, unpublished data). The results suggest that the targeting of the MCPs to the swarmer pole is at least in part determined by a conserved stretch of amino acids. Currently, the precise mechanism by which localization is achieved is unknown. The localization of the Caulobacter MCP to a discrete portion of the cell membrane raises several questions regarding the mechanism of protein targeting in bacteria. Is the MCP protein localized to a distinct polar cellular structure or compartment, akin to the periseptal annulus described in E. coli [44]? Does the E. coli MCP localize to the pole in the E. coli cell? More generally it is also uncertain whether the localization of MCP is unique or whether other proteins may be subject to subcellular localization. For example, cell division proteins, especially those involved in determining the precise location of the division site, may also be localized to a discrete part of the cell by a similar mechanism.
Conclusions The Caulobacter cell cycle, with its unique developmental program provides a tractable system to explore the problem of site-specific localization. Recent results suggest that both localized transcription and protein targeting direct localization. Similar mechanisms may be involved in other systems, where proteins are localized to a discrete position in a cell.
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