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PubMed1997

An alkB gene homolog is differentially transcribed during the Caulobacter crescentus cell cycle.

A Caulobacter crescentus alkB gene homolog was identified in a clone previously shown to contain the heat shock genes dnaK and dnaJ; the homolog is located upstream of dnaK and is transcribed in the opposite orientation. An analysis of the alkB gene has shown that the deduced amino acid sequence is that of a 21-kDa protein, which is 42% identical and 78% similar to Escherichia coli AlkB. Furthermore, an alkB-null mutant was constructed by gene disruption and was shown to be highly sensitive to the alkylating agent methyl methanesulfonate (MMS). However, the alkB gene of C. crescentus, unlike its E. coli counterpart, is not located downstream of the ada gene, and its transcription is not induced by alkylating agents. In addition, no acquired enhanced resistance to MMS toxicity by treatment with low MMS doses was observed, suggesting that no adaptive response occurs in C. crescentus. Nevertheless, transcription of the alkB gene is cell cycle controlled, with a pattern of expression similar to that of several Caulobacter genes involved in DNA replication.

Adaptation, Physiological↗
PubMed1997

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.

Amino Acid Sequence↗
PubMed1998

The CIRCE element and its putative repressor control cell cycle expression of the Caulobacter crescentus groESL operon.

The groESL operon is under complex regulation in Caulobacter crescentus. In addition to strong induction after exposure to heat shock, under physiological growth conditions, its expression is subject to cell cycle control. Transcription and translation of the groE genes occur primarily in predivisional cells, with very low levels of expression in stalked cells. The regulatory region of groESL contains both a sigma32-like promoter and a CIRCE element. Overexpression of C. crescentus sigma32 gives rise to higher levels of GroEL and increased levels of the groESL transcript coming from the sigma32-like promoter. Site-directed mutagenesis in CIRCE has indicated a negative role for this cis-acting element in the expression of groESL only at normal growth temperatures, with a minor effect on heat shock induction. Furthermore, groESL-lacZ transcription fusions carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an hrcA null strain, carrying a disruption in the gene encoding the putative repressor that binds to the CIRCE element, shows constitutive synthesis of GroEL throughout the Caulobacter cell cycle. These results indicate a negative role for the hrcA gene product and the CIRCE element in the temporal control of the groESL operon.

Bacterial Proteins↗
PubMed1999

Cell cycle expression and transcriptional regulation of DNA topoisomerase IV genes in caulobacter.

DNA replication and differentiation are closely coupled during the Caulobacter crescentus cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the parE and parC genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897-910, 1997). We have examined the cell cycle regulation of parE and parC and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of parE and parC expression is not strictly coordinated, however. The rate of parE transcription increases ca. 20-fold during the G1-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the parC promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through parE expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major Caulobacter sigma factor sigma73. We also present evidence that a conserved 8-mer sequence motif located in the spacers between the -10 and -35 elements of the parE and parC promoters is required for maximum levels of parE transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of parE transcription and the parE and parC promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication.

Amino Acid Sequence↗
PubMed2000

Regulation of stalk elongation by phosphate in Caulobacter crescentus.

In Caulobacter crescentus, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes pstSCAB of Escherichia coli. In E. coli, pst mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the Caulobacter phoB gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While phoB was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both pstS and phoB mutants were deficient in phosphate transport. When a phoB mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of phoB and pst mutants were opposite. phoB mutants were unable to elongate stalks during phosphate starvation, whereas pst mutants made long stalks in both high- and low-phosphate media. Analysis of double pst phoB mutants indicated that the long-stalk phenotype of pst mutants was dependent on phoB. In addition, analysis of a pstS-lacZ transcriptional fusion showed that pstS transcription is dependent on phoB. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB.

Amino Acid Sequence↗
PubMed2001

Conserved promoter motif is required for cell cycle timing of dnaX transcription in Caulobacter.

Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including dnaX, dnaA, dnaN, gyrB, and dnaK, are induced at the G(1)/S-phase transition. By analyzing mutations in the dnaX promoter, we identified a motif between the -10 and -35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

Bacterial Proteins↗
PubMed2002

Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus.

The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle.

Bacterial Proteins↗
PubMed2004

Identification and transcriptional control of Caulobacter crescentus genes encoding proteins containing a cold shock domain.

The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.

Adaptation, Physiological↗
PubMed2004

A temperature-sensitive mutation in the dnaE gene of Caulobacter crescentus that prevents initiation of DNA replication but not ongoing elongation of DNA.

A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant. Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the alpha-catalytic subunit of DNA polymerase III holoenzyme. Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein. This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins. Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation.

Caulobacter crescentus↗
PubMed2006

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Caulobacter crescentus↗
PubMed1995

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.

Base Sequence↗
PubMed2005

Role of core promoter sequences in the mechanism of swarmer cell-specific silencing of gyrB transcription in Caulobacter crescentus.

BACKGROUND: Each Caulobacter crescentus cell division yields two distinct cell types: a flagellated swarmer cell and a non-motile stalked cell. The swarmer cell is further distinguished from the stalked cell by an inability to reinitiate DNA replication, by the physical properties of its nucleoid, and its discrete program of gene expression. Specifically, with regard to the latter feature, many of the genes involved in DNA replication are not transcribed in swarmer cells. RESULTS: We show that for one of these genes involved in DNA replication, gyrB, its pattern of temporal expression depends upon an 80 base pair promoter region with strong resemblance to the Caulobacter crescentus sigma73 consensus promoter sequence; regulation does not appear to be affected by the general strength of the promoter activity, as mutations that increased its conformity with the consensus did not affect its cell-cycle expression pattern. Transcription from the gyrB promoter in vitro required only the presence of the sigma73 RNA polymerase (from E. coli) and the requisite nucleoside triphosphates, although a distinct binding activity, present in crude whole-cell extracts, formed a complex gyrB promoter DNA. We also assayed the effect on gyrB expression in strains containing mutations in either smc or dps, two genes encoding proteins that condense DNA. However we found there was no change in the temporal pattern of gyrB transcription in strains containing deletions in either of these genes. CONCLUSION: These experiments demonstrate that gyrB transcription does not require any auxiliary factors, suggesting that temporal regulation is not dependent upon an activator protein. Swarmer-specific silencing may not be attributable to the observed physical difference in the swarmer cell nucleoid, since mutations in either smc or dps, two genes encoding proteins that condense DNA, did not alter the temporal pattern of gyrB transcription in strains containing deletions in either of these genes. Rather a repressor that specifically recognizes sequences in the gyrB promoter region that are also probably essential for transcription, is likely to be responsible for controlling cell cycle expression.

Base Sequence↗
PubMed2005

Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus: fusion with the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi yields a robust, catalytically active product.

Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-beta-D-cellobioside with a KM similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the kcat value was significantly reduced (0.08 s-1 for Cex/RsaA vs 15.8 s-1 for Cex), likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.

Bacterial Proteins↗
PubMed2006

Low flagellar motor torque and high swimming efficiency of Caulobacter crescentus swarmer cells.

We determined the torque of the flagellar motor of Caulobacter crescentus for different motor rotation rates by measuring the rotation rate and swimming speed of the cell body and found it to be remarkably different from that of other bacteria, such as Escherichia coli and Vibrio alginolyticus. The average stall torque of the Caulobacter flagellar motor was approximately 350 pN nm, much smaller than the values of the other bacteria measured. Furthermore, the torque of the motor remained constant in the range of rotation rates up to those of freely swimming cells. In contrast, the torque of a freely swimming cell for V. alginolyticus is typically approximately 20% of the stall torque. We derive from these results that the C. crescentus swarmer cells swim more efficiently than both E. coli and V. alginolyticus. Our findings suggest that C. crescentus is optimally adapted to low nutrient aquatic environments.

Biomechanical Phenomena↗
PubMed2006

Anti-tumor effects of the bacterium Caulobacter crescentus in murine tumor models.

Caulobacter crescentus is a gram negative, non-pathogenic bacterium, common in aquatic and soil environments. One feature of note is a protein surface layer (S-layer) composed of a single protein, organized as a self-assembled crystalline array that coats the bacterium. In the course of efforts to express cancer-associated peptides as genetic insertions into the S-layer, we noted a tumor suppressive effect of the unmodified bacterium. C. crescentus was examined for anti-tumor activity against three transplantable tumor mouse models: Lewis lung carcinoma cells transfected with the MUC1 gene in C57BL/6, murine mammary carcinoma (EMT-6) in BALB/c (both in prophylactic and therapeutic mode) and murine leukemia cells (L1210) in DBA2. Mice were immunized three times i.p. with C. crescentus (2 x 10(7) cells/mouse). In prophylactic mode, the mice were challenged with tumor cells two weeks after the last immunization. Immunization with live C. crescentus resulted in anti-tumor activity in all three transplantable tumor models, as measured by prolonged survival, reduced tumor mass or reduced number of lung nodules, compared to saline control groups. In the Lewis lung and the EMT-6 mammary carcinoma murine models the number of lung nodules as well as the tumor weight was lower in mice treated with C. crescentus, compared to the control group; for EMT-6, this was observed in prophylactic and therapeutic modes. In the murine leukemia and Lewis lung carcinoma models prolonged survival was observed in the groups of mice immunized with Caulobacters. In most cases the live C. crescentus cells were markedly more efficacious than heat killed or formalin fixed cells, despite the fact that they do not grow or persist in mice. The results suggest that C. crescentus may be a safe, bacterial immunomodulator for the treatment of tumors.

Animals↗
PubMed1989

An Escherichia coli chemoreceptor gene is temporally controlled in Caulobacter.

Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. We demonstrate here that the expression of the Escherichia coli chemoreceptor gene tsr, with 2.6 kilobases of its upstream sequence, is temporally controlled in Caulobacter crescentus. The tsr gene was placed on the chromosome in single copy or on a low-copy-number plasmid. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Nuclease S1 mapping experiments showed that the tsr transcript was also controlled by the cell cycle, suggesting that the E. coli tsr gene is regulated by C. crescentus factors that mediate the timing of transcription initiation. The apparent transcription start site of the E. coli tsr gene was determined in both E. coli and C. crescentus, and we found that in both backgrounds the promoter used conforms to the consensus sequence for the promoters of the flagellar and chemosensory genes of Bacillus subtilis and E. coli. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter.

Bacteria↗
PubMed1979

Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.

Bacteria↗
PubMed1976

Amino acid composition of peptidoglycan in Caulobacter crescentus.

Peptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2 : 1 : 1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shedlarski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.

Alanine↗