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PubMed2006

Mutations in DivL and CckA rescue a divJ null mutant of Caulobacter crescentus by reducing the activity of CtrA.

Polar development and cell division in Caulobacter crescentus are controlled and coordinated by multiple signal transduction proteins. divJ encodes a histidine kinase. A null mutation in divJ results in a reduced growth rate, cell filamentation, and mislocalized stalks. Suppressor analysis of divJ identified mutations in genes encoding the tyrosine kinase (divL) and the histidine kinase (cckA). The divL and cckA suppressor alleles all have single amino acid substitutions, some of which confer a temperature-sensitive phenotype, particularly in a wild-type background. Analysis of transcription levels from several positively regulated CtrA-dependent promoters reveals high expression in the divJ mutant, suggesting that DivJ normally serves to reduce CtrA activity. The divL and cckA suppressors reduce the amount of transcription from promoters positively regulated by CtrA, indicating that the mutations in divL and cckA are suppressing the defects of the divJ mutant by reducing the abnormally high level of CtrA activity. Immunoblotting showed no major perturbations in the CtrA protein level in any of these strains, suggesting that the high amount of CtrA activity seen in the divJ mutant and the reduced amount of activity in the suppressors are regulated at the level of activation and not transcription, translation, or degradation. In vivo phosphorylation assays confirmed that divJ mutants have elevated levels of CtrA phosphorylation and that this level is reduced in the suppressors with mutations in divL.

Bacterial Proteins↗
PubMed1992

Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein.

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98,132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest.

Amino Acid Sequence↗
PubMed1993

An "all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus.

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (delta CenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of delta CenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of delta cenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of delta CenA as markers, a simple assay for periplasmic delta CenA hybrids was developed. This assay indicated that delta CenA activity was largely independent of cellular location. To facilitate the use of delta CenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating delta cenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:delta cenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that delta CenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because delta CenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, delta CenA possessed many of the attributes of an "all-purpose" reporter.

Alkaline Phosphatase↗
PubMed1994

Alkaline phosphatase and a cellulase reporter protein are not exported from the cytoplasm when fused to large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein.

Using a gene fusion approach, hybrid proteins were created by linking alkaline phosphatase (PhoA) or a cellulase reporter (delta CenA) to four large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein (RsaA; 1026 amino acids). Three of the sites (amino acids 189, 220, 315) were selected on the basis of TnphoA experiments that suggested the first 250-350 amino acids of RsaA could mediate export of PhoA from the cytoplasm while the fourth lay only 21 amino acids from the C-terminus. Expression of all fusions except rsaA(315):delta cenA and rsaA(315):phoA was toxic to C. crescentus. None of the gene fusions were toxic when expressed by Escherichia coli DH5 alpha, where all the hybrid proteins accumulated as inclusion bodies. The toxicity of hybrid proteins encoding 189, 220, and 1005 RsaA-derived amino acids was related to the nature of the hybrid protein itself because truncated RsaA peptides lacking their reporter domains were nontoxic. Further study of RsaA(delta C21) showed that this and presumably other truncated RsaA derivatives were neither secreted nor prone to intracellular accumulation. Although C. crescentus tolerated the expression of rsaA(315):delta cenA and rsaA(315):phoA, the encoded hybrid proteins were not exported in significant quantities from the cytoplasm. These results extend and confirm earlier work that large portions of the S-layer protein N-terminus cannot mediate export of passenger proteins from the cytoplasm and that the entire native S-layer protein may be required to properly interact with the RsaA secretion machinery.

Alkaline Phosphatase↗
PubMed1996

The extreme N-terminus of the Caulobacter crescentus surface-layer protein directs export of passenger proteins from the cytoplasm but is not required for secretion of the native protein.

The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane. Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface. To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5'-region of the rsaA gene. This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein. Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (delta CenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid protein export was estimated at 9%. No enzyme activity was detected in the cell-free culture fluids as the result of expressing any gene fusion, indicating that no hybrid protein was completely secreted from the cell. RsaA:PhoA hybrid proteins were also exported from the E. coli cytoplasm, a bacterium not expected to contain the necessary machinery for the secretion of RsaA. Taken together, these data indicate that the secretion pathway of RsaA relies on a C-terminal secretion signal and that once separated from the context of the native protein, the extreme N-terminus of RsaA can act as an inefficient cryptic export signal that is not used during native RsaA secretion.

Alkaline Phosphatase↗
PubMed2002

Control of chromosome replication in caulobacter crescentus.

Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Its chromosome replication origin (Cori) may be prototypical of the large and diverse class of alpha-proteobacteria. Cori has features that both affiliate and distinguish it from the Escherichia coli chromosome replication origin. For example, requirements for DnaA protein and RNA transcription affiliate both origins. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria.

Bacterial Proteins↗
PubMed2003

Spatial and temporal control of differentiation and cell cycle progression in Caulobacter crescentus.

The dimorphic and intrinsically asymmetric bacterium Caulobacter crescentus has become an important model organism to study the bacterial cell cycle, cell polarity, and polar differentiation. A multifaceted regulatory network orchestrates the precise coordination between the development of polar organelles and the cell cycle. One master response regulator, CtrA, directly controls the initiation of chromosome replication as well as several aspects of polar morphogenesis and cell division. CtrA activity is temporally and spatially regulated by multiple partially redundant control mechanisms, such as transcription, phosphorylation, and targeted proteolysis. A multicomponent signal transduction network upstream CtrA, containing histidine kinases CckA, PleC, DivJ, and DivL and the essential response regulator DivK, contributes to the control of CtrA activity in response to cell cycle and developmental cues. An intriguing feature of this signaling network is the dynamic cell cycle-dependent polar localization of its components, which is believed to have a novel regulatory function.

Bacterial Proteins↗
PubMed2006

Intermediate filament-like cytoskeleton of Caulobacter crescentus.

Eukaryotic cytoskeleton consists of three main types of filaments: actin microfilaments, microtubules and intermediate filaments (IFs). Actin and tubulin-like proteins are also found in bacteria where they perform diverse cytoskeletal functions. IFs, however, are considered to be a characteristic constituent of metazoan cells only, where they (among other functions) are involved in determination and maintenance of cell shape and cellular integrity. Surprisingly, a coiled coil-rich protein called crescentin was recently shown to play a key role in determining the complex curved and helical cell shapes of the bacterium Caulobacter crescentus, and to exhibit several characteristic properties of animal IF proteins. First, the arrangement of the coiled coil domains of crescentin closely resembles the tripartite molecular architecture of IF proteins. Second, crescentin also possesses the defining biochemical property of IF proteins to assemble into 10-nm-wide filaments in vitro without cofactors. Furthermore, crescentin forms a higher-order helical structure in vivo, which is localized asymmetrically along the concave side of the cell. In close association with the cell membrane, the crescentin structure promotes the helical growth of the cell and thereby determines a curved or a helical shape, depending on the length of the cell. The unexpected finding of an IF-like element in a bacterium raises several interesting questions concerning, for example, the molecular mechanisms whereby complex and asymmetric cell shapes are generated by different bacteria, or the functional and evolutionary relatedness of crescentin to animal IF proteins.

Bacterial Proteins↗
PubMed2025

Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction.

Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response that is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA box properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA box properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as one of the factors modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.

Promoter Regions, Genetic↗
PubMed2005

Fluctuation analysis of Caulobacter crescentus adhesion.

The aquatic bacterium Caulobacter crescentus divides asymmetrically to a flagellated swarmer cell and a cell with a stalk. At the end of the stalk is an adhesive organelle known as the holdfast, which the stalked cell uses to attach to a solid surface. Often there are two or more cells with their stalks attached to the same holdfast. By analyzing the fluctuations in the stalk angle for a pair of cells attached to a single holdfast, we determine the elastic stiffness of the holdfast. We model the holdfast as three torsional springs in series and find that the effective torsional spring constant for the holdfast is of the order of (10(-17)-10(-18)) Nm, with unequal spring constants. The asymmetry suggests the sequence in which the cells attach to each other, and in some cases suggests that strong crosslinks form between the stalks as they make a shared holdfast.

Bacterial Adhesion↗
PubMed1998

Use of diaminobenzidine to stain for cytochrome c oxidase activity in Caulobacter crescentus and Escherichia coli.

Caulobacter crescentus is an aerobic Gram negative bacterium found in bacterial biofilms. C. crescentus produces a sessile stalked cell during its life cycle that allows it to attach to surfaces. Due to the oxygen gradient found in the bacterial biofilm, we postulated that C. crescentus would localize the terminal cytochrome to the cell body that remains in the aerobic portion of the biofilm. The terminal electron acceptor in the electron transport system was determined using Kovac's oxidase test to be cytochrome c oxidase. We are able to use diaminobenzidine to locate the oxidase histochemically using transmission electron microscopy. Upon examination of sectioned bacteria, we determined that cytochrome c oxidase of C. crescentus is found only in the cell body.

Aminobenzoates↗
PubMed2001

Recombinant vaccines against infectious hematopoietic necrosis virus: production by the Caulobacter crescentus S-layer protein secretion system and evaluation in laboratory trials.

We report the development of an IHNV vaccine produced by a new protein production system based on the bacterium Caulobacter crescentus. The subunit vaccines that were tested contain a 184 amino acid segment of the IHNV glycoprotein in different fusion arrangements with the C. crescentus S-layer protein. Relative percent survival of 26 to 34% was demonstrated in rainbow trout fry for a vaccine that contained the 184 amino acid segment of the IHNV glycoprotein fused to the C-terminal one-quarter of the S-layer protein. Inclusion of the universal mammalian T-cell epitopes developed from the measles fusion protein or the tetanus toxin protein did not increase the effectiveness of the IHNV-G/S-layer recombinant protein.

Adjuvants, Immunologic↗
PubMed2004

A genetic oscillator and the regulation of cell cycle progression in Caulobacter crescentus.

Analyses of cell polarity, division, and differentiation in prokaryotes have identified several regulatory proteins that exhibit dramatic changes in expression and spatial localization over the course of a cell cycle. The dynamic behavior of these proteins is often intrinsically linked to their function as polarity determinants.(1-3) In the alpha-proteobacterium, Caulobacter crescentus, the CtrA global transcriptional regulator exhibits a spatially and temporally dynamic expression pattern across the cell cycle. CtrA plays key roles in asymmetric cell division and in the timing of chromosome replication.(3,4) An additional global regulator, GcrA, has recently been discovered that both regulates and is regulated by CtrA.(5) Together, these regulatory proteins create a genetic circuit in which the cellular concentrations of CtrA and GcrA oscillate spatially and temporally to control daughter cell differentiation and cell cycle progression.

Bacterial Proteins↗
PubMed1991

Proteins of Caulobacter crescentus strongly binding to DNA. Spectroscopic analysis of major histone-like proteins.

The protein composition of deoxyribonucleoprotein (DNP) from Caulobacter crescentus, protected from exogenous nucleases, was analysed. This fraction was obtained by purification of cell lysate on Sephacryl S-400. It contained the following proteins: 13.4 kDa (HCc), 15.2 kDa, 17.5 kDa, 28 kDa and 40 kDa. The strength of protein 15.2 kDa binding to ds-DNA was the same as that of the eukaryotic histones H2A and H2B. Proteins 13.4 kDa (HCc) and 17.5 kDa purified to homogeneity have a UV spectrum identical to protein HU of Escherichia coli which lacks tryptophane and tyrosine. This confirms the classification of protein HCc to the class of HU-like proteins.

Bacterial Proteins↗
PubMed1993

Cis- and trans-acting elements required for regulation of flagellar gene transcription in the bacterium Caulobacter crescentus.

The flagellar genes of the bacterium Caulobacter crescentus are organized into a regulatory hierarchy that consists of four classes or levels of genes, and expression of these genes is restricted to a discrete interval in the cell cycle that begins just prior to flagellum assembly. This paper summarizes data on the promoters and other cis-acting elements that are required for transcription of the level II gene fliF and the level III genes flaN and flbG. Regulated expression of flaN and flbG requires sigma 54 promoters, enhancer-like sequences called ftr, and sequences called ihf that conform to the consensus binding sequence for Escherichia coli integration host factor protein. The fliF regulatory region contains a new type of promoter sequence that is different from other known promoter motifs, and it has a sequence called ftr4 that is a site of negative regulation. ftr4 appears to function as part of a developmental switch that turns fliF transcription off at the correct time in the cell cycle. flbD, the last gene in the fliF operon is a negative regulator of fliF and an activator of both flaN and flbG expression. Evidence that FlbD protein plays a direct role as a transcriptional regulator comes from the finding that it has a DNA binding activity within its carboxy terminus that specifically recognizes ftr4 in fliF and four ftr elements in the flaN-flbG promoter region.

Bacterial Proteins↗
PubMed1996

Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell.

Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle. Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch. The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell.

Amino Acid Sequence↗
PubMed1987

Order of gene replication in Caulobacter crescentus; use of in vivo labeled genomic DNA as a probe.

Two methods for determining the time of gene replication in Caulobacter crescentus using a temperature sensitive DNA synthesis mutant to synchronize chromosome replication are described. Swarmer cells, blocked before DNA initiation at 37 degrees C, initiate chromosome replication within 2 min after releasing the temperature block in 32P-orthophosphate medium, as indicated by the appearance of a small number of unique genomic DNA fragments. The time at which a given chromosome segment replicates was determined by isolating genomic DNA from cells labeled for progressively longer times during the S period of the cell cycle and hybridizing the probes to cloned C. crescentus genes. The time of replication of genetically mapped Tn5 insertions was determined by preparing DNA from the Tn5 insertion mutants that had been labeled with 32P in similar experiments and hybridizing it to lambda::Tn5 DNA. These results furnish the first correlation between the order of chromosome replication and the genetic map of C. crescentus. They also show that the times of replication and expression of the hook protein and the flagellin genes, which require DNA synthesis for their transcription, both occur near mid-S phase.

Bacterial Proteins↗
PubMed1984

Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus.

The ability to reactivate ultraviolet (UV) damaged phage phi CbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage phi CbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2-2 h at 30 degrees C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5-10 J/m2 of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec-requiring DNA repair system analogous to W-reactivation in Escherichia coli. In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.

Bacteria↗