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Bacterial cells exhibit a wide variety of shapes. Recent results indicate that the characteristic crescent shape of Caulobacter crescentus depends upon an inter-mediate filament-like protein that localizes to the concave side of the cell.
The Gram-negative Caulobacter crescentus exports RsaA, the crystalline S-layer subunit protein using a dedicated type I secretion system. The protein and two transporter genes (rsaADE) are located together, comparable to the Escherichia coli type I hemolysin hlyCABD operon, where read through of a stem loop following hlyCA results in reduced transcription of the hlyBD. Using two genetic approaches and a direct assessment of transcription from regions 5' to the genes we learned that rsaD and rsaE were transcribed together as a separate transcript from rsaA. These results are contrary to previous assumptions about the rsaADE type I secretion gene control and add another theme to the area of type I secretion transcription regulation. It may be that to accommodate the high levels of RsaA secretion, the type I transporters must be transcribed independently from rsaA.
A transposon library of 5700 mutants was constructed in Caulobacter crescentus strain NA1000. Thirteen mutant strains were isolated that presented growth deficiency in the presence of 20 microM cadmium chloride, and the disrupted genes were identified by DNA sequencing. Among the predicted products were found two putative transcriptional regulators, one histidine kinase/response regulator hybrid protein, two 2Fe-2S family proteins, and also members of a putative metal efflux system. Certain of these mutants also showed an increased sensitivity to zinc and copper, and the genes encoding the efflux system were induced by cadmium and zinc, indicating that this system may be important for homeostasis of both metal ions.
Development in Caulobacter reflects a level of complexity once thought only to exist in eukaryotic cells. The cell cycle and development are not isolated from each other, but are interdependent processes. Checkpoints are in place to ensure that both cell cycle and developmental processes are completed accurately before the next stage is initiated. The timing of these processes is regulated by signal transduction networks that integrate signals from DNA replication, cell division and development. These signal transduction networks achieve precise timing of the cell cycle and development by regulating temporal gene expression, and protein activity by dynamic spatial localization within the cell and timed proteolysis.
The bacterium Caulobacter crescentus divides asymmetrically, producing daughter cells with differing polar structures, different cell fates and asymmetric regulation of the initiation of chromosome replication. Complex intracellular signaling is required to keep the organelle developmental processes at the cell poles synchronized with other cell cycle events. Two recently characterized switch mechanisms controlling cell cycle progress are triggered by relatively large-scale developmental events in the cell: the progress of the DNA replication fork and the physical compartmentalization of the cell that occurs well before division. These mechanisms invoke rapid, precisely timed and even spatially differentiated regulatory responses at important points in the cell cycle.
The extractable lipids of the gram-negative, stalked, freshwater bacterium Caulobacter bacteroides NP-105 account for about 9.5% by weight of dry cells, polar lipids comprising up to 95% of the total. The polar lipids consist of five glycolipids, namely, 1,2-diacyl-3-alpha-D-glucopyranosyl-sn-glycerol (I) (34% of the total), 1,2-diacyl-3-alpha-D-[6'-(1",2"-diacyl-sn-glycero-3-phospho)]glucopyranosyl-sn-glycerol (II) (7%), 1,2-diacyl-3-a-D-glucuronopyranosyl-sn-glycerol (III) (17%), 1,2-diacyl-3-alpha-D-(6'-sulfo)quinovopyranosyl-sn-glycerol (V) (9%), and 1,2-diacyl-3-alpha-D-[4'-(alpha-D-glucopyranosyl)] glucuronopyranosyl-sn-glycerol (VI) (28%), and one glycerophospholipid, 1,2-diacyl-sn-glycero-3-phosphoglycerol (IV) (5%). The main fatty acyls of the lipids are n-16:0, cis-18:1 omega7, and cis-11,12-methyleneoctadecanoic (lactobacillic) acid residue. Of the 6 lipids listed, only 3 (I, IV, and V) can be treated as widely encountered. However, sulfonic glycolipid V is a characteristic lipid component of photosynthetic organisms rather than non-photosynthetic ones. Phosphatidyl derivatives of glycosyldiacylglycerols of type II normally occur in gram-positive bacteria, among gram-negative bacteria they have been revealed only in two species of the Pseudomonas genus. Glucuronosyl and alpha-glucosyl-a-glucuronosyl diacylglycerols such as III and VI, respectively, are very rare lipids, the latter being found so far only in a Streptomyces strain.
The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange [corrected] rates is that CgtA is a bimodal protein with a C-terminal GTP binding domain and an N-terminal GEF domain. In this study we demonstrate that although the N-terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.
The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange rates is that CgtA is a bimodal protein with a C-terminal GTP binding domain and an N-terminal guanine nucleotide exchange factor (GEF) domain. In this study we demonstrate that although the N-terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.
Caulobacter crescentus 101123 possesses a gene (Mbl1b) encoding a metallo-beta-lactamase with 32% amino acid identity to the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia. The gene was cloned into an expression vector and the enzyme, Mbl1b, was expressed in Escherichia coli. Mbl1b was purified. Catalytic properties for several antibiotics were determined. The enzyme exhibits Michaelis-Menten kinetics for imipenem, meropenem and nitrocefin but substrate inhibition kinetics with cefoxitin, cefaloridine, penicillin G and ampicillin. A homology model predicts Mbl1b has the same structural fold as other metallo-beta-lactamases with a detailed structure very similar to L1 but whereas L1 is a homotetramer, Mbl1b is monomeric. The main differences between Mbl1 and L1 are in the N-terminal region.
The two-component signaling protein CtrA activates or represses the expression of one-quarter of the cell-cycle-regulated genes in Caulobacter crescentus, integrating DNA replication, morphogenesis, and cell division. The activity of this essential protein is controlled by a positive transcriptional feedback loop, cell-cycle-regulated phosphorylation, and rapid proteolysis as cells enter S-phase at the swarmer-to-stalked cell transition and in the stalked portion of the asymmetric predivisional cell. CtrA activity must be removed from cells at the onset of DNA replication, because phosphorylated CtrA binds to and silences the origin of replication. The ClpXP protease is required for CtrA proteolysis but is present throughout the cell-cycle, so the mechanism for activating and deactivating CtrA proteolysis is unknown. Here, we identify a bipartite proteolytic signal in the CtrA response regulator consisting of two determinants that are each necessary but not sufficient for regulated degradation. One determinant is present in the last 15 amino acid residues of CtrA, particularly the terminal Ala-Ala residues, and another is located within the first 56 residues of the CtrA receiver domain. A fusion of the receiver domain and last 15 residues of CtrA to YFP is properly degraded in living cells. Although the N-terminal 56 residues contain the conserved Asp51 phosphorylation site, mutant analyses show that cell-cycle-controlled CtrA proteolysis is insensitive to the CtrA phosphorylation state. The N-terminal proteolytic determinant is predicted to reside on the surface of the receiver domain in beta-sheet 2 and alpha-helix 2.
In most bacteria, chromosome dimers arise from homologous recombination between replicated chromosomes. These dimers are then resolved by the action of the XerC and XerD recombinases, which act on the chromosomal dif site in the presence of the FtsK cell division protein. We have cloned the xerC and xerD genes from Caulobacter crescentus, and overexpressed them as maltose-binding protein fusion proteins. These fusion proteins were purified and used in in vitro DNA-binding assays to the Escherichia coli dif site with each protein individually, and in combination with each other. In addition, combinations of Xer proteins from E. coli were also tested for cooperativity with the corresponding C. crescentus proteins.
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.
The Caulobacter cell cycle exhibits time-dependent expression of differentiation events. These include the morphological transition of a swarmer cell to a replication-competent stalked cell and the subsequent polarized distribution of specific gene products that results in an asymmetric predivisional cell. Cell division then yields a new swarmer cell and a stem-cell-like stalked cell. Two-component signal transduction proteins involved in cell cycle control and proteins required for cell division and flagellar biogenesis have been shown to be regulated temporally and spatially during the cell cycle. The mechanisms underlying this regulation include protein phosphorylation and proteolysis.
The transcriptional regulator CtrA controls DNA replication, DNA methylation and cell division during the Caulobacter cell cycle. Recent work on this master cell cycle regulator has provided insight into its control mechanisms. Feedback regulation of ctrA transcription together with timed proteolysis of CtrA determine the levels of the regulator during the cell cycle. Multiple phosphorylation pathways regulate CtrA activity by a mechanism that includes dynamic subcellular localization of the corresponding sensor kinases.
During the Caulobacter life cycle, the timing of DNA replication, cell division and development is precisely coordinated. Recent work has begun to unravel the complex regulatory networks that couple these processes. A key aspect of these regulatory networks is the dynamic localization of multiple histidine protein kinases that control a master response regulator, thus driving downstream pathways.
In Caulobacter crescentus, morphogenic events, such as cytokinesis, the establishment of asymmetry and the biogenesis of polar structures, are precisely regulated during the cell cycle by internal cues, such as cell division and the initiation of DNA replication. Recent studies have revealed that the converse is also true. That is, differentiation events impose regulatory controls on other differentiation events, as well as on progression of the cell cycle. Thus, there are pathways that sense the assembly of structures or the localization of complexes and then transduce this information to subsequent biogenesis or cell cycle events. In this review, we examine the interplay between flagellar assembly and the C. crescentus cell cycle.
In Caulobacter crescentus, a complex regulatory network integrates temporal and spatial information to control the ordered progression of the cell cycle, and to synchronize cell proliferation with development. Periodicity includes the timed synthesis, activation or destruction of key regulatory proteins, which activate a large number of genes at the appropriate time of the cell cycle. Checkpoints serve to couple cell division and polar development to the replication and segregation state of the chromosome. Asymmetrically positioned regulatory components are involved in the sequential positioning of polar organelles. New work sheds light on the spatial organization of cellular components involved in cell cycle progression and polar differentiation, and starts to define the molecular nature of checkpoints involved in cell cycle control and development.
New research on bacterial cells has demonstrated that they have a dynamic and complex subcellular organization. Work in Caulobacter crescentus shows that essential and nonessential proteins localize to discrete positions in the cell as a function of cell-cycle progression. The flagellum and chemotaxis receptor are asymmetrically localized to a single pole in the predivisional cell by coordinated proteolysis and transcriptional regulation. Cell type- and compartment-specific localization of the CtrA global transcriptional regulator is essential for proper cell-cycle progression, and subcellular localization of key chromosome partitioning proteins is correlated with proper nucleoid segregation. Given this structural complexity, we are driven to ask how localization is achieved, and to what end.