The topoisomerase IV ParC subunit colocalizes with the Caulobacter replisome and is required for polar localization of replication origins.
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The process of bacterial DNA replication generates chromosomal topological constraints that are further confounded by simultaneous transcription. Topoisomerases play a key role in ensuring orderly replication and partition of DNA in the face of a continuously changing DNA tertiary structure. In addition to topological constraints, the cellular position of the replication origin is strictly controlled during the cell cycle. In Caulobacter crescentus, the origin of DNA replication is located at the cell pole. Upon initiation of DNA replication, one copy of the duplicated origin sequence rapidly appears at the opposite cell pole. To determine whether the maintenance of DNA topology contributes to the dynamic positioning of a specific DNA region within the cell, we examined origin localization in cells that express temperature-sensitive forms of either the ParC or ParE subunit of topoisomerase (Topo) IV. We found that in the absence of active Topo IV, replication initiation can occur but a significant percent of replication origins are either no longer moved to or maintained at the cell poles. During the replication process, the ParC subunit colocalizes with the replisome, whereas the ParE subunit is dispersed throughout the cell. However, an active ParE subunit is required for ParC localization to the replisome as it moves from the cell pole to the division plane during chromosome replication. We propose that the maintenance of DNA topology throughout the cell cycle contributes to the dynamic positioning of the origin sequence within the cell. (+info)
Cytokinesis monitoring during development; rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter.
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For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development. (+info)
Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters.
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Several methods for isolation of the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A were evaluated. Treatment of cells with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 2 was the most effective means of selectively removing RsaA from cells, and after neutralization, the protein was capable of reassembling into a paracrystalline structure. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid treatment could also be used to extract RsaA and yielded protein capable of reassembly. The success of the methods was likely related to disruption of calcium-mediated bonding; calcium was required for recrystallization, while magnesium and strontium ions were ineffective. Antibody was raised against purified RsaA and, along with the S-layer extraction techniques, was used to evaluate 42 strains of caulobacters isolated from a variety of aquatic and wastewater treatment locations. A single characteristic protein could be isolated from the 35 strains that produced an S layer; with one exception, no proteins were extracted from strains that had no S layer. The presumed S-layer proteins ranged in size from 100 to 193 kDa. All of these proteins specifically reacted with anti-RsaA serum by Western immunoblot analysis. In strain CB15A, a specific S-layer-associated oligosaccharide has been proposed to be involved in a calcium-mediated attachment of the S layer to the cell surface. This molecule was detected by Western immunoblotting with a specific antiserum and on polyacrylamide gels stained for polysaccharides. A comparable band was found in all S-layer-producing strains and for most, S-layer-associated oligosaccharide-specific antibody reacted with them in Western analysis. Overall, in freshwater caulobacters at least portions of their S-layer structures appear to be strongly conserved entities, as well as the means of attachment to the cell surface. (+info)
The phylogeny of marine and freshwater caulobacters reflects their habitat.
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Caulobacter is a distinctive genus of prosthecate bacteria. Because caulobacters adhere to surfaces and are found in diverse locales, their role in oligotrophic environments and bacterial biofilm communities is of interest. The phylogenetic relationships of a group of marine and freshwater caulobacters were examined in part to address whether the taxonomic grouping of these bacteria (based primarily on morphological characters) was consistent with 16S rRNA sequence divergence. The caulobacters examined (9 marine and 11 freshwater species or strains) were affiliated with the alpha proteobacteria. They made up a diverse yet, with the possible exception of a strain of Caulobacter subvibrioides, coherent assemblage. The diversity was most apparent in a comparison of freshwater and marine isolates; an early divergence within the main caulobacter lineage generally corresponded to strains isolated from freshwater and marine habitats. The marine caulobacter assemblage was not exclusive; it also embraced strains of marine hyphomonads and Rhodobacter capsulatus. We hypothesize that these genera are derived from more ancestral caulobacters. Overall, the data are consistent with the interpretation that all of the caulobacters examined, with the possible exception of C. subvibrioides, are ancestrally related, albeit anciently, and that most often division by terrestrial and marine habitats corresponds to an early evolutionary divergence within the genus. (+info)
A copper(I) protein possibly involved in the assembly of CuA center of bacterial cytochrome c oxidase.
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Sco1 and Cox17 are accessory proteins required for the correct assembly of eukaryotic cytochrome c oxidase. At variance with Sco1, Cox17 orthologs are found only in eukaryotes. We browsed bacterial genomes to search proteins functionally equivalent to Cox17, and we identified a class of proteins of unknown function displaying a conserved gene neighborhood to bacterial Sco1 genes, all sharing a potential metal binding motif H(M)X10MX21HXM. Two members of this group, DR1885 from Deinococcus radiodurans and CC3502 from Caulobacter crescentus, were expressed, and their interaction with copper was investigated. The solution structure and extended x-ray absorption fine structure data on the former protein reveal that the protein binds copper(I) through a histidine and three Mets in a cupredoxin-like fold. The surface location of the copper-binding site as well as the type of coordination are well poised for metal transfer chemistry, suggesting that DR1885 might transfer copper, taking the role of Cox17 in bacteria. On the basis of our results, a possible pathway for copper delivery to the Cu(A) center in bacteria is proposed. (+info)
Two independent spiral structures control cell shape in Caulobacter.
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The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter. (+info)
A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator.
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Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle. (+info)
Cell cycle control in bacteria and yeast: a case of convergent evolution?
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Superficially similar traits in phylogenetically unrelated species often result from adaptation to common selection pressures. Examples of convergent evolution are known at the levels of whole organisms, organ systems, gene networks and specific proteins. The phenotypic properties of living things, on the other hand, are determined in large part by complex networks of interacting proteins. Here we present a mathematical model of the network of proteins that controls DNA synthesis and cell division in the alpha-proteobacterium, Caulobacter crescentus. By comparing the protein regulatory circuits for cell reproduction in Caulobacter with that in budding yeast (Saccharomyces cerevisiae), we suggest that convergent evolution may have created similar molecular reaction networks in order to accomplish the same purpose of coordinating DNA synthesis to cell division. Although the genes and proteins involved in cell cycle regulation in prokaryotes and eukaryotes are very different and (apparently) phylogenetically unrelated, they seem to be wired together in similar regulatory networks, which coordinate cell cycle events by identical dynamical principles. (+info)