Cell Cycle–Dependent Polar Localization of Chromosome Partitioning Proteins in Caulobacter crescentus

AbstractBacterial chromosome replication is regulated from a single replication origin (ori) that receives cell cycle signals. Following replication, bacteria often use theparABSpartition system with a centromere-likeparSlocus to place the chromosomes into the daughter cells. Our knowledge of cell cycle regulation is incomplete and we searched for novel regulators of chromosome replication. Here we show that in the cell cycle modelCaulobacter crescentusa novel DNA-binding protein promotes both the initiation of chromosome replication and the earliest step of chromosome partitioning. We used biochemical fractionation to identify a protein (OpaA) that preferentially binds to mutatedoriDNA that also increasesori-plasmid replicationin vivo. OpaA represents a previously unknown class of DNA-binding proteins.opaAgene expression is essential and sufficient OpaA levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified the genomic binding sites for OpaA, with the strongest associations at theparABSlocus nearori. Using molecular-genetic and fluorescence microscopy experiments, we showed that OpaA also promotes the first step of chromosome partitioning, the initial separation of the duplicatedparSloci followingorireplication. This separation occurs before theparABSmechanism and it coincides with the regulatory step that splits the symmetry of the chromosomes so that they are placed at distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that OpaA coordinates replication with the poorly understood mechanism of early chromosome separation.opaAlethal suppressor and antibiotic experiments argue that future studies be focused on the mechanistic roles for transcription and translation at this critical step of the cell cycle.Author SummaryLike all organisms, bacteria must replicate their chromosomes and move them into the newly dividing cells. Eukaryotes use non-overlapping phases, first for chromosome replication (S-phase) followed by mitosis (M-phase) when the completely duplicated chromosomes are separated. However, bacteria combine both phases so chromosome replication and chromosome separation (termed chromosome “partitioning”) overlap. In many bacteria, includingCaulobacter crescentus, chromosome replication initiates from a single replication origin (ori) and the first duplicated regions of the chromosome immediately begin “partitioning” towards the cell poles long before the whole chromosome has finished replication. This partitioning movement uses the centromere-like DNA called“parS”that is located near theori. Here we identify a completely novel type of DNA-binding protein called OpaA and we show that it acts at bothoriandparS. The timing and coordination of overlapping chromosome replication and partitioning phases is a special regulatory problem for bacteria. We further demonstrate that OpaA is selectively required for the initiation of chromosome replication atoriand likewise that OpaA is selectively required for the initial partitioning ofparS. Therefore, we propose that OpaA is a novel regulator that coordinates chromosome replication with the poorly understood mechanism of early chromosome separation.

Download Full-text

Polar Localization of the CckA Histidine Kinase and Cell Cycle Periodicity of the Essential Master Regulator CtrA in Caulobacter crescentus

Journal of Bacteriology ◽

10.1128/jb.00985-09 ◽

2009 ◽

Vol 192 (2) ◽

pp. 539-552 ◽

Cited By ~ 36

Author(s):

Peter S. Angelastro ◽

Oleksii Sliusarenko ◽

Christine Jacobs-Wagner

Keyword(s):

Cell Cycle ◽

Histidine Kinase ◽

Cell Cycle Progression ◽

Fluorescent Protein ◽

Caulobacter Crescentus ◽

Replication Initiation ◽

Protein Fusion ◽

Cycle Progression ◽

Polar Localization ◽

And Function

ABSTRACT The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two cis-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between β-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression.

Download Full-text

Cell Cycle–Dependent Polar Localization of an Essential Bacterial Histidine Kinase that Controls DNA Replication and Cell Division

Cell ◽

10.1016/s0092-8674(00)80719-9 ◽

1999 ◽

Vol 97 (1) ◽

pp. 111-120 ◽

Cited By ~ 193

Author(s):

Christine Jacobs ◽

Ibrahim J. Domian ◽

Janine R. Maddock ◽

Lucy Shapiro

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Histidine Kinase ◽

Polar Localization ◽

Cycle Dependent

Download Full-text

The trans-acting flagellar regulatory proteins, FliX and FlbD, play a central role in linking flagellar biogenesis and cytokinesis in Caulobacter crescentus

Microbiology ◽

10.1099/mic.0.28174-0 ◽

2005 ◽

Vol 151 (11) ◽

pp. 3699-3711 ◽

Cited By ~ 8

Author(s):

Rachel E. Muir ◽

Jesse Easter ◽

James W. Gober

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Caulobacter Crescentus ◽

Class Ii ◽

Timely Completion ◽

Eventual Loss ◽

Cycle Dependent ◽

Flagellar Biogenesis ◽

Synchronized Cultures ◽

Structure Class

The FliX/FlbD-dependent temporal transcription of late flagellar genes in Caulobacter crescentus requires the assembly of an early, class II-encoded flagellar structure. Class II flagellar-mutant strains exhibit a delay in the completion of cell division, with the accumulation of filamentous cells in culture. It is shown here that this cell-division defect is attributable to an arrest in the final stages of cell separation. Normal cell morphology could be restored in class II mutants by gain-of-function alleles of FliX or FlbD, suggesting that the timely completion of cell division requires these trans-acting factors. In synchronized cultures, inhibition of cell division by depleting FtsZ resulted in normal initial expression of the late, FlbD-dependent fliK gene; however, the cell cycle-regulated cessation of transcription was delayed, indicating that cell division may be required to negatively regulate FlbD activity. Interestingly, prolonged depletion of FtsZ resulted in an eventual loss of FlbD activity that could be bypassed by a constitutive mutant of FlbD, but not of FliX, suggesting the possible existence of a second cell cycle-dependent pathway for FlbD activation.

Download Full-text

Identification of the Protease and the Turnover Signal Responsible for Cell Cycle-Dependent Degradation of the Caulobacter FliF Motor Protein

Journal of Bacteriology ◽

10.1128/jb.186.15.4960-4971.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4960-4971 ◽

Cited By ~ 21

Author(s):

Björn Grünenfelder ◽

Sherif Tawfilis ◽

Stefanie Gehrig ◽

Magne Østerås ◽

Daniel Eglin ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Mutational Analysis ◽

Caulobacter Crescentus ◽

C Terminus ◽

Clpap Protease ◽

Tightly Coupled ◽

Primary Amino ◽

Cycle Dependent ◽

Mutant Forms

ABSTRACT Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.

Download Full-text

Role of the flagellum in cell-cycle-dependent expression of bacteriophage receptor activity in Caulobacter crescentus.

Journal of Bacteriology ◽

10.1128/jb.171.2.1035-1040.1989 ◽

1989 ◽

Vol 171 (2) ◽

pp. 1035-1040 ◽

Cited By ~ 11

Author(s):

R A Bender ◽

C M Refson ◽

E A O'Neill

Keyword(s):

Cell Cycle ◽

Caulobacter Crescentus ◽

Receptor Activity ◽

Cycle Dependent

Download Full-text

Protein Sequences and Cellular Factors Required for Polar Localization of a Histidine Kinase in Caulobacter crescentus

Journal of Bacteriology ◽

10.1128/jb.184.21.6037-6049.2002 ◽

2002 ◽

Vol 184 (21) ◽

pp. 6037-6049 ◽

Cited By ~ 28

Author(s):

Stephen A. Sciochetti ◽

Todd Lane ◽

Noriko Ohta ◽

Austin Newton

Keyword(s):

Cell Cycle ◽

Cell Separation ◽

Fluorescent Protein ◽

Catalytic Domain ◽

Caulobacter Crescentus ◽

Mutant Cell ◽

Sensor Kinase ◽

Polar Localization ◽

Cell Pole ◽

Cellular Factors

ABSTRACT 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.

Download Full-text