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1.  The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti 
Molecular microbiology  2013;90(1):54-71.
SUMMARY
Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria.
We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.
doi:10.1111/mmi.12347
PMCID: PMC3793127  PMID: 23909720
2.  DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria 
PLoS Genetics  2013;9(5):e1003541.
Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.
Author Summary
Methylation of genomic DNA at a specific regulatory site can impact a myriad of processes in eukaryotic cells. In bacteria, methylation at the N6 position of adenosine (m6A) is known to mediate a non-adaptive immunity response to protect cells from foreign DNA. While m6A marks are not known to govern expression of cell cycle genes in Gammaproteobacteria, cell cycle transcription in the model alphaproteobacterium Caulobacter crescentus requires the m6A methyltransferase CcrM that introduces m6A marks at GAnTC sequences and the enigmatic factor GcrA. Investigating if a functional and biochemical relationship exists between CcrM and GcrA, we found that CcrM-dependent m6A marks recruit GcrA to the promoters of cell cycle genes in vitro and in vivo and is required for efficient transcription. GcrA interacts with RNA polymerase, explaining how cell cycle transcription is affected. Importantly, m6A-dependent binding is also seen in GcrA orthologs, indicating that this transcriptional regulatory mechanism by CcrM and GcrA is conserved in Alphaproteobacteria.
doi:10.1371/journal.pgen.1003541
PMCID: PMC3667746  PMID: 23737758
3.  Replicon-Dependent Bacterial Genome Evolution: The Case of Sinorhizobium meliloti 
Genome Biology and Evolution  2013;5(3):542-558.
Many bacterial species, such as the alphaproteobacterium Sinorhizobium meliloti, are characterized by open pangenomes and contain multipartite genomes consisting of a chromosome and other large-sized replicons, such as chromids, megaplasmids, and plasmids. The evolutionary forces in both functional and structural aspects that shape the pangenome of species with multipartite genomes are still poorly understood. Therefore, we sequenced the genomes of 10 new S. meliloti strains, analyzed with four publicly available additional genomic sequences. Results indicated that the three main replicons present in these strains (a chromosome, a chromid, and a megaplasmid) partly show replicon-specific behaviors related to strain differentiation. In particular, the pSymB chromid was shown to be a hot spot for positively selected genes, and, unexpectedly, genes resident in the pSymB chromid were also found to be more widespread in distant taxa than those located in the other replicons. Moreover, through the exploitation of a DNA proximity network, a series of conserved “DNA backbones” were found to shape the evolution of the genome structure, with the rest of the genome experiencing rearrangements. The presented data allow depicting a scenario where the pSymB chromid has a distinctive role in intraspecies differentiation and in evolution through positive selection, whereas the pSymA megaplasmid mostly contributes to structural fluidity and to the emergence of new functions, indicating a specific evolutionary role for each replicon in the pangenome evolution.
doi:10.1093/gbe/evt027
PMCID: PMC3622305  PMID: 23431003
chromid; pangenome; bacteria; selection
4.  Analysis of the CtrA Pathway in Magnetospirillum Reveals an Ancestral Role in Motility in Alphaproteobacteria 
Journal of Bacteriology  2012;194(11):2973-2986.
Developmental events across the prokaryotic life cycle are highly regulated at the transcriptional and posttranslational levels. Key elements of a few regulatory networks are conserved among phylogenetic groups of bacteria, although the features controlled by these conserved systems are as diverse as the organisms encoding them. In this work, we probed the role of the CtrA regulatory network, conserved throughout the Alphaproteobacteria, in the magnetotactic bacterium Magnetospirillum magneticum strain AMB-1, which possesses unique intracellular organization and compartmentalization. While we have shown that CtrA in AMB-1 is not essential for viability, it is required for motility, and its putative phosphorylation state dictates the ability of CtrA to activate the flagellar biosynthesis gene cascade. Gene expression analysis of strains expressing active and inactive CtrA alleles points to the composition of the extended CtrA regulon, including both direct and indirect targets. These results, combined with a bioinformatic study of the AMB-1 genome, enabled the prediction of an AMB-1-specific CtrA binding site. Further, phylogenetic studies comparing CtrA sequences from alphaproteobacteria in which the role of CtrA has been experimentally examined reveal an ancestral role of CtrA in the regulation of motility and suggest that its essential functions in other alphaproteobacteria were acquired subsequently.
doi:10.1128/JB.00170-12
PMCID: PMC3370617  PMID: 22467786
5.  Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus  
The cell-cycle regulator ChpT of C. crescentus is a dimeric histidine phosphotransferase that resembles the typical structure of histidine kinases.
Two-component and phosphorelay signal-transduction proteins are crucial for bacterial cell-cycle regulation in Caulobacter crescentus. ChpT is an essential histidine phosphotransferase that controls the activity of the master cell-cycle regulator CtrA by phosphorylation. Here, the 2.2 Å resolution crystal structure of ChpT is reported. ChpT is a homodimer and adopts the domain architecture of the intracellular part of class I histidine kinases. Each subunit consists of two distinct domains: an N-terminal helical hairpin domain and a C-terminal α/β domain. The two N-terminal domains are adjacent within the dimer, forming a four-helix bundle. The ChpT C-terminal domain adopts an atypical Bergerat ATP-binding fold.
doi:10.1107/S1744309112033064
PMCID: PMC3433190  PMID: 22949187
bacterial cell cycle; Caulobacter crescentus; histidine kinases; histidine phosphotransferases
6.  CONTIGuator: a bacterial genomes finishing tool for structural insights on draft genomes 
Recent developments in sequencing technologies have given the opportunity to sequence many bacterial genomes with limited cost and labor, compared to previous techniques. However, a limiting step of genome sequencing is the finishing process, needed to infer the relative position of each contig and close sequencing gaps. An additional degree of complexity is given by bacterial species harboring more than one replicon, which are not contemplated by the currently available programs. The availability of a large number of bacterial genomes allows geneticists to use complete genomes (possibly from the same species) as templates for contigs mapping.
Here we present CONTIGuator, a software tool for contigs mapping over a reference genome which allows the visualization of a map of contigs, underlining loss and/or gain of genetic elements and permitting to finish multipartite genomes. The functionality of CONTIGuator was tested using four genomes, demonstrating its improved performances compared to currently available programs.
Our approach appears efficient, with a clear visualization, allowing the user to perform comparative structural genomics analysis on draft genomes. CONTIGuator is a Python script for Linux environments and can be used on normal desktop machines and can be downloaded from http://contiguator.sourceforge.net.
doi:10.1186/1751-0473-6-11
PMCID: PMC3133546  PMID: 21693004
Genomics; Genome finishing; Software; Structural genomics
7.  Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti 
BMC Genomics  2011;12:235.
Background
Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains.
Results
With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains.
Conclusions
In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.
doi:10.1186/1471-2164-12-235
PMCID: PMC3164228  PMID: 21569405
Sinorhizobium meliloti; nodulation; symbiosis; comparative genomics; pangenome; panregulon
8.  Spread of the group II intron RmInt1 and its insertion sequence target sites in the plant endosymbiont Sinorhizobium meliloti 
Mobile Genetic Elements  2011;1(1):2-7.
RmInt1 is a mobile group II intron from Sinorhizobium meliloti that is exceptionally abundant in this bacterial species. We compared the presence of RmInt1 and two of its insertion sequence homing sites (ISRm2011-2 and ISRm10-2) in two phylogenetic clusters (I and II) identified by AFLP analysis in a collection of S. meliloti field isolates from Italy. Both clusters contained several copies of the ISRm2011-2 element, which is present at high copy number in almost all S. meliloti isolates. By contrast, isolates from cluster I harbored no copies of ISRm10-2 and only a truncated copy of RmInt1, despite the absence of constraints on intron mobility in this genetic background, whereas cluster II strains harbored several copies of this intron. The absence of ISRm10-2 from one of the strains of this cluster suggests that this element was acquired more recently than the other two elements. Furthermore, studies of insertional polymorphisms in cluster II strains revealed the acquisition of ISRm10-2 and subsequent retrohoming of RmInt1 to this homing site. These results highlight the role of intron homing sites (ISs) in facilitating intron dispersal and the dynamic and ongoing nature of the spread of the group II intron RmInt1 in S. meliloti.
doi:10.4161/mge.1.1.15316
PMCID: PMC3190276  PMID: 22016840
group II introns; IS elements; lateral gene transfer; retroelements; Sinorhizobium meliloti; Medicago sativa
9.  Dynamics of Two Phosphorelays Controlling Cell Cycle Progression in Caulobacter crescentus▿ †  
Journal of Bacteriology  2009;191(24):7417-7429.
In Caulobacter crescentus, progression through the cell cycle is governed by the periodic activation and inactivation of the master regulator CtrA. Two phosphorelays, each initiating with the histidine kinase CckA, promote CtrA activation by driving its phosphorylation and by inactivating its proteolysis. Here, we examined whether the CckA phosphorelays also influence the downregulation of CtrA. We demonstrate that CckA is bifunctional, capable of acting as either a kinase or phosphatase to drive the activation or inactivation, respectively, of CtrA. By identifying mutations that uncouple these two activities, we show that CckA's phosphatase activity is important for downregulating CtrA prior to DNA replication initiation in vivo but that other phosphatases may exist. Our results demonstrate that cell cycle transitions in Caulobacter require and are likely driven by the toggling of CckA between its kinase and phosphatase states. More generally, our results emphasize how the bifunctional nature of histidine kinases can help switch cells between mutually exclusive states.
doi:10.1128/JB.00992-09
PMCID: PMC2786585  PMID: 19783630
10.  The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis 
BMC Systems Biology  2010;4:52.
Background
In the bacterium Caulobacter crescentus, CtrA coordinates DNA replication, cell division, and polar morphogenesis and is considered the cell cycle master regulator. CtrA activity varies during cell cycle progression and is modulated by phosphorylation, proteolysis and transcriptional control. In a phosphorylated state, CtrA binds specific DNA sequences, regulates the expression of genes involved in cell cycle progression and silences the origin of replication. Although the circuitry regulating CtrA is known in molecular detail in Caulobacter, its conservation and functionality in the other alpha-proteobacteria are still poorly understood.
Results
Orthologs of Caulobacter factors involved in the regulation of CtrA were systematically scanned in genomes of alpha-proteobacteria. In particular, orthologous genes of the divL-cckA-chpT-ctrA phosphorelay, the divJ-pleC-divK two-component system, the cpdR-rcdA-clpPX proteolysis system, the methyltransferase ccrM and transcriptional regulators dnaA and gcrA were identified in representative genomes of alpha-proteobacteria. CtrA, DnaA and GcrA binding sites and CcrM putative methylation sites were predicted in promoter regions of all these factors and functions controlled by CtrA in all alphas were predicted.
Conclusions
The regulatory cell cycle architecture was identified in all representative alpha-proteobacteria, revealing a high diversification of circuits but also a conservation of logical features. An evolutionary model was proposed where ancient alphas already possessed all modules found in Caulobacter arranged in a variety of connections. Two schemes appeared to evolve: a complex circuit in Caulobacterales and Rhizobiales and a simpler one found in Rhodobacterales.
doi:10.1186/1752-0509-4-52
PMCID: PMC2877005  PMID: 20426835
11.  Metabolic Capacity of Sinorhizobium (Ensifer) meliloti Strains as Determined by Phenotype MicroArray Analysis▿ † 
Applied and Environmental Microbiology  2009;75(16):5396-5404.
Sinorhizobium meliloti is a soil bacterium that fixes atmospheric nitrogen in plant roots. The high genetic diversity of its natural populations has been the subject of extensive analysis. Recent genomic studies of several isolates revealed a high content of variable genes, suggesting a correspondingly large phenotypic differentiation among strains of S. meliloti. Here, using the Phenotype MicroArray (PM) system, hundreds of different growth conditions were tested in order to compare the metabolic capabilities of the laboratory reference strain Rm1021 with those of four natural S. meliloti isolates previously analyzed by comparative genomic hybridization (CGH). The results of PM analysis showed that most phenotypic differences involved carbon source utilization and tolerance to osmolytes and pH, while fewer differences were scored for nitrogen, phosphorus, and sulfur source utilization. Only the variability of the tested strain in tolerance to sodium nitrite and ammonium sulfate of pH 8 was hypothesized to be associated with the genetic polymorphisms detected by CGH analysis. Colony and cell morphologies and the ability to nodulate Medicago truncatula plants were also compared, revealing further phenotypic diversity. Overall, our results suggest that the study of functional (phenotypic) variability of S. meliloti populations is an important and complementary step in the investigation of genetic polymorphism of rhizobia and may help to elucidate rhizobial evolutionary dynamics, including adaptation to diverse environments.
doi:10.1128/AEM.00196-09
PMCID: PMC2725449  PMID: 19561177
12.  Allosteric Regulation of Histidine Kinases by Their Cognate Response Regulator Determines Cell Fate 
Cell  2008;133(3):452-461.
SUMMARY
The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulator that switches PleC from a phosphatase into an autokinase state and thereby mediates a cyclic di-GMP-dependent morphogenetic program. Through allosteric activation of the DivJ autokinase, DivK also stimulates its own phosphorylation and polar localization. These data suggest that DivK is the central effector of an integrated circuit that operates via spatially organized feedback loops to control asymmetry and cell fate determination in C. crescentus. Thus, single domain response regulators can facilitate crosstalk, feedback control, and long-range communication among members of the two-component network.
doi:10.1016/j.cell.2008.02.045
PMCID: PMC2804905  PMID: 18455986
13.  Two-Component Signal Transduction Pathways Regulating Growth and Cell Cycle Progression in a Bacterium: A System-Level Analysis 
PLoS Biology  2005;3(10):e334.
Two-component signal transduction systems, comprised of histidine kinases and their response regulator substrates, are the predominant means by which bacteria sense and respond to extracellular signals. These systems allow cells to adapt to prevailing conditions by modifying cellular physiology, including initiating programs of gene expression, catalyzing reactions, or modifying protein–protein interactions. These signaling pathways have also been demonstrated to play a role in coordinating bacterial cell cycle progression and development. Here we report a system-level investigation of two-component pathways in the model organism Caulobacter crescentus. First, by a comprehensive deletion analysis we show that at least 39 of the 106 two-component genes are required for cell cycle progression, growth, or morphogenesis. These include nine genes essential for growth or viability of the organism. We then use a systematic biochemical approach, called phosphotransfer profiling, to map the connectivity of histidine kinases and response regulators. Combining these genetic and biochemical approaches, we identify a new, highly conserved essential signaling pathway from the histidine kinase CenK to the response regulator CenR, which plays a critical role in controlling cell envelope biogenesis and structure. Depletion of either cenK or cenR leads to an unusual, severe blebbing of cell envelope material, whereas constitutive activation of the pathway compromises cell envelope integrity, resulting in cell lysis and death. We propose that the CenK–CenR pathway may be a suitable target for new antibiotic development, given previous successes in targeting the bacterial cell wall. Finally, the ability of our in vitro phosphotransfer profiling method to identify signaling pathways that operate in vivo takes advantage of an observation that histidine kinases are endowed with a global kinetic preference for their cognate response regulators. We propose that this system-wide selectivity insulates two-component pathways from one another, preventing unwanted cross-talk.
Histidine kinases and their (sensory) response regulators are screened for in C. crescentus. Follow-up experiments determine several essential components, including one pair critical for cell envelope biogenesis and structure.
doi:10.1371/journal.pbio.0030334
PMCID: PMC1233412  PMID: 16176121

Results 1-13 (13)