The Diversity and Evolution of Cell Cycle Regulation in Alpha-Proteobacteria: a Comparative Genomic Analysis

Brilli et al. BMC Systems Biology 2010, 4:52 http://www.biomedcentral.com/1752-0509/4/52 RESEARCH ARTICLE Open Access TheResearch diversity article and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis Matteo Brilli1,2, Marco Fondi1, Renato Fani1, Alessio Mengoni1, Lorenzo Ferri1, Marco Bazzicalupo1 and Emanuele G Biondi*1 Abstract 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. Background teria subdivision is a very heterogeneous group of Living cells continuously receive and process signals bacteria and includes symbionts of plants (Rhizobia), coming from their environment, and by integrating this pathogens for animals (Brucella, Rickettsia) and plants information into their own internal state, are able to react (Agrobacterium), photosynthetic bacteria (Rhodobacter) with appropriate responses which coordinate each func- and also several genera metabolizing C1-compounds tion in the cell in order to divide and produce progeny. (Methylobacterium). Regulation of cell cycle progression needs to be a robust Together with this diversity of life styles and ecological but versatile process that integrates different exogenous niches, the alpha-proteobacteria subdivision is also one and endogenous signals and that guarantees fidelity and of the bacterial groups in which cell cycle regulation has controlled progression throughout the different phases. been studied in more detail and one of its members, Cau- Bacteria have evolved different regulation systems for lobacter crescentus, has recently become a model organ- cell cycle coordination, probably due to different ecologi- ism in these studies [3-6]. In this organism each cell cal and evolutionary constraints [1,2]. Alpha-proteobac- division is asymmetric—producing two functionally and morphologically different cells, the replicating "stalked" * Correspondence: [email protected] 1 Department of Evolutionary Biology, University of Florence, via Romana, 17, cell type and the vegetative "swarmer" type. After each Florence, Italy initiation of DNA replication, the replication fork is kept Full list of author information is available at the end of the article © 2010 Brilli et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At- BioMed Central tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Brilli et al. BMC Systems Biology 2010, 4:52 Page 2 of 16 http://www.biomedcentral.com/1752-0509/4/52 blocked so that the Caulobacter cell cycle can follow a because when phosphorylated, it indirectly inactivates pattern of once-and-only-once replication per division CtrA and thus promotes DNA replication. Two histidine (G1, S, and G2 phases are temporally distinguished). kinases are known to interact with DivK: PleC and DivJ Many factors are known to regulate cell cycle progres- [22-25]. Bacterial histidine kinases can have alternatively sion and most of them are members of the family of two- both kinase and phosphatase activities and these opposite component signal transduction proteins, comprised of activities are modulated by conformational changes of histidine kinases and their response regulator substrates the protein [26]. A null Caulobacter pleC mutant pro- [6]. Among those proteins CtrA is the master regulator of duces almost symmetric cells at the division and displays the Caulobacter cell cycle, an essential response regulator abnormal polar development. The DivJ histidine kinase whose activity as a transcription factor varies as a func- plays a role in controlling the length and location of the tion of the cell cycle [7-9]. CtrA controls various func- stalk and cell division. PleC and DivJ are considered the tions during cell cycle progression by activating or principal phosphatase and kinase, respectively, of DivK repressing gene expression. CtrA also blocks the initia- and they are in opposite locations during cell cycle pro- tion of DNA replication through binding of the replica- gression [27,28]. DivJ activity is also positively controlled tion origin [7]. Among genes regulated by CtrA we can by the TacA/SpmX pathway, which is transcriptionally find those involved in cell division (ftsZ, ftsA, ftsQ and activated by CtrA [10,29]. ftsW), the protease encoding gene clpP which is essential ChpT also transfers the phosphate to a second response in Caulobacter, the DNA methylase gene ccrM, flagellar regulator named CpdR, which, together with RcdA, is a biogenesis genes, stalk biogenesis regulatory genes, pili factor involved in CtrA proteolysis mediated by ClpP- biogenesis genes such as pilA, and chemotaxis genes [10- ClpX protease [30-32]. CtrA is degraded at the stalked 15]. pole at the G1/S transition when the origin of replication CtrA activity and stability varies during the cell cycle. needs to be cleared and also in the stalked compartment, Oscillation of CtrA levels, peaking at the predivisional where initiation of DNA replication occurs immediately stage before cell division, is achieved by different mecha- after cell division [33,34]. nisms: transcription, proteolysis and phosphorylation All these regulations are schematized in Figure 1 where control as discussed in detail below. we illustrate the multilevel regulation of the Caulobacter DnaA and GcrA, and the DNA methyltransferase cell cycle. Two main oscillators are working during cell CcrM are involved in controlling ctrA transcription cycle progression: (i) the transcriptional and epigenetic [11,16]. DnaA is a key element in cell cycle regulation circuit (CtrA-DnaA-GcrA-CcrM); (ii) the phosphoryla- because it is required for the initiation of DNA replication/proteolysis and transcription circuit (CckA-CtrA- tion; it also controls the transcription of about 40 genes DivK). The latter also involves coordination of CtrA pro- involved in nucleotide biogenesis, cell division, and polar teolysis and cell division through regulation of DivK morphogenesis [17,18], and it activates the transcription activity. of the gcrA gene [19]. GcrA controls the transcription of Several of these regulatory mechanisms are at least par- ctrA and genes involved in DNA metabolism and chro- tially redundant, and it has been demonstrated that only mosome segregation, including those encoding DNA phosphorylation of CtrA is indispensable during cell gyrase, DNA helicase, DNA primase, and DNA poly- cycle progression; in fact, cell cycle regulated transcrip- merase III [19]. As a consequence of this genetic circuit, tion of ctrA can be substituted by constitutive transcrip- CtrA accumulates out-of-phase with GcrA [19]. The tion [20] and proteolysis can also be removed. transcriptional loop of ctrA is closed by CcrM. CtrA acti- It has recently been demonstrated that asymmetric vates the transcription of ccrM, which encodes for a DNA division also takes place in Agrobacterium tumefaciens, methyltransferase whose abundance is cell cycle depen- Sinorhizobium meliloti and Brucella abortus [35], indicat- dent. CcrM is able to activate dnaA promoter region ing that at least some of the features governing cell cycle through methylation, closing the positive feedback com- progression in Caulobacter might also be present in other posed by CtrA, DnaA and GcrA. species. For example, in Rhodobacter capsulatus, CtrA A second essential regulatory control on CtrA is carried and CckA are not essential but are required for the out by phosphorylation. In fact, CtrA must be phospho- expression of the GTA, a system for genetic exchanges rylated to bind DNA and its phosphorylation depends on [36,37]. CtrA in Brucella controls cellular events similar cell cycle progression. An essential phosphorelay, com- to those controlled by CtrA in Caulobacter,

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