The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis

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.

Figure 1

Cell cycle regulation in Caulobacter. Scheme of cell cycle regulation in Caulobacter with all factors analyzed in this paper. See the "introduction" for details.

Figure 2

Orthologous genes in alpha-proteobacteria. Presence (black) or absence (white) of genes (found using bidirectional best blast hit, BBH) described in Figure 1 and involved in cell cycle regulation in the alpha-proteobacterial genomes. Proteins are indicated in the uppermost row and they are ordered on the basis of their co-occurrence patterns (see Material and Methods). We show here a reduced dataset comprising one species for each considered genus, for a total of 37 out of 65 alpha-proteobacteria analyzed (the complete results are listed in Table S2, Additional file 4). Organisms are ordered on the basis of their phylogenetic relationships as assessed by using a 5000-residue long concatamer of universal proteins [65], the Neighbor-Joining method with a Dayhoff evolutionary model and 100 bootstrap replicates. A tree representation where nodes with a statistical support of less than 75% have been collapsed (Figure 2, left) was chosen; Dark gray cells indicate the presence of a BBH with respect to the protein corresponding to that column. Medium dark gray cells correspond to DivK proteins in those organisms that have an ambiguous position in the phylogenetic tree (Additional file 3, Figure S1); light gray cells correspond to organisms that have PleC but where DivK is absent or in doubt.

Figure 3

Transcriptional control of CtrA on cell cycle genes. Transcriptional control of CtrA on factors involved in regulation of cell cycle progression (see also Additional file 7, Table S4). Color bar represents p-values of the Z-score transformation of motif scores (Methods section for details).

Figure 4

Functions controlled by CtrA among alpha-proteobacteria. Enrichment of genes controlled by CtrA in COG categories (see also Additional file 8, Table S5). The scale corresponds to the p-value of the functional enrichment calculated as described in the Material and Methods. The p-value is inversely correlated to the strength of the functional enrichment of each regulon, i.e. a lower p-value indicates stronger enrichment.

Figure 5

Transcriptional control of GcrA and DnaA on cell cycle genes. GcrA and DnaA binding sites on factors involved in cell cycle regulation among alpha-proteobacteria (see also Additional file 7, Table S4).

Figure 6

Verification of CtrA and DnaA binding site prediction. A. Number of genes putatively controlled by CtrA (dark gray) and DnaA (light gray) across alpha-proteobacteria. B. Distribution of p-values assigned to each gene with respect to the CtrA binding and DnaA binding for three alpha-proteobacteria (CCRE = C. crescentus, BABO = B. abortus, SMELI = S. meliloti) and species, possessing only DnaA (ECOLI = E. coli, BSUB = B. subtilis). The distributions of CCRE, BABO, SMELI for CtrA start to diverge from the 'background' distributions represented by organisms not possessing CtrA at a p < 0.05, while for DnaA this distribution is uniform among all five organisms. C. Consensus sequences of DnaA and CtrA in different bacteria found experimentally elsewhere (see references near the sequences) compared with our PMWs. M = T or G, W = A or T.

Figure 7

Regulatory circuits of clusters A, B, C, E. See the "Results" and "Discussion" sections for more details. Interactions via phosphorylation, as well as proteolysis, were suggested only considering the interaction demonstrated in Caulobacter. Moreover, DivK inhibition on CckA was considered, as in Biondi (2006), only in Caulobacter [20]. The presence of binding sites of transcription factors CtrA, DnaA and GcrA is shown as a continuous line if predicted binding sites were present in at least 90% of the gene promoters of a cluster. In contrast, the connection is shown as a dotted line for binding sites present in ca. 70%. The Caulobacter-like group corresponds to B. japonicum, P. lavamentivorans and M. maris.