Diego Gonzalez

The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach

DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle

Significance Caulobacter crescentus, a bacterium with an inherent asymmetric cell division, uses dynamic changes in chromosome methylation state to synchronize chromosome replication with cell-cycle regulation. We identified the N6-methyladenine and 5-methylcytosine methylation state of every base pair at five times in the cell cycle to show that 4,515 GANTC sites, recognized by the CcrM methyltransferase, change from full- to hemimethylation upon passage of the replication fork. Significantly, 27 of the GANTC sites are protected from methylation at all times. We also identified four previously unknown methylation motifs and the cognate methyltransferase for two of these motifs. The ability to track the state of the methylome in exquisite temporal detail will be invaluable to investigations of microbial epigenetic regulation. The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N6-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus

DNA methylation regulates many processes, including gene expression, by superimposing secondary information on DNA sequences. The conserved CcrM enzyme, which methylates adenines in GANTC sequences, is essential to the viability of several Alphaproteobacteria. In this study, we find that Caulobacter crescentus cells lacking the CcrM enzyme accumulate low levels of the two conserved FtsZ and MipZ proteins, leading to a severe defect in cell division. This defect can be compensated by the expression of the ftsZ gene from an inducible promoter or by spontaneous suppressor mutations that promote FtsZ accumulation. We show that CcrM promotes the transcription of the ftsZ and mipZ genes and that the ftsZ and mipZ promoter regions contain a conserved CGACTC motif that is critical to their activities and to their regulation by CcrM. In addition, our results suggest that the ftsZ promoter has the lowest activity when the CGACTC motif is non‐methylated, an intermediate activity when it is hemi‐methylated and the highest activity when it is fully methylated. The regulation of ftsZ expression by DNA methylation may explain why CcrM is essential in a subset of Alphaproteobacteria.

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.

Regulation of the Activity of the Dual-Function DnaA Protein in Caulobacter crescentus

DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase

ABSTRACT CcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group of Alphaproteobacteria. In Caulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, that C. crescentus can significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrM populations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulated ftsZ expression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrM mutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulates ftsZ and mipZ transcription, uncovering a previously unsuspected link between metabolic regulation and cell division in Alphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrM cells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM. IMPORTANCE In bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness in Caulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of the ftsZ cell division gene. This finding suggests that the most critical function of CcrM in C. crescentus is to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to other Alphaproteobacteria. In bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness in Caulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of the ftsZ cell division gene. This finding suggests that the most critical function of CcrM in C. crescentus is to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to other Alphaproteobacteria.

Lifestyle transitions and adaptive pathogenesis of Pseudomonas aeruginosa

Pseudomonas aeruginosa acute and chronic infections are of great concern to human health, especially in hospital settings. It is currently assumed that P. aeruginosa has two antagonistic pathogenic strategies that parallel two different lifestyles; free-living cells are predominantly cytotoxic and induce an acute inflammatory reaction, while biofilm-forming communities cause refractory chronic infections. Recent findings suggest that the planktonic-to-sessile transition is a complex, reversible and overall dynamic differentiation process. Here, we examine how the Gac/Rsm regulatory cascade, a key player in this lifestyle switch, endows P. aeruginosa with both a permissive lifecycle in nature and flexible virulence strategy during infection.

Caulobacter crescentus CdnL is a non-essential RNA polymerase-binding protein whose depletion impairs normal growth and rRNA transcription

CdnL is an essential RNA polymerase (RNAP)-binding activator of rRNA transcription in mycobacteria and myxobacteria but reportedly not in Bacillus. Whether its function and mode of action are conserved in other bacteria thus remains unclear. Because virtually all alphaproteobacteria have a CdnL homolog and none of these have been characterized, we studied the homolog (CdnLCc) of the model alphaproteobacterium Caulobacter crescentus. We show that CdnLCc is not essential for viability but that its absence or depletion causes slow growth and cell filamentation. CdnLCc is degraded in vivo in a manner dependent on its C-terminus, yet excess CdnLCc resulting from its stabilization did not adversely affect growth. We find that CdnLCc interacts with itself and with the RNAP β subunit, and localizes to at least one rRNA promoter in vivo, whose activity diminishes upon depletion of CdnLCc. Interestingly, cells expressing CdnLCc mutants unable to interact with the RNAP were cold-sensitive, suggesting that CdnLCc interaction with RNAP is especially required at lower than standard growth temperatures in C. crescentus. Our study indicates that despite limited sequence similarities and regulatory differences compared to its myco/myxobacterial homologs, CdnLCc may share similar biological functions, since it affects rRNA synthesis, probably by stabilizing open promoter-RNAP complexes.

The pUltra plasmid series: A robust and flexible tool for fluorescent labeling of Enterobacteria

Fluorescent labeling has been an invaluable tool for the study of living organisms and bacterial species are no exception to this. Here we present and characterize the pUltra plasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP) from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad range of Enterobacteria. The amount of expressed fluorophore from these genetic constructs is such, that the contours of the cells can be delineated on the basis of the fluorescent signal only. In addition, labeling through the pUltra plasmids can be used successfully for fluorescence and confocal microscopy while unambiguous distinction of cells labeled with different colors can be carried out efficiently by microscopy or flow cytometry. We compare the labeling provided by the pUltra plasmids with that of another plasmid series encoding fluorescent proteins and we show that the pUltra constructs are vastly superior in signal intensity and discrimination power without having any detectable growth rate effects for the bacterial population. We also use the pUltra plasmids to produce mixtures of differentially labeled pathogenic Escherichia, Shigella and Salmonella species which we test during infection of mammalian cells. We find that even inside the host cell, different strains can be distinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltra plasmids are a powerful labeling tool especially useful for complex biological experiments such as the visualization of ecosystems of different bacterial species or of enteric pathogens in contact with their hosts.

Costs and benefits of provocation in bacterial warfare

Significance Bacteria live in dense environments where competition for space and resources is fierce. For this reason, they often use diffusible toxins to eliminate closely related strains. Some toxins trigger systematic retaliation, raising the question of the role of provocation in bacterial warfare. We combine mathematical modeling and experiments to study the costs and benefits of provocation. In one-to-one encounters, provocation is costly as it leads to strong counterattacks. However, with three or more strains present, provocation can provide benefits via a “divide-and-conquer” effect, whereby a strain forces its opponents to wipe each other out. This effect could be harnessed as a targeted antibacterial approach; adding low levels of certain antibiotics to communities can promote warfare and cross-elimination between strains. Competition in animals involves a wide variety of aggressive behaviors. One of the most sophisticated strategies for a focal actor is to provoke a competitor into uncontrolled aggression toward other competitors. Like animals, bacteria rely on a broad spectrum of molecular weapons, some of which provoke potential rivals by triggering retaliation. While bacterial provocation is well documented, its potential adaptive value has received little attention. Here, we examine the costs and benefits of provocation using mathematical modeling and experiments with Escherichia coli strains encoding colicin toxins. We show that provocation is typically costly in one-to-one encounters because a provoking strain receives a strong reciprocal attack compared with nonprovoking strains. By contrast, provocation can be strongly beneficial in communities including more than two toxin-producing strains, especially when the provoker is shielded from, or resistant to, its opponents’ toxins. In these scenarios, we demonstrate that the benefit of provocation derives from a “divide-and-conquer” effect by which aggression-provoking toxin producers force their competitors into increased reciprocal aggression, leading to their cross-elimination. Furthermore, we show that this effect can be mimicked by using antibiotics that promote warfare among strains in a bacterial community, highlighting the potential of provocation as an antimicrobial approach.