Animals have evolved a wide diversity of aggressive behavior often based upon the careful monitoring of other individuals. Bacteria are also capable of aggression, with many species using toxins to kill or inhibit their competitors. Like animals, bacteria also have systems to monitor others during antagonistic encounters, but how this translates into behavior remains poorly understood. Here, we use colonies of Escherichia coli carrying colicin-encoding plasmids as a model for studying antagonistic behavior. We show that in the absence of threat, dispersed cells with low reproductive value produce colicin toxins spontaneously, generating efficient pre-emptive attacks. Cells can also respond conditionally to toxins released by clonemates via autoinduction or other genotypes via competition sensing. The strength of both pre-emptive and responsive attacks varies widely between strains. We demonstrate that this variability occurs easily through mutation by rationally engineering strains to recapitulate the diversity in naturally occurring strategies. Finally, we discover that strains that can detect both competitors and clonemates are capable of massive coordinated attacks on competing colonies. This collective behavior protects established colonies from competitors, mirroring the evolution of alarm calling in the animal world.

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.

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.

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 N 6 -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 | SMRT sequencing | methylome D NA methylation involves the addition of a methyl group to either adenine or cytosine by a site-specific DNA methyltransferase. The role of this epigenetic mechanism in the regulation of multiple bacterial processes is described in several reviews (1-5). Two adenine methyltransferases, Dam in the γ-proteobacterium Escherichia coli and CcrM in the α-proteobacterium Caulobacter crescentus, have regulatory functions and are not part of a restriction-modification system. Notably, the synthesis of CcrM, but not Dam, is cell cycle-regulated. CcrM plays an essential role in Caulobacter cell-cycle control (6-8) and in Agrobacterium tumifaciens (9), and it is involved in infective processes in Brucella abortus (10) and in the plant-microbe symbiotic relationship in Mesorhizobium meliloti (11), whereas Dam is involved in virulence of numerous bacterial species (1).In α-proteobacteria, CcrM methylates adenines in GANTC sites (12). In Caulobacter, CcrM is expressed for only a brief time late in chromosome replication (12), and the Lon protease then rapidly degrades CcrM, with a half-life of about 10 min (13). Before the initiation of DNA replication, the chromosome is in the fully methylated state at GANTC sites. Upon bidirectional progression of the replication forks, newly replicated DNA becomes hemimethylated; each copy contains one parental methylated strand and one daughter unmethylated strand. It was predicted that newly replic...

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.

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.

SignificanceBacteria 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.

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.