Zinc-finger domain transcriptional regulators regulate a myriad of functions in eukaryotes. Interestingly, ancestral versions (MucR) from Alpha-proteobacteria control bacterial virulence/symbiosis. Whether virulence regulators can also control cell cycle transcription is unknown. Here we report that MucR proteins implement a hitherto elusive primordial S→G1 transcriptional switch. After charting G1-specific promoters in the cell cycle model Caulobacter crescentus by comparative ChIP-seq, we use one such promoter as genetic proxy to unearth two MucR paralogs, MucR1/2, as constituents of a quadripartite and homeostatic regulatory module directing the S→G1 transcriptional switch. Surprisingly, MucR orthologues that regulate virulence and symbiosis gene transcription in Brucella, Agrobacterium or Sinorhizobium support this S→G1 switch in Caulobacter. Pan-genomic ChIP-seq analyses in Sinorhizobium and Caulobacter show that this module indeed targets orthologous genes. We propose that MucR proteins and possibly other virulence regulators primarily control bacterial cell cycle (G1-phase) transcription, rendering expression of target (virulence) genes periodic and in tune with the cell cycle.

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. 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.

What are the minimal requirements to sustain an asymmetric cell cycle? Here we use mathematical modelling and forward genetics to reduce an asymmetric cell cycle to its simplest, primordial components. In the Alphaproteobacterium Caulobacter crescentus, cell cycle progression is believed to be controlled by a cyclical genetic circuit comprising four essential master regulators. Unexpectedly, our in silico modelling predicted that one of these regulators, GcrA, is in fact dispensable. We confirmed this experimentally, finding that ΔgcrA cells are viable, but slow-growing and elongated, with the latter mostly due to an insufficiency of a key cell division protein. Furthermore, suppressor analysis showed that another cell cycle regulator, the methyltransferase CcrM, is similarly dispensable with simultaneous gcrA/ccrM disruption ameliorating the cytokinetic and growth defect of ΔgcrA cells. Within the Alphaproteobacteria, gcrA and ccrM are consistently present or absent together, rather than either gene being present alone, suggesting that gcrA/ccrM constitutes an independent, dispensable genetic module. Together our approaches unveil the essential elements of a primordial asymmetric cell cycle that should help illuminate more complex cell cycles. Cell cycle regulation is remarkably complex and the fundamental principles difficult to understand, even in simple cells. The bacterium Caulobacter crescentus is a popular model organism to study cell cycle regulation due to the two different daughter cells resulting from cell division: a mobile “swarmer” cell and a “stalked” cell that adheres to surfaces. Here, we use mathematical modelling and genetic experiments to identify the core components of the asymmetric cell cycle of these bacteria. Using our mathematical model we predicted and confirmed experimentally that the transcription factor and cell cycle regulator, GcrA, hitherto thought to be essential, is in fact dispensable. We also identified another master regulator, the methyltransferase, CcrM as dispensable. Furthermore, simultaneous deletion of both GcrA and CcrM removes the severe cell division defects observed on either single deletion, returning cells to near wild-type morphology. We found that GcrA and CcrM constitute an independent, dispensable, genetic module that regulates transcription of cytokinetic proteins during the cell cycle. Phylogenetically, the module is conserved in Alphaproteobacteria, the class of Caulobacter, but is not present in the tree root of the class, suggesting that we have identified the primordial core of the asymmetric cell cycle regulatory circuit in the Alphaproteobacteria.

In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA) that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ). Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a ‘nutritional override’ system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells. Bacteria predominantly exist within surface-attached communities that facilitate metabolic cooperation, sharing of genetic information, and protect cells against stress. The freshwater bacterium, Caulobacter crescentus elaborates an adhesive structure known as the holdfast, which enables surface attachment. We have discovered a novel mechanism that controls holdfast development in response to cell cycle and environmental cues. This regulatory mechanism involves a small protein inhibitor, HfiA, which targets a conserved holdfast synthesis enzyme and ensures that the holdfast is produced at the appropriate stage of cell development and under the appropriate environmental conditions. To our knowledge, the regulatory system we report here is unprecedented, and provides a mechanism for integrative control of bacterial cell adhesion in response to cell cycle and environmental signals.

Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger—bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)—that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 → S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMB^s loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603–1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division. The pathways involved in bacterial surface assembly are critical for cell morphogenesis and serve as attractive targets for antibiotic development. Bacteria in the suborder Corynebacterineae, which includes important pathogens like Mycobacterium tuberculosis, possess a unique multilayered surface structure. In addition to the common peptidoglycan cell wall, they have an attached polysaccharide layer called arabinogalactan and an outer membrane made of mycolic acids. To enhance our understanding of cell surface biogenesis in these bacteria, we performed a global genetic analysis of gene function in the model system Corynebacterium glutamicum (Cglu) using transposon sequencing. In addition to defining the essential gene set in this organism, our analysis also identified SteA and SteB as components of the cytokinetic ring. These factors are conserved among the Corynebacterineae, and our results reveal that they play a critical role in the final stages of cytokinesis by promoting remodeling of the peptidoglycan layer at the division site.