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Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of high-throughput spatiotemporal databases including data from gene expression profiling experiments (microarrays, RNA-seq, ChIP-seq, ribosome profiling, LC-ms proteomics), gene essentiality studies (Tn-seq), genome wide protein localization studies, and global chromosome methylation analyses (SMRT sequencing). A major challenge involves the integration of these diverse data sets into one comprehensive community resource. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource.

Summary Upon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the ΔHU1ΔHU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.

A cyclical control circuit composed of four master regulators drives the Caulobacter cell cycle. We report that SciP, a helix-turn-helix transcription factor, is an essential component of this circuit. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the α-proteobacteria. SciP is expressed late in the cell cycle and accumulates preferentially in the daughter swarmer cell. At least 58 genes, including many flagellar and chemotaxis genes, are regulated by a type 1 incoherent feedforward motif in which CtrA activates sciP , followed by SciP repression of ctrA and CtrA target genes. We demonstrate that SciP binds to DNA at a motif distinct from the CtrA binding motif that is present in the promoters of genes co-regulated by SciP and CtrA. SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. The type 1 incoherent feedforward circuit motif enhances the pulse-like expression of the downstream genes, and the negative feedback to ctrA expression reduces peak CtrA accumulation. The presence of SciP in the control network enhances the robustness of the cell cycle to varying growth rates.

Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

The dynamic range of a bacterial species’ natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. The cell cycle control circuitry is tied closely to chromosome replication and morphogenesis by multiple feedback pathways from the modular functions that implement the cell cycle. The sophistication of the genetic regulatory circuits and the elegant integration of temporally controlled transcription and protein synthesis with spatially dynamic phosphosignaling and proteolysis pathways, and epigenetic regulatory mechanisms, form a remarkably robust living system.

The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G 1 and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. The timing of DnaA accumulation was found to be regulated by the methylation state of the dnaA promoter, which in turn depends on the chromosomal position of dnaA near the origin of replication and restriction of CcrM synthesis to the end of the cell cycle. The dnaA gene is preferentially transcribed from a fully methylated promoter. DnaA initiates DNA replication and activates the transcription of the next cell-cycle regulator, GcrA. With the passage of the replication fork, the dnaA promoter becomes hemimethylated, and DnaA accumulation drops. GcrA then activates the transcription of the next cell-cycle regulator, CtrA, once the replication fork passes through the ctrA P1 promoter, generating two hemimethylated copies of ctrA . The ctrA gene is preferentially transcribed from a hemimethylated promoter. CtrA then activates the transcription of ccrM , to bring the newly replicated chromosome to the fully methylated state, promoting dnaA transcription and the start of a new cell cycle. We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control.

The recent proliferation of protein interaction networks has motivated research into network alignment: the cross-species comparison of conserved functional modules. Previous studies have laid the foundations for such comparisons and demonstrated their power on a select set of sparse interaction networks. Recently, however, new computational techniques have produced hundreds of predicted interaction networks with interconnection densities that push existing alignment algorithms to their limits. To find conserved functional modules in these new networks, we have developed Græmlin, the first algorithm capable of scalable multiple network alignment. Græmlin's explicit model of functional evolution allows both the generalization of existing alignment scoring schemes and the location of conserved network topologies other than protein complexes and metabolic pathways. To assess Græmlin's performance, we have developed the first quantitative benchmarks for network alignment, which allow comparisons of algorithms in terms of their ability to recapitulate the KEGG database of conserved functional modules. We find that Græmlin achieves substantial scalability gains over previous methods while improving sensitivity.

The level of DnaA, a key bacterial DNA replication initiation factor, increases during the Caulobacter swarmer-to-stalked transition just before the G1/S transition. We show that DnaA coordinates DNA replication initiation with cell cycle progression by acting as a global transcription factor. Using DnaA depletion and induction in synchronized cell populations, we have analysed global transcription patterns to identify the differential regulation of normally co-expressed genes. The DnaA regulon includes genes encoding several replisome components, the GcrA global cell cycle regulator, the PodJ polar localization protein, the FtsZ cell division protein, and nucleotide biosynthesis enzymes. In cells depleted of DnaA, the G1/S transition is temporally separated from the swarmer-to-stalked cell differentiation, which is normally coincident. In the absence of DnaA, the CtrA master regulator is cleared by proteolysis during the swarmer-to-stalked cell transition as usual, but DNA replication initiation is blocked. In this case, expression of gcrA, which is directly repressed by CtrA, does not increase in conjunction with the disappearance of CtrA until DnaA is subsequently induced, showing that gcrA expression requires DnaA. DnaA boxes are present upstream of many genes whose expression requires DnaA, and His6-DnaA binds to the promoters of gcrA, ftsZ and podJ in vitro. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage.

The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites.

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle., Author Summary The generation of diverse cell types occurs through two fundamental processes; asymmetric cell division and cell differentiation. Cells progress through these developmental changes guided by complex and layered genetic programs that lead to differential expression of the genome. To explore how a genetic program directs cell cycle progression, we examined the global activity of promoters at distinct stages of the cell cycle of the bacterium Caulobacter crescentus, that undergoes cellular differentiation and divides asymmetrically at each cell division. We found that approximately 21% of transcription start sites are cell cycle-regulated, driving the transcription of both mRNAs and non-coding and antisense RNAs. In addition, 102 cell cycle-regulated genes are transcribed from multiple promoters, allowing multiple regulatory inputs to control the logic of gene activation. We found combinatorial control by the five master transcription regulators that provide the core regulation for the genetic circuitry controlling the cell cycle. Much of this combinatorial control appears to be directed at refinement of temporal expression of various genes over the cell cycle, and at tighter control of asymmetric gene expression between the swarmer and stalked daughter cells.

Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression.

Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. We provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5′-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5′-RACE. Additionally, using ribosome profiling and LC-MS, we mapped translation start sites and coding regions with near complete coverage. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the coding DNA sequence (CDS). These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Overall 19% of the transcribed and translated genomic elements were newly identified or significantly improved by this approach, providing a valuable genomic resource to elucidate the complete C. crescentus genetic circuitry that controls asymmetric cell division., Author Summary Caulobacter crescentus is a model system for studying asymmetric cell division, a fundamental process that, through differential gene expression in the two daughter cells, enables the generation of cells with different fates. To explore how the genome directs and maintains asymmetry upon cell division, we performed a coordinated analysis of multiple genomic and proteomic datasets to identify the RNA and protein coding features in the C. crescentus genome. Our integrated analysis identifies many new genetic regulatory elements, adding significant regulatory complexity to the C. crescentus genome. Surprisingly, 75.4% of protein coding genes lack a canonical translation initiation sequence motif (the Shine-Dalgarno site) which hybridizes to the 3′ end of the ribosomal RNA allowing translation initiation. We find Shine-Dalgarno sites primarily inside of genes where they cause translating ribosomes to pause, possibly allowing nascent proteins to correctly fold. With our detailed map of genomic transcription and translation elements, a systems view of the genetic network that controls asymmetric cell division is within reach.

New technologies made possible by this sequence data, such as DNA microarrays, in combination with the small size and ease of genetic manipulation of bacteria, now make it possible to identify the complete genetic regulatory circuitry that controls the bacterial cell. Analysis of the global gene expression profile of the bacterial cell during its cell cycle, under conditions of environmental challenge, and during pathogen invasion of host organisms will provide an unprecedented understanding of the bacterial cell as an integrated system. This chapter addresses the use of microarrays for study of a wide range of microbiological problems with emphasis on the profoundly different results that this genome-wide technique provides relative to the analysis of single genes and conventional forward genetics. By assaying the response of all genes to a given genetic or environmental perturbation in parallel and simultaneously, the microarray results identify whole pathways or subroutines of the organism’s genetic regulatory circuitry. The immobilized arrays, or spots, of DNA are typically the products of PCR that generate amplicons ranging from a few hundred base pairs to several kilobases in length. Application of microarray-based genomic analysis to study the cell cycle of Caulobacter crescentus, has recently led to a dramatic increase in one's understanding of regulation of the bacterial cell cycle. Although microarrays were initially developed to analyze RNA levels, they can also be used to examine DNA samples.

This report presents full-genome evidence that bacterial cells use discrete transcription patterns to control cell cycle progression. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. We conclude that in bacteria, as in yeast, (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding proteins that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA member of the two-component signal transduction family, is directly or indirectly involved in the control of 26% of the cell cycle–regulated genes.

▪ Abstract Biochemical and genetic approaches have identified the molecular mechanisms of many genetic reactions, particularly in bacteria. Now a comparably detailed understanding is needed of how groupings of genes and related protein reactions interact to orchestrate cellular functions over the cell cycle, to implement preprogrammed cellular development, or to dynamically change a cell's processes and structures in response to environmental signals. Simulations using realistic, molecular-level models of genetic mechanisms and of signal transduction networks are needed to analyze dynamic behavior of multigene systems, to predict behavior of mutant circuits, and to identify the design principles applicable to design of genetic regulatory circuits. When the underlying design rules for regulatory circuits are understood, it will be far easier to recognize common circuit motifs, to identify functions of individual proteins in regulation, and to redesign circuits for altered functions.

In cellular regulatory networks, genetic activity is controlled by molecular signals that determine when and how often a given gene is transcribed. In genetically controlled pathways, the protein product encoded by one gene often regulates expression of other genes. The time delay, after activation of the first promoter, to reach an effective level to control the next promoter depends on the rate of protein accumulation. We have analyzed the chemical reactions controlling transcript initiation and translation termination in a single such “genetically coupled” link as a precursor to modeling networks constructed from many such links. Simulation of the processes of gene expression shows that proteins are produced from an activated promoter in short bursts of variable numbers of proteins that occur at random time intervals. As a result, there can be large differences in the time between successive events in regulatory cascades across a cell population. In addition, the random pattern of expression of competitive effectors can produce probabilistic outcomes in switching mechanisms that select between alternative regulatory paths. The result can be a partitioning of the cell population into different phenotypes as the cells follow different paths. There are numerous unexplained examples of phenotypic variations in isogenic populations of both prokaryotic and eukaryotic cells that may be the result of these stochastic gene expression mechanisms.

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.

Two recent papers report the de novo design of a functioning biological circuit using well-characterized genetic elements.(1,2) Gardner et al. designed and constructed a genetic toggle switch while Elowitz and Leibler built an oscillating genetic circuit. Both circuits were designed with the aid of mathematical models. These papers demonstrate progress towards the unification of theory and experiment in the study of genetic circuits. Comparison of the predicted and observed behavior of the circuits, however, shows that the models explain only some of the circuits' properties. Further study of the observed behaviors not predicted by the model would lead to new insight into the properties of genetic networks. BioEssays 22:507-509, 2000.

Genetic circuits can now be engineered that perform moderately complicated switching functions or exhibit predictable dynamical behavior. These ‘forward engineering’ techniques may have to be combined with directed evolution techniques to produce robustness comparable with naturally occurring circuits.

Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of an entire bacterial cell cycle system functioning as a state machine. Oscillating levels of a few temporally-controlled master regulator proteins in a cyclical circuit drive cell cycle progression. To a striking degree, the cell cycle regulation is a whole cell phenomenon. Phospho-signaling proteins and proteases dynamically deployed to specific locations on the cell wall are vital. An essential phospho-signaling system integral to the cell cycle circuitry is central to accomplishing asymmetric cell division.

The bacterial cell has less internal structure and genetic complexity than cells of eukaryotic organisms, yet it is a highly organized system that uses both temporal and spatial cues to drive its cell cycle. Key insights into bacterial regulatory programs that orchestrate cell cycle progression have come from studies of Caulobacter crescentus, a bacterium that divides asymmetrically. Three global regulatory proteins cycle out of phase with one another and drive cell cycle progression by directly controlling the expression of 200 cell-cycle-regulated genes. Exploration of this system provided insights into the evolution of regulatory circuits and the plasticity of circuit structure. The temporal expression of the modular subsystems that implement the cell cycle and asymmetric cell division is also coordinated by differential DNA methylation, regulated proteolysis, and phosphorylation signaling cascades. This control system structure has parallels to eukaryotic cell cycle control architecture. Remarkably, the transcriptional circuitry is dependent on three-dimensional dynamic deployment of key regulatory and signaling proteins. In addition, dynamically localized DNA-binding proteins ensure that DNA segregation is coupled to the timing and cellular position of the cytokinetic ring. Comparison to other organisms reveals conservation of cell cycle regulatory logic, even if regulatory proteins, themselves, are not conserved.

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show that Caulobacter crescentus makes use of and requires a dedicated mechanism to initiate chromosome segregation. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement.

Small non-coding RNAs (sRNAs) are active in many bacterial cell functions, including regulation of the cell's response to environmental challenges. We describe the identification of 27 novel Caulobacter crescentus sRNAs by analysis of RNA expression levels assayed using a tiled Caulobacter microarray and a protocol optimized for detection of sRNAs. The principal analysis method involved identification of sets of adjacent probes with unusually high correlation between the individual intergenic probes within the set, suggesting presence of a sRNA. Among the validated sRNAs, two are candidate transposase gene antisense RNAs. The expression of 10 of the sRNAs is regulated by either entry into stationary phase, carbon starvation, or rich versus minimal media. The expression of four of the novel sRNAs changes as the cell cycle progresses. One of these shares a promoter motif with several genes expressed at the swarmer-to-stalked cell transition; while another appears to be controlled by the CtrA global transcriptional regulator. The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome.

Using 62 probe-level datasets obtained with a custom-designed Caulobacter crescentus microarray chip, we identify transcriptional start sites of 769 genes, 53 of which are transcribed from multiple start sites. Transcriptional start sites are identified by analyzing probe signal cross-correlation matrices created from probe pairs tiled every 5 bp upstream of the genes. Signals from probes binding the same message are correlated. The contribution of each promoter for genes transcribed from multiple promoters is identified. Knowing the transcription start site enables targeted searching for regulatory-protein binding motifs in the promoter regions of genes with similar expression patterns. We identified 27 motifs, 17 of which share no similarity to the characterized motifs of other C. crescentus transcriptional regulators. Using these motifs, we predict coregulated genes. We verified novel promoter motifs that regulate stress-response genes, including those responding to uranium challenge, a stress-response sigma factor and a stress-response noncoding RNA.

A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus . The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

Analysis of genome-wide codon bias shows that only two parameters effectively differentiate the genome-wide codon bias of 100 eubacterial and archaeal organisms. The first parameter correlates with genome GC content, and the second parameter correlates with context-dependent nucleotide bias. Both of these parameters may be calculated from intergenic sequences. Therefore, genome-wide codon bias in eubacteria and archaea may be predicted from intergenic sequences that are not translated. When these two parameters are calculated for genes from nonmammalian eukaryotic organisms, genes from the same organism again have similar values, and genome-wide codon bias may also be predicted from intergenic sequences. In mammals, genes from the same organism are similar only in the second parameter, because GC content varies widely among isochores. Our results suggest that, in general, genome-wide codon bias is determined primarily by mutational processes that act throughout the genome, and only secondarily by selective forces acting on translated sequences.

Many molecules that control genetic regulatory circuits act at extremely low intracellular concentrations. Resultant fluctuations (noise) in reaction rates cause large random variation in rates of development, morphology and the instantaneous concentration of each molecular species in each cell. To achieve regulatory reliability in spite of this noise, cells use redundancy in genes as well as redundancy and extensive feedback in regulatory pathways. However, some regulatory mechanisms exploit this noise to randomize outcomes where variability is advantageous.

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.

Correctly localizing proteins is essential for many biological processes. Since bacteria lack organelles compartmentalizing transcription and translation, it is possible that the site of protein synthesis is organized around the location of the corresponding gene. This would provide a mechanism to create a high local concentration of a specific protein. Past work supports this hypothesis of complete spatial and temporal overlap between the positions of active ribosomes and the corresponding gene (model 1). However, other results support a conflicting model with separation (on the order of hundreds of nanometers) between the gene and its site of translation (model 2). The latter case also implies that transcription and translation do not occur simultaneously. The small size of bacteria prohibits using conventional fluorescence imaging to determine the positions of individual ribosomes. To overcome this, we apply the method of 3D super-resolution microscopy which reveals individual ribosome position with tens of nanometer precision. In order to distinguish between models 1 and 2, we combine 3D super-resolution microscopy with the translation level of individual genes mapped to cell position on a population of Caulobacter crescentus cells in the same developmental state. According to model 1, high spatial density of ribosomes correlates with the positions of highly translated genes on the chromosome. We quantify the level of correlation for individual cells and compare the correlation with simulated ribosome positions from model probability distributions. Combining high resolution single molecule information with translation profiling data reveals the underlying spatial heterogeneity of ribosome organization on a well-defined system.