Harley H. McAdams

It’s a noisy business! Genetic regulation at the nanomolar scale

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.

Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle

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.

Why and how bacteria localize proteins.

Despite their small size, bacteria have a remarkably intricate internal organization. Bacteria deploy proteins and protein complexes to particular locations and do so in a dynamic manner in lockstep with the organized deployment of their chromosome. The dynamic subcellular localization of protein complexes is an integral feature of regulatory processes of bacterial cells.

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.

The essential genome of a bacterium

Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non‐coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper‐saturated transposon mutagenesis coupled with high‐throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non‐coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti‐sigma factor. We identified all essential promoter elements for the cell cycle‐regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high‐resolution strategy used here is applicable to high‐throughput, full genome essentiality studies and large‐scale genetic perturbation experiments in a broad class of bacterial species.

High-throughput identification of transcription start sites, conserved promoter motifs and predicted regulons

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 Dynamically Localized Protease Complex and a Polar Specificity Factor Control a Cell Cycle Master Regulator

Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.

The evolution of genetic regulatory systems in bacteria

The genomes of bacterial species show enormous plasticity in the function of individual genes, in genome organization and in regulatory organization. Over millions of years, both bacterial genes and their genomes have been extensively reorganized and adapted so that bacteria occupy virtually every environmental niche on the earth. In addition, changes have occurred in the regulatory circuitry that controls cell operations, cell-cycle progression and responses to environmental signals. The mechanisms that underlie the adaptation of the bacterial regulatory circuitry are crucial for understanding the bacterial biosphere and have important roles in the emergence of antibiotic resistance.

A DNA methylation ratchet governs progression through a bacterial cell cycle

The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G1 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.

DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus

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.