Search

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.

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.

Principles of modular design are evident in signaling networks that detect and integrate a given signal and, depending on the organism in which the network module is present, transduce this signal to affect different metabolic or developmental pathways. Here we report a global transcriptional analysis of an oxygen sensory/signaling network in Caulobacter crescentus consisting of the sensor histidine kinase FixL, its cognate response regulator FixJ, the transcriptional regulator FixK, and the kinase inhibitor FixT. It is known that in rhizobial bacteria these proteins form a network that regulates transcription of genes required for symbiotic nitrogen fixation, anaerobic and microaerobic respiration, and hydrogen metabolism under hypoxic conditions. We have identified a positive feedback loop in this network and present evidence that the negative feedback regulator, FixT, acts to inhibit FixL by mimicking a response regulator. Overall, the core circuit topology of the Fix network is conserved between the rhizobia and C. crescentus , a free-living aerobe that cannot fix nitrogen, respire anaerobically, or metabolize hydrogen. In C. crescentus , the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. Thus, the Fix network is a conserved sensory/signaling module whose transcriptional output has been adapted to the unique physiologies of C. crescentus and the nitrogen-fixing rhizobia.

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed prefission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

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.

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus , this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.

Transcriptional regulatory circuits provide only a fraction of the signaling pathways and regulatory mechanisms that control the bacterial cell cycle. The CtrA regulatory network, important in control of the Caulobacter cell cycle, illustrates the critical role of nontranscriptional pathways and temporally and spatially localized regulatory proteins. The system architecture of Caulobacter cell-cycle control involves top-down control of modular functions by a small number of master regulatory proteins with cross-module signaling coordinating the overall process. Modeling the cell cycle probably requires a top-down modeling approach and a hybrid control system modeling paradigm to treat its combined discrete and continuous characteristics.

Bacteria are often highly polarized, exhibiting specialized structures at or near the ends of the cell. Among such structures are actin-organizing centers, which mediate the movement of certain pathogenic bacteria within the cytoplasm of an animal host cell; organized arrays of membrane receptors, which govern chemosensory behavior in swimming bacteria; and asymmetrically positioned septa, which generate specialized progeny in differentiating bacteria. This polarization is orchestrated by complex and dynamic changes in the subcellular localization of signal transduction and cytoskeleton proteins as well as of specific regions of the chromosome. Recent work has provided information on how dynamic subcellular localization occurs and how it is exploited by the bacterial cell. The main task of a bacterial cell is to survive and duplicate itself. The bacterium must replicate its genetic material and divide at the correct site in the cell and at the correct time in the cell cycle with high precision. Each kind of bacterium also executes its own strategy to find nutrients in its habitat and to cope with conditions of stress from its environment. This involves moving toward food, adapting to environmental extremes, and, in many cases, entering and exploiting a eukaryotic host. These activities often involve processes that take place at or near the poles of the cell. Here we explore some of the schemes bacteria use to orchestrate dynamic changes at their poles and how these polar events execute cellular functions. In spite of their small size, bacteria have a remarkably complex internal organization and external architecture. Bacterial cells are inherently asymmetric, some more obviously so than others. The most easily recognized asymmetries involve surface structures, e.g., flagella, pili, and stalks that are preferentially assembled at one pole by many bacteria. “New” poles generated at the cell division plane differ from old poles from the previous round of cell division. Even in Escherichia coli , which is generally thought to be symmetrical, old poles are more static than new poles with respect to cell wall assembly ( 1 ), and they differ in the deposition of phospholipid domains ( 2 ). There are many instances of differential polar functions; among these is the preferential use of old poles when attaching to host cells as in the interaction of Bradyrhizobium with plant root hairs ( 3 ) or the polar pili-mediated attachment of the Pseudomonas aeruginosa pathogen to tracheal epithelia ( 4 ). An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus . The gliding motility of this bacterium is propelled by a nozzle-like structure that squirts a polysaccharide-containing slime from the pole of the cell ( 5 ). Interestingly, M. xanthus , which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells.

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.

Environmental inorganic phosphorus is frequently the growth-limiting nutrient for aquatic microorganisms, and there is fierce interspecies competition for the available supply. What are the limiting factors in effective bacterial scavenging of environmental phosphorus? It depends on the concentration. When the supply is relatively high, the limit is determined by the number of phosphate transporters on the surface times their individual throughput. At very low concentrations, however, the physics of diffusion at small scales comes into play. A relatively sparse distribution of transporters on the cell surface can capture every molecule that approaches the surface and phosphate influx becomes diffusion-limited. The prosthecate (stalked) bacteria, such as Caulobacter crescentus, have evolved an effective fitness strategy for these low-phosphorus environments that are dissected by Wagner et al. (1) in this issue of PNAS. Long, slender stalks extend the effective surface area of the cell, acting as a sort of nutrient-collecting antenna while adding little to the cell volume (Fig. 1). Thus, the cell’s phosphate collection effectiveness is greatly increased at low cost. This hypothesis for the stalk’s function was suggested by observations 40 years ago that the prosthecate bacteria grow much longer stalks in phosphate-limited media (2, 3).

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.