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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 [alpha]-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 SdP 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. CtrA | feedback | genetic circuit doi/ 10.1073/pnas.1014395107

The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype. doi: 10.1128/JB.00440-10

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. [beta]-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription. doi: 10.1128/JB.00496-10

Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscopy images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor. doi: 10.1128/JB.00607-10

Small noncoding regulatory RNAs (sRNAs) play a key role in the posttranscriptional regulation of many bacterial genes. The genome of Caulobacter crescentus encodes at least 31 sRNAs, and 27 of these sRNAs are of unknown function. An overexpression screen for sRNA-induced growth inhibition along with sequence conservation in a related Caulobacter species led to the identification of a novel sRNA, CrfA, that is specifically induced upon carbon starvation. Twenty-seven genes were found to be strongly activated by CrfA accumulation. One-third of these target genes encode putative TonB-dependent receptors, suggesting CrfA plays a role in the surface modification of C. crescentus, facilitating the uptake of nutrients during periods of carbon starvation. The mechanism of CrfA-mediated gene activation was investigated for one of the genes predicted to encode a TonB-dependent receptor, CC3461. CrfA functions to stabilize the CC3461 transcript. Complementarity between a region of CrfA and the terminal region of the CC3461 5'-untranslated region (5'-UTR) and also the behavior of a deletion of this region and a site-specific base substitution and a 3-base deletion in the CrfA complementary sequence suggest that CrfA binds to a stem-loop structure upstream of the CC3461 Shine-Dalgarno sequence and stabilizes the transcript. doi: 10.1128/JB.00343-10

Despite its fundamental nature, bacterial chromosome segregation remains poorly understood. Viewing segregation as a single process caused multiple proposed mechanisms to appear in conflict and failed to explain how asymmetrically dividing bacteria break symmetry to move only one of their chromosomes. Here, we demonstrate that the ParA ATPase extends from one cell pole and pulls the chromosome by retracting upon association with the ParB DNA-binding protein. Surprisingly, ParA disruption has a specific effect on chromosome segregation that only perturbs the latter stages of this process. Using quantitative high-resolution imaging, we demonstrate that this specificity results from the multistep nature of chromosome translocation. We propose that Caulobacter chromosome segregation follows an ordered pathway of events with distinct functions and mechanisms. Initiation releases polar tethering of the origin of replication, distinction spatially differentiates the two chromosomes, and commitment irreversibly translocates the distal centromeric locus. Thus, much as eukaryotic mitosis involves a sequence of distinct subprocesses, Caulobacter cells also segregate their chromosomes through an orchestrated series of steps. We discuss how the multistep view of bacterial chromosome segregation can help to explain and reconcile outstanding puzzles and frame future investigation. Par system | quantitative image analysis | bacterial cell biology doi/ 10.1073/pnas.1005274107

Caulobacter crescentus initiates a single round of DNA replication during each cell cycle. Following the initiation of DNA replication, the essential CckA histidine kinase is activated by phosphorylation, which (via the ChpT phosphotransferase) enables the phosphorylation and activation of the CtrA global regulator. CtrA~P then blocks the reinitiation of replication while regulating the transcription of a large number of cell cycle-controlled genes. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Because CckA~P promotes the activation of CtrA, we addressed the question of what controls the temporal activation of CckA. We found that the initiation of DNA replication is a prerequisite for remodeling the new cell pole, which includes the localization of the DivL protein kinase to that pole and, consequently, the localization, autophosphorylation, and activation of CckA at that pole. Thus, CckA activation is dependent on polar remodeling and a DNA replication initiation checkpoint that is tightly integrated with the polar phospho-signaling cascade governing cell cycle progression. doi: 10.1128/JB.00468-10

The dimorphic bacterium Caulobacter crescentus has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between C. crescentus strain CB15 and its derivative NAI000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus, zwf, between lab-adapted C. crescentus and clinical isolates of Pseudomonas aeruginosa that have adapted to a human host during chronic infection. doi: 10.1128/JB.00255-10

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant Opolysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis. doi: 10.1128/JB.01371-09

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 pill 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 pill 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. automated fluorescence microscopy | high content screening | asymmetric cell division | systems biology | Caulobacter crescentus www.pnas.org/cgi/doi/10.1073/pnas.1000846107

The outer membrane of Gram-negative bacteria is an essential compartment containing a specific complement of lipids and proteins that constitute a protective, selective permeability barrier. Outer membrane [beta]-barrel proteins are assembled into the membrane by the essential hetero-oligomeric BAM complex, which contains the lipoprotein BamE. We have identified a homologue of BamE, encoded by CC1365, which is located in the outer membrane of the stalked alpha-proteobacterium Caulobacter crescentus. BamE associates with proteins whose homologues in other bacteria are known to participate in outer membrane protein assembly: BamA (CC1915), BamB (CC1653) and BamD (CC1984). Caulobacter cells lacking BamE grow slowly in rich medium and are hypersensitive to anionic detergents, some antibiotics and heat exposure, which suggest that the membrane integrity of the mutant is compromised. Membranes of the [DELTA]bamE mutant have normal amounts of the outer membrane protein RsaF, a TolC homologue, but are deficient in CpaC*, an aggregated form of the outer membrane secretin for type IV pili. [DELTA]bamE membranes also contain greatly reduced amounts of three TonB-dependent receptors that are abundant in wild-type cells. Cells lacking BamE have short stalks and are delayed in stalk outgrowth during the cell cycle. Based on these findings, we propose that Caulobacter BamE participates in the assembly of outer membrane [beta]-barrel proteins, including one or more substrates required for the initiation of stalk biogenesis. DOI 10.1099/mic.0.035055-0

In a developmental strategy designed to efficiently exploit and colonize sparse oligotrophic environments, Caulobacter crescentus cells divide asymmetrically, yielding a motile swarmer cell and a sessile stalked cell. After a relatively fixed time period under typical culture conditions, the swarmer cell differentiates into a replicative stalked cell. Since differentiation into the stalked cell type is irreversible, it is likely that environmental factors such as the availability of essential nutrients would influence the timing of the decision to abandon motility and adopt a sessile lifestyle. We measured two different parameters in nutrient-limited chemostat cultures, biomass concentration and the ratio of nonstalked to stalked cells, over a range of flow rates and found that nitrogen limitation significantly extended the swarmer cell life span. The transcriptional profiling experiments described here generate the first comprehensive picture of the global regulatory strategies used by an oligotroph when confronted with an environment where key macronutrients are sparse. The pattern of regulated gene expression in nitrogen- and carbon-limited cells shares some features in common with most copiotrophic organisms, but critical differences suggest that Caulobacter, and perhaps other oligotrophs, have evolved regulatory strategies to deal distinctly with their natural environments. We hypothesize that nitrogen limitation extends the swarmer cell lifetime by delaying the onset of a sequence of differentiation events, which when initiated by the correct combination of external environmental cues, sets the swarmer cell on a path to differentiate into a stalked cell within a fixed time period. doi: 10.1128/JB.01240-09

The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two c/s-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between [beta]-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression. doi: 10.1128/JB.00985-09

Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA~P exhibits high affinity (dissociation constant [[K.sub.d], < 10 nM) for consensus TTAA-N7-TrAA binding sites with 'typical' (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity ([K.sub.d], >500 nM) CtrA binding sites with 'atypical' (N [not equal to] 7) spacing. Footprints demonstrated that phosphorylated CtrA ~P does not exhibit increased affinity for 'atypical' sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 [micro]M) accumulate, which can drive CtrA binding to 'atypical' sites. In vivo cross-linking showed that when the stable CtrAA3 protein persists during the cell cycle, the 'atypical' sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA PI and P2 transcription is not altered by persistent CtrA[delta]3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA ~P phosphorylation that provides the dominant 'activation' signal. Protein dimerization is one potential means of 'activation.' The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the 'atypical' sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how 'atypical' sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

Caulobacter crescentus (CB15) initiates chromosome replication only in stalked cells and not in swarmers. To better understand this dimorphic control of chromosome replication, we isolated replication origins (oris) from freshwater Caulobacter (FWC) and marine Caulobacter (MCS) species. Previous studies implicated integration host factor (IHF) and CcrM DNA methylation sites in replication control. However, ori IHF and CcrM sites identified in the model FWC CB15 were only conserved among closely related FWCs. DnaA boxes and CtrA binding sites are established CB15 ori components. CtrA is a two-component regulator that blocks chromosome replication selectively in CB1 5 swarmers. DnaA boxes and CtrA sites were found in five FWC and three MCS oris. Usually, a DnaA box and a CtrA site were paired, suggesting that CtrA binding regulates DnaA activity. We tested this hypothesis by site-directed mutagenesis of an MCS10 ori which contains only one CtrA binding site overlapping a critical DnaA box. This overlapping site is unique in the whole MCS10 genome. Selective DnaA box mutations decreased replication, while selective CtrA binding site mutations increased replication of MCS10 ori plasmids. Therefore, both FWC and MCS oris use CtrA to repress replication. Despite this similarity, phylogenetic analysis unexpectedly shows that CtrA usage evolved separately among these Caulobacter oris. We discuss consensus oris and convergent ori evolution in differentiating bacteria.

Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated.

Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.

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. centromere | parS | ParA

Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter erescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.

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 methylationstate 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. Caulobacter | model | regulatory circuit | hybrid system | symbolic model checking

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alpha-proteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTC[N.sub.7]GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

In the oligotrophic freshwater bacterium Caulobacter crescentus, D-xylose induces expression of over 50 genes, including the xyl operon, which encodes key enzymes for xylose metabolism. The promoter ([P.sub.xylE]) controlling expression of the xyl operon is widely used as a tool for inducible heterologous gene expression in C. crescentus. We show here that [P.sub.xylX] and at least one other promoter in the xylose regulon ([P.sub.xylE]) are controlled by the CC3065 (xylR) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of D-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xyiose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding D-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as xylR mutants exhibit reduced expression of the Eutner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.

The branching order and coherence of the alphaproteobacterial orders have not been well established, and not all studies have agreed that mitochondria arose from within the Rickettsiales. A species tree for 72 alphaproteobacteria was produced from a concatenation of alignments for 104 well-behaved protein families. Coherence was upheld for four of the five orders with current standing that were represented here by more than one species. However, the family Hyphomonadaceae was split from the other Rhodobacterales, forming an expanded group with Caulobacterales that also included Parvularcula. The three earliest-branching alphaproteobacterial orders were the Rickettsiales, followed by the Rhodospirillales and then the Sphingomonadales. The principal uncertainty is whether the expanded Caulobacterales group is more closely associated with the Rhodobacterales or the Rhizobiales. The mitochondrial branch was placed within the Rickettsiales as a sister to the combined Anaplasmataceae and Rickettsiaceae, all subtended by the Pelagibacter branch. Pelagibacter genes will serve as useful additions to the bacterial outgroup in future evolutionary studies of mitochondrial genes, including those that have transferred to the eukaryotic nucleus.

Genetic data suggest that the oligotrophic freshwater bacterium Caulobacter crescentus metabolizes D-xylose through a pathway yielding [alpha]-ketoglutarate, comparable to the recently described L-arabinose degradation pathway of Azospirillum brasilense. Enzymes of the C. crescentus pathway, including an [NAD. sup.+] -dependent xylose dehydrogenase, are encoded in the xylose-inducible xylXABCD operon (CC0823-CC0819).

The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first ~225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaceharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first ~225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.

The article discusses the genetic network and integrated three-dimensional sensor/response systems, regulating the cell cycle and asymmetric cell division in the bacterium Caulobacter crecentus.

Misfolding and aggregation of protein molecules are major threats to all living organisms. Therefore, cells have evolved quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins. DnaK/DnaJ and GroES/ GroEL are the best-characterized molecular chaperone systems in bacteria. In Caulobacter crescentus these chaperone machines are the products of essential genes, which are both induced by heat shock and cell cycle regulated. In this work, we characterized the viabilities of conditional dnaKJ and groESL mutants under different types of environmental stress, as well as under normal physiological conditions. We observed that C. crescentus cells with GroES/EL depleted are quite resistant to heat shock, ethanol, and freezing but are sensitive to oxidative, saline, and osmotic stresses. In contrast, cells with DnaK/J depleted are not affected by the presence of high concentrations of hydrogen peroxide, NaCl, and sucrose but have a lower survival rate after heat shock, exposure to ethanol, and freezing and are unable to acquire thermotolerance. Cells lacking these chaperones also have morphological defects under normal growth conditions. The absence of GroE proteins results in long, pinched filamentous cells with several Z-rings, whereas cells lacking DnaK/J are only somewhat more elongated than normal predivisional cells, and most of them do not have Z-rings. These findings indicate that there is cell division arrest, which occurs at different stages depending on the chaperone machine affected. Thus, the two chaperone systems have distinct roles in stress responses and during cell cycle progression in C. crescentus.

The dimorphic prosthecate bacteria (DPB) are [alpha]-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

The terminus region of the Caulobacter crescentus chromosome and the dif chromosome dimer resolution site were characterized. The Caulobacter genome contains skewed sequences that abruptly switch strands at dif and may have roles in chromosome maintenance and segregation. Absence of dif or the XerCD recombinase results in a chromosome segregation defect. The Caulobacter terminus region is unusual, since it contains many essential or highly expressed genes.

Significant strides were made towards understanding the molecular basis of cell cycle progression in the model bacterium Caulobacter crescentus, which is an alpha proteobacterium that dwells in all types of aqueous environments, including lakes, streams and oceans. Cell cycle regulation in Caulobacter is driven by complex genetic circuitry controlling the abundance or activity of master regulators via controlled synthesis and degradation.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Bacterial chromosome partitioning and cell division are tightly connected cellular processes. We show here that the Caulobacter crescentus FtsK protein localizes to the division plane, where it mediates multiple functions involved in chromosome segregation and cytokinesis. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. These results suggest that the interdependence between chromosome partitioning and cell division in Caulobacter is mediated, in part, by the FtsK protein.

The predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter. actin | MreB | MreC | Pbp2

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.

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated [Fe.sup.3+] and vitamin [B.sub.12]--the substrates hitherto known to be transported by TonB-dependent transporters--the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-[K.sub.d]a protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [[sup.14]C]maltodextrins from [[sup.14]C]maltose to [[sup.14]C]maltopentaose. [[sup.14]C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a [K.sub.d] of 0.2 [micro]M, while the second transport had a [K.sub.d] of 5 [micro]M. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 [micro]M carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [[sup.14]C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on [Fe.sup.3+]-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since [Fe.sup.3+]-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamb mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with [K.sub.d] values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin [B.sub.12] and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin [B.sub.12] has been demonstrated.

The FliX/FlbD-dependent temporal transcription of late flagellar genes in Caulobacter crescentus requires the assembly of an early, class II-encoded flagellar structure. Class II flagellar-mutant strains exhibit a delay in the completion of cell division, with the accumulation of filamentous cells in culture. It is shown here that this cell-division defect is attributable to an arrest in the final stages of cell separation. Normal cell morphology could be restored in class II mutants by gain-of-function alleles of FliX or FlbD, suggesting that the timely completion of cell division requires these trans-acting factors. In synchronized cultures, inhibition of cell division by depleting FtsZ resulted in normal initial expression of the late, FlbD-dependent fliK gene; however, the cell cycle-regulated cessation of transcription was delayed, indicating that cell division may be required to negatively regulate FlbD activity. Interestingly, prolonged depletion of FtsZ resulted in an eventual loss of FlbD activity that could be bypassed by a constitutive mutant of FlbD, but not of FliX, suggesting the possible existence of a second cell cycle-dependent pathway for FlbD activation.

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.

The impossibility of obtaining a rho null mutant and sensitivity to bicyclomyein have indicated that rho is essential for the viability of Caulobacter crescentus. Transcription gene fusions of sequences with serial deletions of the rho 5' untranslated region (5'-UTR) with a lacZ reporter gene indicated that rho is autoregulated at the level of attenuation of transcription in the 5'-UTR.

In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the [[sigma].sup.54]-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the [beta]-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The [DELTA]ihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a wild-type cell cycle-regulated pattern of expression in the absence of IHF. Thus, IHF is required for maximal transcription of several late flagellar genes. The [DELTA]ihfB strain was found to express significantly reduced amounts of the class 1V flageilin, FljL, as a consequence of reduced transcriptional activity. Our results indicate that the motility defect exhibited by the [DELTA]ihfB strain is most likely attributable to its failure to accumulate the class IV-encoded 27-kDa flagellin subunit, FljL.

Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes [rsaF.sub.a] and [rsaF.sub.a] were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The [rsaF.sub.a] gene is located several kilobases downstream of the other transporter genes, while [rsaF.sub.b] is completely unlinked. An [rsaF.sub.a] knockout had ~56% secretion compared to wild-type levels, while the [rsaF.sub.b] knockout reduced secretion levels to ~79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of ~9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaF, (with normal [rsaF.sub.b] levels) in concert with overexpression of [rsa.sub.A] resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.

A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a [[sigma].sup.32]-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.

NMR structure and [Ca.sup.2+] binding properties of a [beta][gamma]-crystallin domain, hahellin from Hahella chejuensis are described. The study identified a new class of natively unfolded [beta][gamma]-crystallins and provided insight into the commencement of the possible functional roles of such proteins in hahellin superfamily.

Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 are cloned, expressed and purified to homogeneity. The structure of Cc2672 from Caulobacter crecentus, an evolutionarily related protein, is determined as a complex with the N-methylphosphonate derivative of L-arginine.

A distinct UDP-2,3-diacylglucosamine pyrophosphatase (LpxI) is described, which has no sequence similarity to LpxH but has generated the same products by a different route. When the Caulobacter crescentus LpxI (CcLpxI) reaction is conducted in the presence of [H.sub.2][super 18]O, the [super 18]O is incorporated into the 2,3-diacylglucosamine 1-phosphate product, indicating that CcLpxI has catalyzed the attack of water on the [beta]-P atom of UDP-2,3-diacylglucosamine.

A study was conducted to examine the ability of Caulobacter crescentus to utilize lactose and to gain insight into the metabolic capacities of oligotrophic bacteria. The involvement of dehydrogenase in the metabolism of varied sugars provides insight into the ability of C. crescentus to scavenge for scarce nutrients and suggested that degradation of lactose and other sugars in C. crescentus might resemble a proposed pathway in Agrobacterium tumefaciens.

A crystal structure of a chromosomally encoded ParD-ParE complex (family of toxin-antitoxin (TA) system) from Caulobacter crescentus at 2.6 [Angstrom] resolution is presented. The dimeric, stably folded, and higly helical state of ParD antitoxin when not bound to ParE toxin indicated that the paradigmatic model in which antitoxin undergoes a disorder-to-order transition upon toxin binding does not apply to the chromosomal ParD-ParE TA system.