A novel nucleoid-associated protein coordinates chromosome replication and chromosome partition.

We searched for regulators of chromosome replication in the cell cycle model Caulobacter crescentus and found a novel DNA-binding protein (GapR) that selectively aids the initiation of chromosome replication and the initial steps of chromosome partitioning. The protein binds the chromosome origin of replication (Cori) and has higher-affinity binding to mutated Cori-DNA that increases Cori-plasmid replication in vivo. gapR gene expression is essential for normal rapid growth and sufficient GapR levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified dynamic DNA-binding distributions for GapR, with the strongest associations at the partitioning (parABS) locus near Cori. Using molecular-genetic and fluorescence microscopy experiments, we showed that GapR also promotes the first steps of chromosome partitioning, the initial separation of the duplicated parS loci following replication from Cori. This separation occurs before the parABS-dependent partitioning phase. Therefore, this early separation, whose mechanisms is not known, coincides with the poorly defined mechanism(s) that establishes chromosome asymmetry: C. crescentus chromosomes are partitioned to distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that GapR coordinates chromosome replication with asymmetry-establishing chromosome separation, noting that both roles are consistent with the phylogenetic restriction of GapR to asymmetrically dividing bacteria.

The Caulobacter crescentus Homolog of DnaA (HdaA) Also Regulates the Proteolysis of the Replication Initiator Protein DnaA.

UNLABELLED: It is not known how diverse bacteria regulate chromosome replication. Based on Escherichia coli studies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatory inactivation of DnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly related Caulobacter crescentus homolog HdaA also prevents extra chromosome replication (J. Collier and L. Shapiro, J Bacteriol 191:5706-5715, 2009, http://dx.doi.org/10.1128/JB.00525-09). To further study the HdaA/RIDA mechanism, we created a C. crescentus strain that shuts off hdaA transcription and rapidly clears HdaA protein. We confirm that HdaA prevents extra replication, since cells lacking HdaA accumulate extra chromosome DNA. DnaA binds nucleotides ATP and ADP, and our results are consistent with the established E. coli mechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlike E. coli DnaA, C. crescentus DnaA is also regulated by selective proteolysis. C. crescentus cells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-type C. crescentus cells remove all DnaA protein when they enter stationary phase. However, cells lacking HdaA retain stable DnaA protein even when they stop growing in nutrient-depleted medium that induces complete DnaA proteolysis in wild-type cells. Additional experiments argue for a distinct HdaA-dependent mechanism that selectively removes DnaA prior to stationary phase. Related freshwater Caulobacter species also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis. IMPORTANCE: Bacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based on Escherichia coli studies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly related Caulobacter crescentus homolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike the E. coli Hda, the C. crescentus HdaA also regulates DnaA proteolysis. Furthermore, this HdaA proteolysis acts in logarithmically growing and in stationary-phase cells and therefore in two very different physiological states. We argue that HdaA acts to help time chromosome replications in logarithmically growing cells and that it is an unexpected component of the programed entry into stationary phase.

The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites.

The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control.

Mycobacterium tuberculosis origin of replication and the promoter for immunodominant secreted antigen 85B are the targets of MtrA, the essential response regulator.

Efficient proliferation of Mycobacterium tuberculosis (Mtb) inside macrophage requires that the essential response regulator MtrA be optimally phosphorylated. However, the genomic targets of MtrA have not been identified. We show by chromatin immunoprecipitation and DNase I footprinting that the chromosomal origin of replication, oriC, and the promoter for the major secreted immunodominant antigen Ag85B, encoded by fbpB, are MtrA targets. DNase I footprinting analysis revealed that MtrA recognizes two direct repeats of GTCACAgcg-like sequences and that MtrA approximately P, the phosphorylated form of MtrA, binds preferentially to these targets. The oriC contains several MtrA motifs, and replacement of all motifs or of a single select motif with TATATA compromises the ability of oriC plasmids to maintain stable autonomous replication in wild type and MtrA-overproducing strains, indicating that the integrity of the MtrA motif is necessary for oriC replication. The expression of the fbpB gene is found to be down-regulated in Mtb cells upon infection when these cells overproduce wild type MtrA but not when they overproduce a nonphosphorylated MtrA, indicating that MtrA approximately P regulates fbpB expression. We propose that MtrA is a regulator of oriC replication and that the ability of MtrA to affect apparently unrelated targets, i.e. oriC and fbpB, reflects its main role as a coordinator between the proliferative and pathogenic functions of Mtb.

CtrA response regulator binding to the Caulobacter chromosome replication origin is required during nutrient and antibiotic stress as well as during cell cycle progression.

The Caulobacter crescentus chromosome replication origin (Cori) has five binding sites for CtrA, an OmpR/PhoB family 'response regulator'. CtrA is degraded in replicating 'stalked' cells but is abundant in the non-replicating 'swarmer' cells, where it was proposed to repress replication by binding to Cori. We systematically mutated all Cori CtrA binding sites, and examined their consequences in the contexts of autonomous Cori-plasmid replication and in the natural chromosome locus. Remarkably, the C. crescentus chromosome tolerates severe mutations in all five CtrA binding sites, demonstrating that CtrA is not essential for replication. Further physiological and cell cycle experiments more rigorously supported the original hypothesis that CtrA represses replication. However, our experiments argued against another hypothesis that residual and/or replenished CtrA protein in stalked cells might prevent extra or unscheduled chromosome replication before cell division. Surprisingly, we also demonstrated that Cori CtrA binding sites are very advantageous and can become essential when cells encounter nutrients and antibiotics. Therefore, the CtrA cell cycle regulator co-ordinates replication with viable cell growth in stressful and rapidly changing environments. We argue that this new role for CtrA provided the primary selective pressure for evolving control by CtrA.

CtrA, a global response regulator, uses a distinct second category of weak DNA binding sites for cell cycle transcription control in Caulobacter crescentus.

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA approximately P exhibits high affinity (dissociation constant [K(d)], <10 nM) for consensus TTAA-N7-TTAA binding sites with "typical" (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (K(d), >500 nM) CtrA binding sites with "atypical" (N not equal 7) spacing. Footprints demonstrated that phosphorylated CtrA approximately P does not exhibit increased affinity for "atypical" sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 microM) accumulate, which can drive CtrA binding to "atypical" sites. In vivo cross-linking showed that when the stable CtrADelta3 protein persists during the cell cycle, the "atypical" sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrADelta3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA approximately 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.

Crosstalk Regulation Between Bacterial Chromosome Replication and Chromosome Partitioning.

Despite much effort, the bacterial cell cycle has proved difficult to study and understand. Bacteria do not conform to the standard eukaryotic model of sequential cell-cycle phases. Instead, for example, bacteria overlap their phases of chromosome replication and chromosome partitioning. In "eukaryotic terms," bacteria simultaneously perform "S-phase" and "mitosis" whose coordination is absolutely required for rapid growth and survival. In this review, we focus on the signaling "crosstalk," meaning the signaling mechanisms that advantageously commit bacteria to start both chromosome replication and chromosome partitioning. After briefly reviewing the molecular mechanisms of replication and partitioning, we highlight the crosstalk research from Bacillus subtilis, Vibrio cholerae, and Caulobacter crescentus. As the initiator of chromosome replication, DnaA also mediates crosstalk in each of these model bacteria but not always in the same way. We next focus on the C. crescentus cell cycle and describe how it is revealing novel crosstalk mechanisms. Recent experiments show that the novel nucleoid associated protein GapR has a special role(s) in starting and separating the replicating chromosomes, so that upon asymmetric cell division, the new chromosomes acquire different fates in C. crescentus's distinct replicating and non-replicating cell types. The C. crescentus PopZ protein forms a special cell-pole organizing matrix that anchors the chromosomes through their centromere-like DNA sequences near the origin of replication. We also describe how PopZ anchors and interacts with several key cell-cycle regulators, thereby providing an organized subcellular environment for more novel crosstalk mechanisms.

Structures of GapR reveal a central channel which could accommodate B-DNA.

GapR is a nucleoid-associated protein required for the cell cycle of Caulobacter cresentus. We have determined new crystal structures of GapR to high resolution. As in a recently published structure, a GapR monomer folds into one long N-terminal α helix and two shorter α helices, and assembles into a tetrameric ring with a closed, positively charged, central channel. In contrast to the conclusions drawn from the published structures, we observe that the central channel of the tetramer presented here could freely accommodate B-DNA. Mutation of six conserved lysine residues lining the cavity and electrophoretic mobility gel shift experiments confirmed their role in DNA binding and the channel as the site of DNA binding. Although present in our crystals, DNA could not be observed in the electron density maps, suggesting that DNA binding is non-specific, which could be important for tetramer-ring translocation along the chromosome. In conjunction with previous GapR structures we propose a model for DNA binding and translocation that explains key published observations on GapR and its biological functions.

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

Glutamate at the phosphorylation site of response regulator CtrA provides essential activities without increasing DNA binding.

The essential response regulator CtrA controls the Caulobacter crescentus cell cycle and phosphorylated CtrA approximately P preferentially binds target DNA in vitro. The CtrA aspartate to glutamate (D51E) mutation mimics phosphorylated CtrA approximately P in vivo and rescues non-viable C.crescentus cells. However, we observe that the CtrA D51E and the unphosphorylated CtrA wild-type proteins have identical DNA affinities and produce identical DNase I protection footprints inside the C.crescentus replication origin. There fore, D51E promotes essential CtrA activities separate from increased DNA binding. Accordingly, we argue that CtrA protein recruitment to target DNA is not sufficient to regulate cell cycle progression.

Redefining bacterial origins of replication as centralized information processors.

In this review we stress the differences between eukaryotes and bacteria with respect to their different cell cycles, replication mechanisms and genome organizations. One of the most basic and underappreciated differences is that a bacterial chromosome uses only one ori while eukaryotic chromosome uses multiple oris. Consequently, eukaryotic oris work redundantly in a cell cycle divided into separate phases: First inactive replication proteins assemble on eukaryotic oris, and then they await conditions (in the separate "S-phase") that activate only the ori-bound and pre-assembled replication proteins. S-phase activation (without re-assembly) ensures that a eukaryotic ori "fires" (starts replication) only once and that each chromosome consistently duplicates only once per cell cycle. This precise chromosome duplication does not require precise multiple ori firing in S-phase. A eukaryotic ori can fire early, late or not at all. The single bacterial ori has no such margin for error and a comparable imprecision is lethal. Single ori usage is not more primitive; it is a totally different strategy that distinguishes bacteria. We further argue that strong evolutionary pressures created more sophisticated single ori systems because bacteria experience extreme and rapidly changing conditions. A bacterial ori must rapidly receive and process much information in "real-time" and not just in "cell cycle time." This redefinition of bacterial oris as centralized information processors makes at least two important predictions: First that bacterial oris use many and yet to be discovered control mechanisms and second that evolutionarily distinct bacteria will use many very distinct control mechanisms. We review recent literature that supports both predictions. We will highlight three key examples and describe how negative-feedback, phospho-relay, and chromosome-partitioning systems act to regulate chromosome replication. We also suggest future studies and discuss using replication proteins as novel antibiotic targets.

Comparative analysis of Caulobacter chromosome replication origins.

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 CB15 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.

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus.

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.

Control of chromosome replication in caulobacter crescentus.

Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Its chromosome replication origin (Cori) may be prototypical of the large and diverse class of alpha-proteobacteria. Cori has features that both affiliate and distinguish it from the Escherichia coli chromosome replication origin. For example, requirements for DnaA protein and RNA transcription affiliate both origins. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria.

A dual binding site for integration host factor and the response regulator CtrA inside the Caulobacter crescentus replication origin.

The response regulator CtrA controls chromosome replication by binding to five sites, a, b, c, d, and e, inside the Caulobacter crescentus replication origin (Cori). In this study, we demonstrate that integration host factor (IHF) binds Cori over the central CtrA binding site c. Surprisingly, IHF and CtrA share DNA recognition sequences. Rather than promoting cooperative binding, IHF binding hinders CtrA binding to site c and nearby site d. Unlike other CtrA binding sites, DNA mutations in the CtrA c/IHF site uniquely impair autonomous Cori plasmid replication. These mutations also alter transcription from distant promoters more than 100 bp away. When the CtrA c/IHF site was deleted from the chromosome, these cells grew slowly and became selectively intolerant to a CtrA phosphor-mimic allele (D51E). Since CtrA protein concentration decreases during the cell cycle as IHF protein concentration increases, we propose a model in which IHF displaces CtrA in order to bend Cori and promote efficient chromosome replication.

Conserved response regulator CtrA and IHF binding sites in the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii chromosomal replication origins.

CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA. CzcR expression partially compensates for developmental defects in ctrA mutant C. crescentus cells, and CzcR binds to all five CtrA binding sites in the C. crescentus replication origin. Conversely, CtrA binds to five similar sites in the putative R. prowazekii replication origin (oriRp). Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp. Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R. prowazekii.