Noriko Ohta

Complete genome sequence of Caulobacter crescentus

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living α-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Sialic acid contents and controls of normal and malignant cells

The sialic acid contents of normal and malignant cells have been determined by two procedures. Three lines of virus-transformed cells had consistently lower contents than normal cells. These same lines showed clear differences in contact inhibition of growth. The first enzyme of sialic acid biosynthesis, l-glutamine: d-fructose-6-phosphate aminotransferase (EC 2.6.1.16) was determined also. No significant differences between the kinetic parameters for activity or feedback inhibition by uridine diphospho-N-acetylglucosamine was found. It is concluded that there is no positive correlation between sialic acid content and loss of contact inhibition in the cell lines examined.

The Core Dimerization Domains of Histidine Kinases Contain Recognition Specificity for the Cognate Response Regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.

Signal transduction in the cell cycle regulation of Caulobacter differentiation.

Caulobacter crescentus differentiates to form a new cell type during asymmetric cell division. Recent results indicate that signal transduction pathways mediated by protein kinases and essential response regulators play a central role in the regulation of development and cell division in response to cell cycle checkpoints.

Protein Sequences and Cellular Factors Required for Polar Localization of a Histidine Kinase in Caulobacter crescentus

The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle.

Crystallographic and Biochemical Studies of DivK Reveal Novel Features of an Essential Response Regulator in Caulobacter crescentus.

DivK is an essential response regulator in the Gram-negative bacterium Caulobacter crescentus and functions in a complex phosphorelay system that precisely controls the sequence of developmental events during the cell division cycle. Structure determinations of this single domain response regulator at different pH values demonstrated that the five-stranded α/β fold of the DivK protein is fully defined only at acidic pH. The crystal structures of the apoprotein and of metal-bound DivK complexes at higher pH values revealed a synergistic pH- and cation binding-induced flexibility of the β4-α4 loop and of the α4 helix. This motion increases the solvent accessibility of the single cysteine residue in the protein. Solution state studies demonstrated a 200-fold pH-dependent increase in the affinity of manganese for the protein between pH 6.0 and 8.5 that seems to involve deprotonation of an acido-basic couple. Taken together, these results suggest that flexibility of critical regions of the protein, ionization of the cysteine 99 residue and improved K D values for the catalytic metal ion are coupled events. We propose that the molecular events observed in the isolated protein may be required for DivK activation and that they may be achieved in vivothrough the specific protein-protein interactions between the response regulator and its cognate kinases.

Regulation of the Caulobacter crescentus rpoN gene and function of the purified σ54 in flagellar gene transcription

The sequential transcription off lagellar (fla) genes in the Caulobacter crescentus cell cycle is controlled by the organization of these genes in a regulatory hierarchy of four levels (I–IV). Level III and level IV genes at the bottom of the hierarchy are dependent on level II genes and are transcribed late in the cell cycle from σ54-dependent promoters. To study the regulation of genes at levels III and IV, we have isolated and sequenced the rpoN gene in order to analyze its expression, purified the rpoN gene product, and examined the role of the RpoN protein in initiation of transcription from σ54-dependent promoters. We report here epistasis experiments that show rpoN is required for transcription of level III genes, but that the expression of the rpoN gene itself is not dependent on any of the fla genes examined; these results place rpoN at level II near the top of the hierarchy. Consistent with this conclusion were nuclease S 1 assays that mapped the rpoN transcription start site and identified a sequence centered at −24, GTTA/TACCA/TT, which is similar to the core consensus sequence of the level IIB fliF, fliL, and fliQ promoters. We purified the full-length rpoN gene product to near homogeneity and demonstrated that the RpoN protein is required for transcription from the well-characterized σ54-dependent glnAp2 promoter of Escherichia coli and specifically recognizes the level III flbG gene promoter of C. crescentus. These last results confirm that rpoN encodes the C. crescentus σ54 factor and opens the way for the biochemical analysis of transcriptional regulation of level III and IV fla genes.

The role of polar localization in the function of an essential Caulobacter crescentus tyrosine kinase.

DivL is an essential tyrosine kinase in Caulobacter crescentus that controls an early step in the cell division cycle. We show here that DivL dynamically localizes to the stalk‐distal cell pole and less frequently to the stalked cell pole during the S‐phase. The kinase is subsequently released from the cell poles late in division and remains dispersed in the newly divided progeny stalk and swarmer cells. Mutational analysis of DivL in a DivL–GFP fusion protein demonstrated that the extreme C‐terminus and residues in the conserved four‐helix bundle, which is the phosphorylation–dimerization domain, are important for localization. We speculate that the four‐helix bundle of the core catalytic domain may serve as a recognition site for the ‘localization machinery’. Unexpectedly, a DivL protein with mutations in the C‐terminal localization sequence, and an intact catalytic domain, efficiently complemented a divL null mutation. Thus, subcellular localization of DivL is not essential to its function in cell division regulation. Regulation of cell division by DivL does, however, depend on its localization in the cell membrane.

Thermosensing Function of the Escherichia coli Redox Sensor Aer

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain).

Purification, Characterization, and Reconstitution of DNA-dependent RNA Polymerases from Caulobacter crescentus

Cell differentiation in the Caulobacter crescentus cell cycle requires differential gene expression that is regulated primarily at the transcriptional level. Until now, however, a defined in vitro transcription system for the biochemical study of developmentally regulated transcription factors had not been available in this bacterium. We report here the purification of C. crescentus RNA polymerase holoenzymes and resolution of the core RNA polymerase from holoenzymes by chromatography on single-stranded DNA cellulose. The three RNA polymerase holoenzymes Eς54, Eς32, and Eς73 were reconstituted exclusively from purifiedC. crescentus core and sigma factors. Reconstituted Eς54 initiated transcription from the ς54-dependent fljK promoter ofC. crescentus in the presence of the transcription activator FlbD, and active Eς32 specifically initiated transcription from the ς32-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the Eς73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coliand purified the full-length ς73 protein. The reconstituted Eς73 recognized the ς70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the ς73-dependent housekeeping promoters of theC. crescentus pleC and rsaA genes. The ability of the C. crescentus Eς73 RNA polymerase to recognize E. coliς70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.