Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (Pvan) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (Pxyl). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

Temporally and spatially controlled master regulators drive the Caulobacter cell cycle by regulating the expression of >200 genes. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. We show here that a previously unidentified single domain-response regulator, CpdR, when in the unphosphorylated state, binds to ClpXP and, thereby, causes its localization to the cell pole. We further show that ClpXP localization is required for CtrA proteolysis. When CpdR is phosphorylated, ClpXP is delocalized, and CtrA is not degraded. Both CtrA and CpdR are phosphorylated via the same CckA histidine kinase phospho-signaling pathway, providing a reinforcing mechanism that simultaneously activates CtrA and prevents its degradation by delocalizing the CpdR͞ClpXP complex. In swarmer cells, CpdR is in the phosphorylated state, thus preventing ClpXP localization and CtrA degradation. As swarmer cells differentiate into stalked cells (G1͞S transition), unphosphorylated CpdR accumulates and is localized to the stalked cell pole, where it enables ClpXP localization and CtrA proteolysis, allowing the initiation of DNA replication. Dynamic protease localization mediated by a phosphosignaling pathway is a novel mechanism to integrate spatial and temporal control of bacterial cell cycle progression.Caulobacter ͉ ClpXP ͉ phosphorylation ͉ proteolysis ͉ temporal control

Using 62 probe-level datasets obtained with a custom-designed Caulobacter crescentus microarray chip, we identify transcriptional start sites of 769 genes, 53 of which are transcribed from multiple start sites. Transcriptional start sites are identified by analyzing probe signal cross-correlation matrices created from probe pairs tiled every 5 bp upstream of the genes. Signals from probes binding the same message are correlated. The contribution of each promoter for genes transcribed from multiple promoters is identified. Knowing the transcription start site enables targeted searching for regulatory-protein binding motifs in the promoter regions of genes with similar expression patterns. We identified 27 motifs, 17 of which share no similarity to the characterized motifs of other C. crescentus transcriptional regulators. Using these motifs, we predict coregulated genes. We verified novel promoter motifs that regulate stress-response genes, including those responding to uranium challenge, a stress-response sigma factor and a stress-response noncoding RNA.

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

Caulobacter crescentus integrates phospho-signaling pathways and transcription factor regulatory cascades to drive the cell cycle. Despite the essential role of the CckA histidine kinase in the control of cell cycle events, the factors that signal its activation at a specific time in the cell cycle have remained elusive. A conditional genetic screen for CckA mislocalization mutants, using automated fluorescence microscopy and an image processing platform, revealed that the essential DivL protein kinase promotes CckA localization, autophosphorylation, and activity at the new cell pole. The transient accumulation of DivL at the new cell pole, but not its kinase activity, is required for the localization and activation of CckA. Because DivL and CckA accumulate at the same cell pole after the initiation of DNA replication and were found to interact in vivo, we propose that DivL recruits CckA to the pole, thereby promoting its autophosphorylation and activity.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond. These lipids are found in animals and some bacteria and have proposed membrane organization, signaling, and antioxidant roles. We discovered the plasmanylethanolamine desaturase activity that is essential for vinyl ether bond formation in a bacterial enzyme, CarF, which is a homolog of the human enzyme TMEM189. CarF mediates light-induced carotenogenesis in Myxococcus xanthus, and plasmalogens participate in sensing photooxidative stress through singlet oxygen. TMEM189 and other animal homologs could functionally replace CarF in M. xanthus, and knockout of TMEM189 in a human cell line eliminated plasmalogens. Discovery of the human plasmanylethanolamine desaturase will spur further study of plasmalogen biogenesis, functions, and roles in disease.

Conditional expression of a gene is a powerful tool to study its function and is typically achieved by placing the gene under the control of an inducible promoter. There is, however, a dearth of such inducible systems in Myxococcus xanthus, a well-studied prokaryotic model for multicellular development, cell differentiation, motility, and light response and a promising source of secondary metabolites. The few available systems have limitations, and exogenously based ones are unavailable. Here, we describe two new, versatile inducible systems for conditional expression of genes in M. xanthus. One employs isopropyl-␤-D-thiogalactopyranoside (IPTG) as an inducer and is inspired by those successfully applied in some other bacteria. The other requires vanillate as an inducer and is based on the system developed originally for Caulobacter crescentus and recently adapted for mammalian cells. Both systems are robust, with essentially no expression in the absence of an inducer. Depending on the inducer and the amounts added, expression levels can be modulated such that either system can conditionally express genes, including ones that are essential and are required at high levels such as ftsZ. The two systems operate during vegetative growth as well as during M. xanthus development. Moreover, they can be used to simultaneously induce expression of distinct genes within the same cell. The conditional expression systems we describe substantially expand the genetic tool kit available for studying M. xanthus gene function and cellular biology.

Chromosome segregation is an essential cellular function in eukaryotic and prokaryotic cells. The ParABS system is a fundamental player for a mitosis-like process in chromosome partitioning in many bacterial species. This work shows that the social bacterium Myxococcus xanthus also uses the ParABS system for chromosome segregation. Its large prokaryotic genome of 9.1 Mb contains 22 parS sequences near the origin of replication, and it is shown here that M. xanthus ParB binds preferentially to a consensus parS sequence in vitro. ParB and ParA are essential for cell viability in M. xanthus as in Caulobacter crescentus, but unlike in many other bacteria. Absence of ParB results in anucleate cells, chromosome segregation defects and loss of viability. Analysis of ParA subcellular localization shows that it clusters at the poles in all cells, and in some, in the DNA-free cell division plane between two chromosomal DNA masses. This ParA localization pattern depends on ParB but not on FtsZ. ParB inhibits the nonspecific interaction of ParA with DNA, and ParA colocalizes with chromosomal DNA only when ParB is depleted. The subcellular localization of ParB suggests a single ParB-parS complex localized at the edge of the nucleoid, next to a polar ParA cluster, with a second ParB-parS complex migrating after the replication of parS takes place to the opposite nucleoid edge, next to the other polar ParA cluster.