The dynamic interplay between a cell fate determinant and a lysozyme homolog drives the asymmetric division cycle of <i>Caulobacter crescentus</i>

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 transposongenerated 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 pili 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 pili 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

Section: Discussionsupporting

confidence: 50%

Section: Hierarchical Clustering Identifies Functional Protein Localimentioning

confidence: 99%

High-throughput identification of protein localization dependency networks

Christen

Fero

Hillson

et al. 2010

“…Concomitant with the G1→S transition, the PleC-bearing swarmer pole is remodeled into a stalked pole, and polar PleC is substituted with DivJ. Thus, perturbations in the spatiotemporal dynamics of this system lead to alterations in the relative DivK∼P levels and a commensurate cell fate dysfunction (5). Such perturbations occur when the localization factor SpmX is inactivated (5).…”

mentioning

confidence: 99%

“…Thus, perturbations in the spatiotemporal dynamics of this system lead to alterations in the relative DivK∼P levels and a commensurate cell fate dysfunction (5). Such perturbations occur when the localization factor SpmX is inactivated (5). SpmX localizes to the stalked pole during the G1→S transition and recruits DivJ to this site, enhancing its activity and therefore promoting DivK∼P levels ( Fig.…”

mentioning

confidence: 99%

In-phase oscillation of global regulons is orchestrated by a pole-specific organizer

Janakiraman

Mignolet

Narayanan

et al. 2016

Self Cite

Cell fate determination in the asymmetric bacterium Caulobacter crescentus (Caulobacter) is triggered by the localization of the developmental regulator SpmX to the old (stalked) cell pole during the G1→S transition. Although SpmX is required to localize and activate the cell fate-determining kinase DivJ at the stalked pole in Caulobacter, in cousins such as Asticcacaulis, SpmX directs organelle (stalk) positioning and possibly other functions. We define the conserved σ 54 -dependent transcriptional activator TacA as a global regulator in Caulobacter whose activation by phosphorylation is indirectly down-regulated by SpmX. Using a combination of forward genetics and cytological screening, we uncover a previously uncharacterized and polarized component (SpmY) of the TacA phosphorylation control system, and we show that SpmY function and localization are conserved. Thus, SpmX organizes a site-specific, ancestral, and multifunctional regulatory hub integrating the in-phase oscillation of two global transcriptional regulators, CtrA (the master cell cycle transcriptional regulator A) and TacA, that perform important cell cycle functions.cell cycle | Caulobacter crescentus | σ 54 | SpmX | α-proteobacteria

“…Dissecting the underlying molecular mechanisms for localization is facilitated by the availability of suitable proteins that can be used as molecular probes. In the dimorphic Gramnegative bacterium Caulobacter crescentus localization probes for cellular organelles such as the medial cytokinetic apparatus, the cell-fate signaling hub at the old pole, and surface organelles positioned at the new pole (pili or the flagellum) led to the establishment of molecular localization hierarchies (2)(3)(4)(5). C. crescentus also features another polar surface organelle, the stalk, whose molecular anatomy is not well characterized.…”

mentioning

confidence: 99%

Protein localization and dynamics within a bacterial organelle

Hughes

Huitema

Pritchard

et al. 2010

Self Cite

Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium Caulobacter crescentus, the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that (i) the StpX transmembrane domain enables access to the stalk organelle, (ii) the N-terminal periplasmic domain mediates retention in the stalk, and (iii) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk.Caulobacter | fluorescence loss in photobleaching/fluorescence recovery after photobleaching | polar organelle | protein localization | protein mobility M echanisms exist in prokaryotic and eukaryotic cells to direct specialized proteins to distinct subcellular sites where they execute topologically constrained functions, for example, morphogenesis (1). Dissecting the underlying molecular mechanisms for localization is facilitated by the availability of suitable proteins that can be used as molecular probes. In the dimorphic Gramnegative bacterium Caulobacter crescentus localization probes for cellular organelles such as the medial cytokinetic apparatus, the cell-fate signaling hub at the old pole, and surface organelles positioned at the new pole (pili or the flagellum) led to the establishment of molecular localization hierarchies (2-5). C. crescentus also features another polar surface organelle, the stalk, whose molecular anatomy is not well characterized. Until recently, no molecular probes for the study of the stalk ultrastructure and biogenesis were known. Conducting a cytological survey of 75% of the predicted translation products of Caulobacter, Werner et al. uncovered candidate proteins that appear associated with the stalk (6). The stalk is a cylindrical protrusion of all envelope layers (inner membrane, periplasm, outer membrane, and S-layer) at the old cell pole and encloses cytoplasmic material that is free of chromosomal DNA, ribosomes, and most cytoplasmic proteins (7,8). The absence of ribosomes precludes a cotranslational protein targeting mechanism within the stalk. Instead, mobile molecules might diffuse into the stalk where they are subsequently retained, i.e.,...