The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites. Bacterial chromosomes are not static structures. They undergo dynamic topological changes during DNA replication, segregation, and transcription (1-5). For bacteria with circular chromosomes, replication initiates at a single origin of replication (ori) and proceeds bidirectionally toward the terminus of replication (ter) (6). During replication, the DNA double helix is unwound locally, introducing compensatory superhelicity and entanglements that are relieved by the action of topoisomerases (1). Before newly replicated DNA can be segregated, the two sister chromosomes are unlinked by topoisomerases, resolvases, and recombinases (2,7,8). After the completion of replication and segregation, each half of the predivisional cell contains one sister chromosome. The mechanism whereby the chromosomes are moved, positioned, and finally restructured before cell division is poorly understood.Fluorescence in situ hybridization (FISH) has been used to visualize distinct chromosomal loci in fixed bacterial cells (9). In addition, a technique to label chromosomal loci in live cells has been developed by using a lac repressor GFP hybrid protein (LacI-GFP) that binds to arrays of lac operator (lacO) sequences inserted at specific sites on the chromosome (10-12). By using this labeling technique along with time-lapse fluorescence microscopy (FM), both the Escherichia coli and the Bacillus subtilis ori have been shown to move rapidly toward the cell poles once DNA replication has initiated (13-15).Chromosome replication and segregation is coordinated with other events during the cell cycle, such as polar morphogenesis and cell division. In Caulobacter, DNA replication initiates once and only once per cell cycle and proceeds bidirectionally from a single origin (16,17). Caulobacter cell division is asymmetric, yielding a replicative stalked cell and a nonreplicative swarmer cell with polar pili and a polar flagellum (18)(19...

A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a ⌬podJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a twocomponent system.

Caulobacter crescentus divides asymmetrically into a swarmer cell and a stalked cell, a process that is governed by the imbalance in phosphorylated levels of the DivK cell fate determinant in the two cellular compartments. The asymmetric polar localization of the DivJ kinase results in its specific inheritance in the stalked daughter cell where it phosphorylates DivK. The mechanism for the polar positioning of DivJ is poorly understood. SpmX, an uncharacterized lysozyme homolog, is shown here to control DivJ localization and activation. In the absence of SpmX, DivJ is delocalized and dysfunctional, resulting in developmental defects caused by an insufficiency in phospho-DivK. While SpmX stimulates DivK phosphorylation in the stalked cell, unphosphorylated DivK in the swarmer cell activates an intricate transcriptional cascade that leads to the production of the spmX message. This event primes the swarmer cell for the impending transition into a stalked cell, a transition that is sparked by the abrupt accumulation and localization of SpmX to the future stalked cell pole. Localized SpmX then recruits and stimulates DivJ, and the resulting phospho-DivK implements the stalked cell fate. The dynamic interplay between SpmX and DivK is at the heart of the molecular circuitry that sustains the Caulobacter developmental cycle.[Keywords: Asymmetric cell division; cell fate determinant; polar protein localization; muramidase; kinase] Supplemental material is available at http://www.genesdev.org.

Zinc-finger domain transcriptional regulators regulate a myriad of functions in eukaryotes. Interestingly, ancestral versions (MucR) from Alpha-proteobacteria control bacterial virulence/symbiosis. Whether virulence regulators can also control cell cycle transcription is unknown. Here we report that MucR proteins implement a hitherto elusive primordial S→G1 transcriptional switch. After charting G1-specific promoters in the cell cycle model Caulobacter crescentus by comparative ChIP-seq, we use one such promoter as genetic proxy to unearth two MucR paralogs, MucR1/2, as constituents of a quadripartite and homeostatic regulatory module directing the S→G1 transcriptional switch. Surprisingly, MucR orthologues that regulate virulence and symbiosis gene transcription in Brucella, Agrobacterium or Sinorhizobium support this S→G1 switch in Caulobacter. Pan-genomic ChIP-seq analyses in Sinorhizobium and Caulobacter show that this module indeed targets orthologous genes. We propose that MucR proteins and possibly other virulence regulators primarily control bacterial cell cycle (G1-phase) transcription, rendering expression of target (virulence) genes periodic and in tune with the cell cycle.