Chromosome replication and segregation govern the biogenesis and inheritance of inorganic polyphosphate granules

Abstract

Prokaryotes and eukaryotes synthesize long chains of orthophosphate, known as polyphosphate (polyP), which form dense granules within the cell. PolyP regulates myriad cellular functions and is often localized to specific subcellular addresses through mechanisms that remain undefined. In this study, we present a molecular-level analysis of polyP subcellular localization in the model bacterium Caulobacter crescentus. We demonstrate that biogenesis and localization of polyP is controlled as a function of the cell cycle, which ensures regular partitioning of granules between mother and daughter. The enzyme polyphosphate kinase 1 (Ppk1) is required for granule production, colocalizes with granules, and dynamically localizes to the sites of new granule synthesis in nascent daughter cells. Localization of Ppk1 within the cell requires an intact catalytic active site and a short, positively charged tail at the C-terminus of the protein. The processes of chromosome replication and segregation govern both the number and position of Ppk1/polyP complexes within the cell. We propose a multistep model in which the chromosome establishes sites of polyP coalescence, which recruit Ppk1 to promote the in situ synthesis of large granules. These findings underscore the importance of both chromosome dynamics and discrete protein localization as organizing factors in bacterial cell biology.

FIGURE 1:

ppk1 is required for polyP granule production and survival in stationary phase. (A) Representative micrographs showing DAPI-stained log-phase WT C. crescentus and ∆ppk1 cells. Scale bars for this and subsequent micrographs, 1 μm. DAPI (polyP) images were taken with the same exposure and equally scaled for comparison. (B) Loss in viability of WT and ∆ppk1 complemented with xylose-inducible ppk1 (ppk1++) or an empty vector (EV) in M2X minimal medium after 24-h shaking in stationary phase. CFU/ml for three biological replicates of each strain plated upon entry to stationary phase and after 24-h culture in stationary phase; log change in CFU/ml from the initial plating to the 24-h plating is expressed on the y-axis. Dotted line, limit of detection (5 CFU/ml). Error bars, SE of the mean. *p < 0.001, Student's t test comparing the indicated strain with WT empty vector control (EV). Because zero CFU were isolated from ∆ppk1 EV cultures, error bars represent SE of the initial viability.

FIGURE 2:

Granule production is cell cycle regulated and spatially organized. (A) Log-phase WT cells were synchronized, and samples were taken every 25 min and stained with DAPI for 25 min. Representative micrographs show cells at t = 25, 75, and 125 min postsynchrony, which includes staining time. (B) DAPI-staining foci per cell were manually enumerated from micrographs using ImageJ at each time point for two independent biological replicates. Error bars, SE of the mean. (C) MicrobeTracker was used to quantify the position of each granule within all cells in an image series. Each data point represents the position of a single DAPI-staining granule, plotted as a function of its displacement from the mid-cell along the cell's long axis vs. the total cell length. Dashed lines, the maximum position a granule can occupy, that is, the farthest possible distance from the mid-cell before leaving the detected cell boundary. Positive or negative displacements are arbitrary designations, as poles were not differentiated in this experiment. Times postsynchrony are given in minutes. For each frame, N = 1000 cells. (D) Representative micrographs of two additional DAPI-stained Alphaproteobacteria, A. biprosthecum and R. capsulatus.

FIGURE 3:

Venus-Ppk1 forms clusters, colocalizes with polyP granules, and dynamically localizes over the cell cycle. (A) Representative micrographs of DAPI-stained C. crescentus expressing venus-ppk1 from the native chromosomal locus, as well as DAPI-stained E. coli ∆ppk1 expressing a plasmid-borne venus-ppk1(CC) fusion from an IPTG-inducible promoter (E. coli ∆ppk1 venus-ppk1++). In the merged image, Venus is depicted in the red channel, and DAPI (polyP) is depicted in green. (B) Time-lapse merged phase and fluorescence images of unstained venus-ppk1 growing on nutrient-agar pads. Black arrows, unambiguous Venus-Ppk1 foci, which are depicted in the red channel.

FIGURE 4:

Catalysis and a C-terminal tail are required for normal Venus-Ppk1 localization. (A) Representative micrographs of venus-ppk1(H434A)++ expressed in the WT or ∆ppk1 background. For merged images in A and C, Venus is depicted in the red channel and DAPI (polyP) in the green channel. (B) Positions of Venus-Ppk1 or Venus-Ppk1(H434A) expressed from the native locus in populations of cells 120 min postsynchrony. For A and B, N = 299 cells with detected foci. (C) Representative micrographs of venus-ppk1(∆CT16)++ expressed in the WT or ∆ppk1 background. (D) Positions of Venus-Ppk1(∆CT16) in populations of cells grown in M2G. Cells were imaged at 120 min postsynchrony. For C and D, N = 352 cells with detected foci.

FIGURE 5:

Chromosome replication and segregation are required for normal granule biogenesis and positioning. (A) Representative TEM micrographs of synchronized WT cells treated with either MMC or HU or untreated. Black arrows, polyP granules. Scale bars, 1 μm. (B) Manually measured distances between the stalked pole and the center of each granule, following the center of the long axis of the cell. For the untreated group, N = 106 cells; MMC, N = 112 cells; HU, N = 118 cells. Pie graphs represent the percentage of cells in each treatment group containing one (dark gray), two (light gray), or three or more granules (off-white), as observed by TEM. (C) Position of DAPI-staining granules in synchronized populations of cells at t = 125 min postsynchrony, using a temperature-sensitive allele of holB to block DNA synthesis. Left, N = 764 cells; right, N = 742 cells. (D) Position of DAPI-staining granules in synchronized populations of cells at t = 125 min postsynchrony, using a dominant-negative allele of parA to block chromosome segregation. Left, N = 914 cells; right, N = 813 cells.

FIGURE 6:

A model of polyP granule biogenesis in C. crescentus. (A) At the molecular level, Ppk1 enzyme (red circles) forms focal clusters. The positively charged Ppk1 C-terminal tail (+) and polyP catalytic activity are both required to properly localize these enzyme clusters, which in turn produce large, visible polyP granules (phosphate chains depicted as black Ps; PolyP granules depicted as green spheres). Groups of red circles represent Ppk1 clusters of unspecified stoichiometry. (B) At the cellular level, Ppk1 is localized to an existing granule in swarmer cells. As swarmers differentiate into stalked cells, the chromosome (blue ribbons) replicates, and one copy segregates to the nascent daughter cell. On segregation Ppk1 localizes to a new, chromosomally determined niche. A second granule is subsequently synthesized at this new subcellular address. Ppk1 can associate with either or both of these granules.