DivIVA Controls Progeny Morphology and Diverse ParA Proteins Regulate Cell Division or Gliding Motility in Bdellovibrio bacteriovorus

Abstract

The predatory bacterium B. bacteriovorus grows and divides inside the periplasm of Gram-negative bacteria, forming a structure known as a bdelloplast. Cell division of predators inside the dead prey cell is not by binary fission but instead by synchronous division of a single elongated filamentous cell into odd or even numbers of progeny cells. Bdellovibrio replication and cell division processes are dependent on the finite level of nutrients available from inside the prey bacterium. The filamentous growth and division process of the predator maximizes the number of progeny produced by the finite nutrients in a way that binary fission could not. To learn more about such an unusual growth profile, we studied the role of DivIVA in the growing Bdellovibrio cell. This protein is well known for its link to polar cell growth and spore formation in Gram-positive bacteria, but little is known about its function in a predatory growth context. We show that DivIVA is expressed in the growing B. bacteriovorus cell and controls cell morphology during filamentous cell division, but not the number of progeny produced. Bacterial Two Hybrid (BTH) analysis shows DivIVA may interact with proteins that respond to metabolic indicators of amino-acid biosynthesis or changes in redox state. Such changes may be relevant signals to the predator, indicating the consumption of prey nutrients within the sealed bdelloplast environment. ParA, a chromosome segregation protein, also contributes to bacterial septation in many species. The B. bacteriovorus genome contains three ParA homologs; we identify a canonical ParAB pair required for predatory cell division and show a BTH interaction between a gene product encoded from the same operon as DivIVA with the canonical ParA. The remaining ParA proteins are both expressed in Bdellovibrio but are not required for predator cell division. Instead, one of these ParA proteins coordinates gliding motility, changing the frequency at which the cells reverse direction. Our work will prime further studies into how one bacterium can co-ordinate its cell division with the destruction of another bacterium that it dwells within.

Keywords: Bdellovibrio; DivIVA; ParAB; cell-morphology; predatory bacteria; septation.

FIGURE 1

Genomic context of the B. bacteriovorus divIVA_Bd gene and protein alignment of DivIVA_Bd with Gram-positive DivIVA homologs. (A) Map of the B. bacteriovorus divIVA gene and neighboring region. The divIVA_Bd gene is downstream of genes encoding a predicted yggS homolog (Bd0466 accession: NP_967454.1) and pyrroline-5-carboxylate reductase (Bd0465 accession: NP_967453.1) and upstream of a gene encoding an YggU homolog (Bd0463 accession: NP_967451.1). (B) Alignment of DivIVA_Bb with DivIVA homologs in Streptococcus pneumoniae (DivIVA_Spn; accession: AAC95445.1) and Bacillus subtilis (DivIVA_Bsub; accession: P71021.1). Sequences are colored and annotated based on the DivIVA_Bsub crystal structure. The blue bar under the sequences indicates the N terminal domain and the yellow bar, the C terminal domain. The A78 residue, conserved in DivIVA_Bd, was shown to be important for DivIVA function in B. subtilis, and an A78T substitution in S. pneumoniae DivIVA disturbed protein localization and perturbed interactions with FtsK and late-stage divisome components. Residues F17 and R18 are highlighted by an orange box and arrow underneath the green cross-link indicator.

FIGURE 2

(A) Phase and epifluorescence microscopy displaying the location of DivIVA_Bd tagged with mCherry. Localization was observed during invasion and growth within E. coli prey cells. The Bdellovibrio cytoplasm is constitutively fluorescent with Bd0064-mCerulean to visualize the cell within the bdelloplast. DivIVA-mCherry localizes to the poles of the Bdellovibrio, except at the 3–4 h timepoints, where it is less clear due to the growing filament extending and twisting in three dimensions as it elongates (see Supplementary Figure S1). Images are representative of three biological repeats. Scale bars are 2 μm. (B) Cellular position maps of fluorescent foci of DivIVA-mCherry detected by MicrobeJ. All of the cells detected (including free swimming attack phase cells) were measured at time 0, but only cells within bdelloplasts were measured at the other timepoints. Data are pooled from three independent experiments (N values of cells at each timepoint: T0- 845, T30- 110, T45–65, T1h- 226, and T2h- 120).

FIGURE 3

Morphological changes in the attack phase cells in B. bacteriovorusΔdivIVA_Bd deletion strain grown with E. coli S17-1. TEM images of (A) Wild-type HD100 (pSUP404.2) cells are long and slender, whilst ΔdivIVA_Bd (pSUP404.2) cells (B) are shorter and wider. (C) A rare a doublet cell of ΔdivIVA_Bd with pinched septum. (D) B. bacteriovorus HD100 wild-type attack phase cell. Cells were stained with 0.5% uranyl acetate. Scale bars are 2 μm.

FIGURE 4

ΔdivIVA_Bd strains are shorter and wider than wild type, and are partially restored through complementation. Bar charts showing the mean lengths (A) and widths (B) of B. bacteriovorus strains containing a pSUP404.2 plasmid either empty, or encoding DivIVA_Bd, or a mutant DivIVA_Bd(A78T). ΔdivIVA_Bd strains are significantly shorter (P < 0.001) than wild type, with partial restoration of length when divIVA_Bd is introduced on plasmid pSUP404.2 (P < 0.001). Average width of ΔdivIVA_Bd strains is significantly greater than wild type (P < 0.001) with partial restoration when complemented with divIVA_Bd (P < 0.05) or divIVA_Bd(A78T) (P < 0.01). Width measurements show that wild type HD100 containing a plasmid with divIVA_Bd are thinner (P < 0.01), and wider with divIVA_Bd(A78T) (P < 0.001), suggesting that in trans expression levels may perturb DivIVA function. n = 75 for each population and data are from three biological repeats (all significance calculated as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, using a t-test). Images were acquired through transmission electron microscopy and analyzed in SimplePCI.

FIGURE 5

Expression pattern of B. bacteriovorus HD100 parA-like genes, parB, bactofilin, divIVA_Bd, and its upstream genomic neighbors bd0465 and bd0466. Expression was assessed alongside control gene dnaK throughout the predatory life cycle using RT-PCR with transcript specific primers. RNA was prepared from identical volumes of infection cultures with B. bacteriovorus HD100 predator and E. coli S17-1 prey at time-points throughout the predatory life cycle. AP: RNA from attack-phase B. bacteriovorus, 15–45: RNA from 15–45 min time-points through the predatory cycle, 1–4 h: RNA from 1–4 h through the time-course, E. coli: E. coli S17-1 only template RNA, -ve: no template negative control, Gen: B. bacteriovorus HD100 genomic DNA template as positive control. Images are representative of two biological repeats.

FIGURE 6

β-galactosidase assay results for pairwise and library screening bacterial two hybrid interactions. Points represent average Miller Units of cotransformants (n = 16), error bar represent standard error. (A) shows significant interactions between DivIVA_Bd (Bd0464) and Bd0465, Bd0465 and ParA3 (Bd3906), and Bd0465 and Bd0466 (all P < 0.001) when compared to the negative control. (B) shows significant interactions between protein fragments encoded from BTH library plasmids, specifically partial proteins of Bd0548, Bd2106, Bd2107, and Bd3538, with DivIVA_Bd plasmids (all P < 0.001). (C) shows results for interactions between DivIVA_Bd and full length proteins Bd0548, Bd2106, Bd2107, and Bd3538 (***P < 0.001, **P < 0.01, and *P < 0.05). Data are from two biological repeats. Individual data points are presented in Supplementary Figure S5.

FIGURE 7

Cell length distributions for attack phase cells of wild type and ParA3-mTFP strains. (A) Distribution of cell lengths for B. bacteriovorus fliC1 merodiploid strain (“wild type” equivalent, green) and HD100 ParA3-mTFP (yellow) populations (n = 100). The mean is denoted by the dotted line. Representative TEM images of both populations are shown in (B). Scale bars are 1 μm and data are from three biological repeats.

FIGURE 8

Plot of prey luminescence decrease over time comparing the predation rates of B. bacteriovorus cells expressing C terminally, fluorescently tagged ParA1, ParA2, or ParA3. Predation rate is measured as the drop in luminescence from the prey as they are preyed upon by Bdellovibrio, compared to wild type equivalent strain B. bacteriovorus FliC1 merodiploid (blue) (n = 12 technical replicates per strain for three biological repeats). Only the tagged ParA3 strain (orange) is delayed in causing a logarithmic drop in luminescence (which represents killing of the luminescent prey) compared to the other 3 strains. Control: E. coli prey only. ***p < 0.001, **p < 0.01, *p < 0.05, and NS not significant by Mann-Whitney U test.