Binary or Nonbinary Fission? Reproductive Mode of a Predatory Bacterium Depends on Prey Size

Abstract

Most bacteria, including model organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, reproduce by binary fission. However, some bacteria belonging to various lineages, including antibiotic-producing Streptomyces and predatory Bdellovibrio, proliferate by nonbinary fission, wherein three or more chromosome copies are synthesized and the resulting multinucleoid filamentous cell subdivides into progeny cells. Here, we demonstrate for the first time that the predatory bacterium Bdellovibrio bacteriovorus reproduces through both binary and nonbinary fission inside different prey bacteria. Switching between the two modes correlates with the prey size. In relatively small prey cells, B. bacteriovorus undergoes binary fission; the FtsZ ring assembles in the midcell, and the mother cell splits into two daughter cells. In larger prey cells, B. bacteriovorus switches to nonbinary fission and creates multiple asynchronously assembled FtsZ rings to produce three or more daughter cells. Completion of bacterial cell cycle critically depends on precise spatiotemporal coordination of chromosome replication with other cell cycle events, including cell division. We show that B. bacteriovorus always initiates chromosome replication at the invasive pole of the cell, but the spatiotemporal choreography of subsequent steps depends on the fission mode and/or the number of progeny cells. In nonbinary dividing filaments producing five or more progeny cells, the last round(s) of replication may also be initiated at the noninvasive pole. Altogether, we find that B. bacteriovorus reproduces through bimodal fission and that extracellular factors, such as the prey size, can shape replication choreography, providing new insights about bacterial life cycles. IMPORTANCE Most eukaryotic and bacterial cells divide by binary fission, where one mother cell produces two progeny cells, or, rarely, by nonbinary fission. All bacteria studied to date use only one of these two reproduction modes. We demonstrate for the first time that a predatory bacterium, Bdellovibrio bacteriovorus, exhibits bimodal fission and the mode of division depends on the size of the prey bacterium inside which B. bacteriovorus grows. This work provides key insights into the mode and dynamics of B. bacteriovorus proliferation in different pathogens that pose a major threat to human health due to their emerging antibiotic resistance (Proteus mirabilis, Salmonella enterica, and Shigella flexneri). The use of predatory bacteria such as B. bacteriovorus is currently regarded as a promising strategy to kill antibiotic-resistant pathogens. We find that B. bacteriovorus employs different chromosome replication choreographies and division modes when preying on those pathogens. Our findings may facilitate the design of efficient pathogen elimination strategies.

Keywords: Bdellovibrio bacteriovorus; FtsZ; cell cycle; chromosome replication; predator.

FIG 1

Number of progeny cells of B. bacteriovorus formed in different preys (n = 100 for each prey).

FIG 2

Spatiotemporal analysis of chromosome replication in a B. bacteriovorus cell growing in P. mirabilis. Time-lapse analysis of a representative B. bacteriovorus cell showing the localization of replisome(s) (green) in a predatory cell growing inside the P. mirabilis bdelloplast. (A) Attachment of B. bacteriovorus to a P. mirabilis cell. (B) Bdelloplast formation; time = 0 min. (C) Appearance of the first DnaN-mNeonGreen focus at the invasive pole of a B. bacteriovorus cell, indicating initiation of chromosome replication. (D to G) Further steps of chromosome replication, including splitting of replication forks (the appearance of the second DnaN-mNeonGreen focus at the former flagellar pole) and merging of replication forks at the midcell. (H) Termination of chromosome replication (disassembly of replisomes) and formation of two progeny cells. Photos represent merged bright-field and green fluorescence images. Scale bar = 1 μm. The full time-lapse is shown in Video S1 at https://figshare.com/authors/Jolanta_Zakrzewska-Czerwinska/14854612.

FIG 3

Time-lapse analysis of chromosome replication and oriC segregation in a B. bacteriovorus cell growing in P. mirabilis. Localization of replisome(s) (red) and oriC (i.e., ParB complex) (green) in a predatory B. bacteriovorus cell growing inside a P. mirabilis bdelloplast. (A) Attachment of B. bacteriovorus to a P. mirabilis cell. (B) Bdelloplast formation; time = 0 min. (C) Appearance of the first DnaN-mCherry focus at the invasive pole of a B. bacteriovorus cell, indicating initiation of chromosome replication. (D to G) Duplication of the oriC region (i.e., mNeonGreen-ParB focus) at the invasive pole; thereafter, one of the two oriC regions migrates to the opposite pole and remains there until the end of the cell cycle. Further steps of chromosome replication include splitting of replication forks (the appearance of the second DnaN-mCherry focus at the former flagellar pole) and merging of replication forks at the midcell. (H) Termination of chromosome replication (disassembly of replisomes) and formation of two progeny cells. Photos represent merged bright-field and fluorescence (red and green) images. Scale bar = 1 μm. The full time-lapse is shown in Video S2 at https://figshare.com/authors/Jolanta_Zakrzewska-Czerwinska/14854612.

FIG 4

Conversion of the flagellated pole of the mother cell into an invasive pole inherited by a daughter cell during B. bacteriovorus proliferation in P. mirabilis. Time-lapse microscopy of B. bacteriovorus growing inside P. mirabilis shows that the flagellated pole of the mother cell (violet arrow) becomes an invasive pole (orange arrow) that is inherited by the daughter cell. The newly formed invasive pole has a visible ParB complex (i.e., oriC region) and is functional since the daughter cell attacks the next P. mirabilis cell through that pole. Photos represent merged bright-field images. The B. bacteriovorus cell and the bdelloplast are marked by yellow and blue dotted lines, respectively. Scale bar = 1 μm. The full time-lapse is shown in Video S3 at https://figshare.com/authors/Jolanta_Zakrzewska-Czerwinska/14854612.

FIG 5

Dynamics of chromosome replication and oriC segregation in a B. bacteriovorus cell growing in S. enterica: formation of three daughter cells. (A) Attachment of B. bacteriovorus to a S. enterica cell. (B) Bdelloplast formation; time = 0 min. (C) Appearance of the first DnaN-mCherry focus at the invasive pole of a B. bacteriovorus cell, indicating initiation of chromosome replication. (D) Duplication of the oriC region (i.e., mNeonGreen-ParB focus) at the invasive pole; thereafter, one of the two oriC regions migrates to the opposite pole and one of the two DnaN-mCherry foci follows behind the newly replicated oriC region that is already attached to the opposite pole. (E and F) Reinitiation of DNA replication from the mother chromosome located at the invasive pole, migration of both replisomes toward the opposite pole, and the appearance of the third ParB focus at the middle of the filament. (G) Disappearance of the DnaN-mCherry focus located near the former flagellar pole, indicating termination of the first-round DNA replication. (H) Termination of the second round of chromosome replication and formation of three daughter cells. Photos represent merged bright-field and fluorescence (red and green) images. The B. bacteriovorus cell and the bdelloplast are marked by yellow and blue dotted lines, respectively. Scale bar = 1 μm. The full time-lapse is shown in Video S4 at https://figshare.com/authors/Jolanta_Zakrzewska-Czerwinska/14854612.

FIG 6

Dynamics of chromosome replication and oriC segregation in a B. bacteriovorus cell during proliferation in S. flexneri. (A) Attachment of B. bacteriovorus to an S. flexneri cell. (B) Bdelloplast formation; time = 0 min. (C) Appearance of the first DnaN-mCherry focus at the invasive pole of a B. bacteriovorus cell, indicating initiation of chromosome replication. (D and E) Replisome follows the newly replicated oriC region, indicating the progress of DNA replication. The appearance of a second DnaN-mCherry focus (reinitiation of chromosome replication), movement of the two replisomes toward opposite poles, and development of the third mNeonGreen-ParB focus in the middle of the filament. (F and G) Reinitiation of DNA replication from the chromosome located at the former flagellar pole. Appearance of another ParB signals reflects the emergence of a newly replicated oriC region. (H) Termination of DNA replication. Photos represent merged bright-field and fluorescence (red and green) images. The B. bacteriovorus cell and the bdelloplast are marked by yellow and blue dotted lines, respectively. Scale bar = 1 μm. The full time-lapse is shown in Video S6 at https://figshare.com/authors/Jolanta_Zakrzewska-Czerwinska/14854612.

FIG 7

Replisomes dynamics in relation to cell division in a B. bacteriovorus cell growing inside different preys. Localization of replisome(s) (red) and divisiome(s) (i.e., FtsZ) (green) in a B. bacteriovorus cell growing inside P. mirabilis, S. enterica, and S. flexneri. Time = 0 min; bdelloplast formation. Photos represent merged bright-field and fluorescence (red and green) images. Scale bar = 1 μm. The full time-lapse is shown in Video S7 to S9 at https://figshare.com/authors/Jolanta_Zakrzewska-Czerwinska/14854612.

FIG 8

Binary and nonbinary proliferation of Bdellovibrio bacteriovorus in preys differing in size. (A) Choreography of DNA replication initiation and modes of cell division. B. bacteriovorus depending on a prey size divides either by binary (in small prey cells) or by nonbinary fission (in larger prey cells) producing two or more daughter cells, respectively. The chromosome replication, regardless of the mode of cell division, is always initiated at the invasive pole. In larger preys (when 5 or more progeny cells are formed), the last round(s) of chromosome replication may also be initiated at the cell pole, which is opposite to the invasive pole. (B) Cell cycle of B. bacteriovorus dividing by binary fission, highlighting the dynamics of chromosome replication and cell division. The chromosome replication is initiated at the invasive pole and then one of the newly replicated oriC regions is segregated toward the flagellated pole. During the progress of DNA replication, the replication forks split (one replisome follows the segregated oriC region) and merge at the midcell and then disassembled. The FtsZ ring assembly is initiated at the midcell before the termination of DNA replication, and next the filament is divided into two progeny cells.