Challenges Faced by Highly Polyploid Bacteria with Limits on DNA Inheritance

Abstract

Most studies of bacterial reproduction have centered on organisms that undergo binary fission. In these models, complete chromosome copies are segregated with great fidelity into two equivalent offspring cells. All genetic material is passed on to offspring, including new mutations and horizontally acquired sequences. However, some bacterial lineages employ diverse reproductive patterns that require management and segregation of more than two chromosome copies. Epulopiscium spp., and their close relatives within the Firmicutes phylum, are intestinal symbionts of surgeonfish (family Acanthuridae). Each of these giant (up to 0.6 mm long), cigar-shaped bacteria contains tens of thousands of chromosome copies. Epulopiscium spp. do not use binary fission but instead produce multiple intracellular offspring. Only ∼1% of the genetic material in an Epulopiscium sp. type B mother cell is directly inherited by its offspring cells. And yet, even in late stages of offspring development, mother-cell chromosome copies continue to replicate. Consequently, chromosomes take on a somatic or germline role. Epulopiscium sp. type B is a strict anaerobe and while it is an obligate symbiont, its host has a facultative association with this intestinal microorganism. Therefore, Epulopiscium sp. type B populations face several bottlenecks that could endanger their diversity and resilience. Multilocus sequence analyses revealed that recombination is important to diversification in populations of Epulopiscium sp. type B. By employing mechanisms common to others in the Firmicutes, the coordinated timing of mother-cell lysis, offspring development and congression may facilitate the substantial recombination observed in Epulopiscium sp. type B populations.

Keywords: Epulopiscium; comparative genomics; endospore; fish gut microbiota; intracellular offspring.

Fig. 1

Epulopiscium sp. type B is an obligate symbiont of the surgeonfish Naso tonganus. (A) Epulopiscium sp. type B, also known as “Ca. Epulopiscium viviparus.” The two large structures within this Epulopiscium are offspring. (B) The surgeonfish Naso tonganus inhabits tropical coral reef systems and its native range includes the Great Barrier Reef. Epulopiscium sp. type B is often, but not always, found in high numbers in the intestinal tract of N. tonganus. The photo of N. tonganus was taken by Kendall Clements.

Fig. 2

DNA in Epulopiscium sp. type B forms a network beneath the cytoplasmic membrane. These images show two different focal planes of the same DAPI-stained cell. The image on the left is a medial focal plane. To the right is a focal plane just under the cell surface. In large cells, most of the DNA is located in a layer at the cytoplasmic periphery, just under the cell membrane. The two bright structures at the poles of this cell are offspring. Note that only a portion of the mother-cell DNA is directly inherited. Scale bar represents 50 µm. Reprinted with permission from (Robinow and Angert 1998).

Fig. 3

The Epulopiscium spp. life cycle produces internal offspring once every 24 h. (A) The two polar offspring primordia are engulfed by the mother cell. (B) The polar offspring are contained within a membrane-bound compartment in the cytoplasm of the mother cell. (C) Offspring cells grow within the mother cell until they nearly fill the mother cell. (D) The offspring are released through a tear in the mother-cell envelop. The mother cell is killed in this process. In Epulopiscium sp. type B, asymmetric division usually occurs in offspring (to produce “granddaughter cells”) prior to their release from the mother cell.

Fig. 4

After intracellular offspring are formed, DNA continues to replicate in the terminally differentiated mother cell. To follow the fate of DNA in Epulopiscium cells, BrdU was introduced to live cells within the fish intestinal tract. Immunolocalization of the BrdU showed DNA replication had occurred within growing offspring cells but replication was also taking place in mother-cell DNA. Shown here is a medial optical section of the tip of a mother cell. BrdU immunolocalization is shown in green.

Fig. 5

Genetic diversity in Epulopiscium sp. type B populations is likely maintained by recombination. A model of inheritance of diverse DNA fragments via congression is shown here. Offspring emerging from their mother cells may be in the process of polar division or harbor pole-associated offspring primordia. Before engulfment is complete, the chromosomes within these primordia would be separated from the environment by only one cell membrane, as illustrated here. If like B. subtilis, DNA uptake proteins (competence proteins) assemble at cell poles, newly acquired DNA would be transported into the polar offspring primordia. In this image, fragments of DNA released by the synchronized lysis of diverse mother cells are shown as different colored double helices. Competence proteins are shown as purple transport complexes bringing in single-stranded DNA into the polar offspring. Cell membranes are show as black outlines, the cell envelop is shown in gray, and Epulopiscium nucleoids are shown as thick light blue lines.