Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 2003513827835Original ArticleIntracellular offspring of EpulopisciumE. R. Angert and K. D. Clements Molecular Microbiology (2004) 51(3), 827–835 doi:10.1046/j.1365-2958.2003.03869.x Initiation of intracellular offspring in Epulopiscium Esther R. Angert1* and Kendall D. Clements2 of surgeonfish (Family Acanthuridae) (Fishelson et al., 1Department of Microbiology, Cornell University, Ithaca, 1985; Montgomery and Pollak, 1988; Clements et al., NY, USA. 1989). The largest Epulopiscium cells are cigar shaped 2School of Biological Sciences, University of Auckland, and reach lengths in excess of 600 mm. Phylogenetic Auckland, New Zealand. analyses based on small subunit rRNA sequence compar- isons revealed that Epulopiscium spp. are members of the low G + C Gram-positive group of bacteria (Angert et al., Summary 1993), affiliated with Cluster XIVb of the clostridia (Collins Epulopiscium spp. are the largest heterotrophic bac- et al., 1994). A diagnostic feature of the genus Epulopis- teria yet described. A distinguishing feature of the cium is the ability of individuals to produce multiple, active, Epulopiscium group is their viviparous production of intracellular offspring. Depending on the strain, an individ- multiple, internal offspring as a means of cellular ual cell (mother cell) can produce 1–7 internal offspring reproduction. Based on their phylogenetic position, (daughter cells), but generally two are produced (Clem- among low G + C Gram-positive endospore-forming ents et al., 1989). Offspring primordia are closely associ- bacteria, and the remarkable morphological similarity ated with the tips of the mother cell but occasionally between developing endospores and Epulopiscium similar structures are seen associated with the internal offspring, we hypothesized that intracellular offspring side wall of a mother cell (Robinow and Angert, 1998). As production in Epulopiscium evolved from endospore offspring develop, they grow and elongate until they com- formation. These observations also raise the pos- pletely fill the mother cell cytoplasm. In a process that sibility that a cell with the capacity to form multiple destroys the mother cell, mature offspring burst through intracellular offspring was the ancestor of all contem- the outer surface layers of the mother cell (Montgomery porary endospore-forming bacteria. In an effort to and Pollak, 1988). Epulopiscium spp. are not available in characterize mechanisms common to both pro- culture. Based on observations of cells collected from host cesses, we describe the earliest stages of offspring fish (Acanthurus nigrofuscus) that were sacrificed at inter- formation in Epulopiscium. First, in anticipation of vals throughout the day and into the night it has been polar division, some of the mother cell DNA coalesces shown that Epulopiscium reproduction follows a circadian at the cell poles. FtsZ then localizes in a bipolar cycle (Montgomery and Pollak, 1988). As a result pattern and the cell divides. A portion of the pole- natural populations are synchronized with respect to associated DNA is trapped within the small cells development. formed by division at both poles. As development One of the closest known relatives of the Epulopiscium progresses, more pole-associated DNA is apparently group is the uncultured, endospore-forming bacterium packaged into the offspring primordia. These results Metabacterium polyspora (Angert et al., 1996). This bac- illustrate three mechanisms, the reorganization of cel- terium has the unusual ability to produce multiple, phase- lular DNA, asymmetric division and DNA packaging, bright endospores (Chatton and Pérard, 1913). Sporula- that are common to both endospore formation in tion in M. polyspora is coordinated with passage of the Bacillus subtilis and the production of active, intrac- bacterium through the gastrointestinal tract of its coproph- ellular offspring in Epulopiscium. Unlike most agous host, the guinea pig. Only mature endospores sur- endospore formers, Epulopiscium partitions only a vive passage through the mouth and stomach of the small proportion of mother cell DNA into the develop- guinea pig, finally germinating in the small intestine. Usu- ing offspring. ally these germinating cells have already initiated the next round of spore formation, bypassing vegetative division (binary fission) altogether. Endospores develop and Introduction mature within the guinea pig and are found in the faeces. Epulopiscium spp. are a predictable and conspicuous The ability of a single M. polyspora cell to produce multiple component of the intestinal microbiota of certain species endospores has become the primary means of propaga- tion for this organism when it is associated with its natural Accepted 8 October, 2003. *For correspondence. E-mail host (Angert and Losick, 1998). Endospore formation is [email protected]; Tel. (+1) 607 254 4778; Fax (+1) 607 255 3904. an essential part of the lifecycle of M. polyspora as it © 2003 Blackwell Publishing Ltd 828 E. R. Angert and K. D. Clements protects these strict anaerobes from the harsh environ- Bath et al., 2000; Errington et al., 2001; Sharp and mental conditions encountered outside the intestinal tract Pogliano, 2002). The forespore is then engulfed by the of the host. larger mother cell, forming a cell within a cell. Through a Daughter cell formation in Epulopiscium may represent series of highly regulated genetic programmes the fore- the next stage in the evolution of a novel form of cellular spore is prepared for dormancy (for review see Piggot and propagation. While each M. polyspora cell produces up to Coote, 1976; Errington, 1993; Stragier and Losick, 1996). nine intracellular offspring in the form of dormant Many of the mechanisms of endospore formation in B. endospores, an Epulopiscium cell produces active, not subtilis appear to be utilized for offspring production in quiescent, offspring (Montgomery and Pollak, 1988). Epulopiscium. If these processes are related, certain Based on phylogenetic position and morphological obser- essential genes involved in endospore formation and their vations we proposed that the process of intracellular off- functions should be conserved in Epulopiscium. Here we spring production in Epulopiscium has its evolutionary report immunolocalization of FtsZ and changes in the roots in endospore formation (Angert et al., 1996). A link distribution of DNA in Epulopiscium cells at the earliest between the production of active intracellular offspring stages in offspring formation. For the morphotype used and endospores is also seen in an unrelated lineage for this study, polar FtsZ rings form in large intracellular within the low G + C Gram-positive bacteria. Uncultured offspring before the release of those offspring from their intestinal symbionts of rodents, referred to as the ‘seg- mother cell. The bipolar localization of Z rings is preceded mented filamentous bacteria’, may alternatively produce by the coalescence of DNA at the poles of the cell. A either multiple live internal offspring or endospores portion of pole-associated DNA is trapped in the small (Chase and Erlandsen, 1976; Ferguson and Birch- cells formed at the poles during division. At later stages, Andersen, 1979; Snel et al., 1995). Early stages in the all pole-associated DNA appears to be packaged into the development of either of these forms of intracellular off- developing offspring. This evidence further supports the spring are morphologically indistinguishable. hypothesis that mechanisms used in endospore formation The process of endospore formation is best character- are conserved in Epulopiscium spp. and perform a similar ized in Bacillus subtilis (Piggot and Coote, 1976; Err- role in multiple offspring production. In addition, only a ington, 1993; Stragier and Losick, 1996). This provisional small portion of parental DNA is partitioned into the newly developmental programme allows B. subtilis and other formed offspring of Epulopiscium. This observation indi- endospore-forming bacteria to produce quiescent, stress- cates that the genome of Epulopiscium is present in many resistant cells thus preserving the genome during less thousands of copies in large cells. Medially positioned Z than favourable environmental conditions. In B. subtilis, rings, or other indicators of binary fission, were not endospores form in response to nutrient deprivation. The observed. process begins with cell remodelling in preparation for a modified form of binary fission. The two chromosomes of the cell consolidate into a sporulation-specific conforma- Results and discussion tion, referred to as the axial filament (Ryter et al., 1966; FtsZ localization Kay and Warren, 1968). This filament stretches along the long axis of the cell, extending from pole to pole. Another Epulopiscium spp. are not available in laboratory culture. key conversion involves changes in the localization of For these studies morphotype B Epulopiscium cells were FtsZ, a tubulin homologue and highly conserved compo- collected from surgeonfish, Naso tonganus, caught in the nent of the cell division apparatus of prokaryotes (Bi and wild (Clements et al., 1989). Remarkably, intracellular off- Lutkenhaus, 1991; Addinall et al., 1996; Margolin, 2000). spring production in morphotype A Epulopiscium cells FtsZ assembles at the future site of division forming a follows a predictable circadian cycle resulting in naturally ring-like structure, the Z ring (Addinall et al., 1996). As