Initiation of Intracellular Offspring in Epulopiscium

Home , Acanthurus nigrofuscus, Epulopiscium

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 propagation 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

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 synchronized populations with respect to offspring initia- endospore formation proceeds, FtsZ switches from a tion and development (Montgomery and Pollak, 1988). medial to a bipolar localization pattern, whereupon an Although a careful circadian developmental study has not asymmetrically positioned septum forms creating two dis- been performed with the B morphotype, these populations similar cells, a small forespore and a larger mother cell also exhibit synchronized reproduction. This allows for the (Beall and Lutkenhaus, 1991; Levin and Losick, 1996). analysis of large numbers of cells at approximately the During division the septum becomes a ligature around the same stage of offspring development from a population axial filament, trapping approximately one-third of one taken from a single host fish. The Epulopiscium popula- chromosome in the smaller forespore (Setlow et al., tion, which contained the Z rings shown in this study 1991). The remainder of the chromosome is translocated (referred to later as ‘sample I’), consisted of cells approx- into the forespore by the septum-localized DNA pump, imately 140–350 mm in length, with widths of 40–95 mm. SpoIIIE (Wu and Errington, 1994; 1997; Wu et al., 1995; These Epulopiscium mother cells each contained 1–5

Intracellular offspring of Epulopiscium 829 large offspring; the majority (80%) contained two off- start of asymmetric division cells with bipolar septa spring. It was these intracellular offspring that possessed appear, indicating that division occurs sequentially at the polar rings of FtsZ (see illustration Fig. 1). Data collected two poles (Pogliano et al., 1999). Similarly, in sporulating from this and four other N. tonganus samples are M. polyspora, we have observed cells with either a single described in more detail below. polar Z ring or a single polar septum although almost all Immunolocalization of the key cell division protein FtsZ cells eventually produce at least two endospores (Angert was used to determine the role of cell division in the and Losick, 1998). Although we never observed similar initiation of offspring in Epulopiscium sp. morphotype B. stages in Epulopiscium we would expect sequential divi- During sporulation in B. subtilis, FtsZ switches from sion at the poles in these cells also. Mother cells with only medial to bipolar localization as a prelude to polar division one intracellular offspring are seen in natural populations (Levin and Losick, 1996). Similarly, FtsZ localizes in a at low frequencies (0–6%) and may be the result of divi- polar pattern during spore formation in M. polyspora sion at only one pole. It is possible that sequential division (Angert and Losick, 1998). We speculated that daughter within Epulopiscium occurs rapidly and we were unable to cell formation in Epulopiscium would require a similar observe the more ephemeral monopolar Z ring and sep- localization pattern. Strikingly, we find that FtsZ localizes tum stage. Alternatively, regulatory mechanisms that hold in a bipolar pattern in Epulopiscium before daughter cell division at the second pole may not be conserved or be formation (Fig. 1). Immunofluorescence microscopy indi- able to function in large Epulopiscium and bipolar division cates that FtsZ forms rings adjacent to both poles of the occurs simultaneously. cell early in the process of reproduction. Only large No medial Z rings were detected in this study. Binary daughter cells, still within an intact mother cell, contained fission has never been observed in the largest Epulopis- Z rings. The ensuing division would therefore initiate the cium morphotypes (Clements et al., 1989), although some formation of the next generation or ‘granddaughter’ cells. reports have suggested that binary fission does occur at In most instances, all cells within a single mother cell night (Montgomery and Pollak, 1988; Bresler et al., 1998). exhibited the same stage of development. That is, if one This proposition is based on surveys of natural assem- daughter cell exhibited polar Z rings, all siblings pos- blages of Epulopiscium symbionts taken from several fish sessed Z rings. Only bipolar localization of Z rings was (Acanthurus nigrofuscus) sacrificed at various time-points observed; a single FtsZ ring at only one pole was never throughout the day and into the night. The tallies show a seen. reduction in estimated mean cell length in samples taken In contrast to B. subtilis in which only one Z ring is used, during the night, which is interpreted as evidence for both are used in Epulopiscium, generally. Division at only binary fission occurring in these populations. Based on one pole was never observed in these Epulopiscium sam- 16S rRNA gene sequence analyses, we found that even ples, which indicates that either synchronous polar divi- the largest Epulopiscium symbionts of A. nigrofuscus sion or very rapid sequential division occurs. In B. subtilis, comprise a complex community of cells (Angert et al., bipolar division occurs in some stage II mutants (Ryter 1993). In situ hybridizations with variant-specific probes et al., 1966). In these sporulating cultures, cells with a demonstrated that multiple strains of Epulopiscium sym- single division septum appear first but within 10 min of the bionts, with rRNA sequences that vary by 1–9%, are nor-

Fig. 1. Polar rings of FtsZ in Epulopiscium. A. Provides an overview of the developmental stage of the cells that harboured Z rings shown in this study. The boxed region illustrates the portion of Epulopiscium shown in the three- dimensional reconstruction in B. Z rings were found only at the poles of large intracellular offspring. B. Polar Z rings of Epulopiscium morphotype B cells are approximately 4 mm in diameter. To view the entire Z ring at high magnification, a Z-series of images encompassing the diameter of the Z ring was obtained. The image stack was deconvolved to remove haze from out- of-focus fluorescent objects and image planes were assembled into a three-dimensional image of the cell pole. The digital reconstruction was then rendered into a two-dimensional image. The fluorescence seen in the cytoplasm of the cell may be due to unassembled FtsZ. Scale bar, 10 mm.

830 E. R. Angert and K. D. Clements mally found in a single A. nigrofuscus taken from either which is responsible for the formation of the axial filament the Red Sea or the South Pacific (Angert, 1995). Bressler and efficient segregation of the forespore chromosome and co-workers treated these communities of closely (Ben-Yehuda et al., 2003). As an asymmetrically posi- related bacteria as a single population. Therefore it is tioned septum is laid down in sporulating B. subtilis, difficult to determine if changes in mean cell length were approximately one-third of one chromosome is trapped a result of binary fission or differences in relative propor- within the forespore. The remainder must be pumped into tions of Epulopiscium strains with different cell lengths. the forespore by a septum-localized, DNA translocating The issue of whether large Epulopiscium spp. undergo protein, SpoIIIE (Errington et al., 2001). In Epulopiscium, binary fission has yet to be resolved. For the studies asymmetric division is anticipated by the coalescence of reported here, we did not sample surgeonfish at night and DNA at the poles of the mother cell and FtsZ rings assem- therefore did not study populations at times when binary ble at both poles. We observed polar Z rings in two forms fission may occur. Potential barriers to the assembly of a in Epulopiscium (Fig. 2). Some Z rings appeared aligned medially located Z ring exist in Epulopiscium cells. Pub- with the outer surface of the cell, whereas others lished electron micrographs of thin sections of various appeared to be exerting force on the pole-associated Epulopiscium morphotypes reveal that some contain a DNA. These Z rings are apparently part of a ligature at highly convoluted cytoplasmic membrane (Fishelson the cell pole, which we interpret as the earliest stages of et al., 1985; Montgomery and Pollak, 1988; Clements and progress in polar division. In either form, the predicted Bullivant, 1991; Robinow and Angert, 1998). The pres- division plane would cleave the pole-associated DNA. In ence of cell membrane invaginations may confound for- cells at a slightly later stage in development, when polar mation of a fully functional, medially located Z ring. septa are present, only some of the polar DNA is con- Membrane convolutions are not as prevalent near the cell tained within the newly formed cells. We estimate that poles, which may better accommodate Z ring assembly. approximately 0.2% of the total DNA, only about one tenth In addition, the progression from medial to polar Z rings of the pole-associated DNA, is initially contained within in B. subtilis involves the formation of a spiral-like inter- each small polar cell. At still later stages in development, mediate comprised of FtsZ (Ben-Yehuda and Losick, more DNA is contained within the primordial offspring and 2002). If this is a general mechanism for redeployment of the amount of pole-associated DNA outside of the off- FtsZ to polar locals in low G + C Gram-positive bacteria, spring diminishes. By the start of engulfment the amount the formation or progression through a potentially mem- of DNA contained within the primordium has increased brane-associated, spiral-like intermediate may be difficult and at least half, if not all, of the polar DNA appears to achieve in Epulopiscium. Clearly Epulopiscium has contained within the offspring. This apparent increase retained functional cell division machinery, but direct may be due to translocation of mother cell DNA into the observations are needed to confirm the use of binary offspring or DNA synthesis within the offspring. In addi- fission by these cells and determine its role in population tion, it appears that degradation or reallocation of mother maintenance. cell DNA occurs. At the completion of engulfment the offspring is a distinct body within the mother cell although it remains closely associated with the mother cell pole. As DNA distribution and cell division it grows and moves away from the mother cell pole a void We followed the pattern of DNA distribution in Epulopis- in the mother cell DNA layer is seen. In the population that cium cells and again found remarkable parallels between contained cells with polar Z rings 648 cells were scored Epulopiscium and B. subtilis. In both instances DNA is for stage of development (Fig. 3). Data collected from four reorganized in anticipation of polar division and subse- other, independent fish samples are presented to illustrate quently the DNA appears to be packaged into the newly the progression of offspring development. Each of these formed offspring or forespore. In a fine-scale time-lapse populations exhibited a very narrow range of developmen- study of B. subtilis, condensation of DNA at the forespore- tal stages. Additional data obtained from these popula- proximal end of a sporulating cell was shown to precede tions, specifically cell length and width measurements, are Z ring assembly (Pogliano et al., 2002). Likewise, before provided (Table 1). the assembly of Z rings in Epulopiscium, DNA coalesces Even after DNA transfer to the offspring and engulfment at the poles of the cell (Fig. 2). Highly condensed, pole- is complete, only a small proportion of the Epulopiscium associated DNA has been observed in other Epulopis- cellular DNA is partitioned into newly formed offspring. cium morphotypes as well (data not shown). This is in stark contrast to chromosome partitioning seen Cytological, genetic and genomic approaches continue in sporulating B. subtilis. One of the two copies of the B. to identify novel proteins involved in earliest stages of subtilis chromosome is partitioned into the forespore while endospore formation in B. subtilis. A recent study identi- the second remains in the mother cell. In B. subtilis fied a sporulation-specific DNA-binding protein, RacA, mutants that produce a disporic sporangium, the two

Fig. 2. Asymmetric division and offspring production in Epulopiscium. A–F, a series of cells representing successive stages of offspring production in Epulopiscium morphotype B. The micrographs focus on cell poles, where offspring are initiated. Vertical columns of micrographs are images of the same cell. The upper row illustrates DAPI fluorescence, middle row contains Nomarski Differential Interference Contrast (DIC) micrographs, and the lower two panels in A and B show the immunofluorescent localization of FtsZ. A cross section through a Z ring is seen in each of these medial focal planes. Each Z ring, indicated by arrowheads, is visible as two bright spots at the pole of the cell. Columns A through D show offspring still contained within parent cells, whereas E and F show cells after release. A¢–F¢, drawings are provided to illustrate the relative position of cell structures in corresponding (A–F) micrographs. In these drawings, black lines represent outer surface of the cell, the division septum and the engulfment membrane, DNA is shown as grey shading within each cell and the relative location of FtsZ is indicated by circles in panels A¢ and B¢. A. DNA coalesces in the tip of a mother cell and Z ring assembles in anticipation of septum formation. B. The FtsZ ring appears to exert force on the pole-associated DNA as division begins. C. Septum formation is complete. The arrow marks the position of the polar septum. D. Illustrates a later stage, engulfment. At this stage the coalesced, polar DNA appears to have been packaged into the newly formed offspring. E. The offspring is now a distinct body within the cytoplasm of the mother cell. Note the absence of DNA at the mother cell pole. F. DNA in the offspring begins to take on a peripheral arrangement. All images are the same magnification. Scale bar, 10 mm.

Fig. 3. Developmental stages of Epulopiscium populations from ﬁve surgeonﬁsh. Drawings represent cross sections through the poles of Epulopiscium cells. This series illustrates successive stages early in offspring development. Black lines delineate the outer surface of the cell, the division septum and the engulfment membrane; grey shading indicates the distribution of DNA. The number of cells of a particular stage and the approximate proportion of the scored population are given under the repre- sentative drawing. For sample I, the population that contained cells with polar Z rings, 648 cells were scored for stage of development. Within this group 108 cells (17%) had Z rings, 78 of these cells contained ‘early’ Z rings as illus- trated in Fig. 2A and 30 had ‘later’ Z rings as in Fig. 2B. For this analysis, all of these cells are grouped with cells that only exhibited coalesced polar DNA.

Table 1. Length and width measurements of Epulopiscium cells just beneath the cytoplasmic membrane (Robinow and undergoing reproduction. Kellenberger, 1994). For this reason we analyse cells in

Length Width several focal planes, which reveals a large quantity of min – max min – max DNA located in the mother cell after offspring are formed, Sample mean ± standard mean ± standard Fig. 2 and see (Robinow and Kellenberger, 1994; Rob- sample size deviation deviation inow and Angert, 1998). It appears that in Bresler et al. sample Ia 113.5–213.9 mm 18.0–40.6 mm (1998) a single medial focal plane is scanned to detect 71 cells 171.8 ± 19.19 27.1 ± 4.24 DNA in Epulopiscium cells. If only a medial plane were sample IIa 155.2–241.0 mm 30.1–54.6 mm 100 cells 193.6 ± 18.62 39.8 ± 5.16 used in these analyses, much of the DNA in the mother sample IIIa 127.8–209.1 mm 22.8–47.4 mm cell would not be detected. 100 cells 167.1 ± 15.36 31.2 ± 3.88 sample IVa 136.7–296.0 mm 19.7–51.8 mm 100 cells 215.2 39.77 34.8 6.22 ± ± Phylogenetic perspective sample IV 1.5–4.9 mm 4.8–10.6 mm 200 primordia 2.7 ± 0.61 7.4 ± 1.1 sample V 118.3–202.6 mm 21.9–35.6 mm Endospore formation in the low G + C Gram-positive 109 cells 158.6 ± 15.98 29.7 ± 3.16 bacteria is a marvel of cellular reorganization and gene sample V 10.9–32.5 mm 3.7–12.6 mm regulation. For B. subtilis, hundreds of genes have a dem- 294 primordia 22.6 ± 4.42 7.0 ± 1.5 onstrated role in endospore formation, with dozens of a. The cell measurements provided are of cells undergoing reproduc- these genes dedicated to the process. This highly suc- tion. Although these cells were still contained within parental cells, cessful survival programme has allowed for the evolution the parent cell dimensions are not presented. Samples correspond to those in Fig. 3. For samples with cells exhib- of innumerable strains of bacteria that are adapted for life iting later stages of reproduction, data for offspring primordia are in a variety of harsh and benign habitats throughout the provided in italics. Length measurements were taken from pole to world. But some lineages have taken this programme and pole and width measurements were taken perpendicular to the length measurement, at the widest part of the cell or primordium. modified it for their own advantage, using the process now as an alternative form of reproduction. A few of the described strains of low G + C Gram- copies of the chromosome are partitioned instead into the positive bacteria can produce more than one endospore. two forespores, leaving the mother cell devoid of any Several Bacillus and Clostridium species occasionally genetic material which leads to the arrest of sporulation. produce two endospores per mother cell. Clostridium oce- In comparison, large Epulopiscium cells contain an enor- anicum, for example, does so by dividing at both poles mous amount of DNA that is distributed about the periph- (Smith, 1970). The two small forespores formed are sub- ery of the cytoplasm (Robinow and Kellenberger, 1994). sequently engulfed by the mother cell and develop into Little DNA is found in the central cytoplasm. Prior to asym- dormant cells. Other bacteria produce two endospores in metric division some of the peripheral DNA layer becomes an alternative fashion. With the segmented filamentous reallocated to poles of the cell. A similar pattern of DNA bacteria (SFB), each cell divides asymmetrically produc- distribution is seen in M. polyspora cells as they undergo ing a single forespore, which undergoes division after endospore formation (Angert and Losick, 1998). We spec- engulfment (Chase and Erlandsen, 1976; Ferguson and ulate that this pattern of peripheral DNA localization Birch-Andersen, 1979). A number of other multiple accommodates the formation of multiple intracellular off- endospore-forming bacteria have been described (Duda spring, either endospores or daughter cells. Only a small et al., 1987; Kunstyr et al., 1988). However, the manner in proportion of the mother cell DNA is translocated into a which multiple forespores are formed in these organisms single offspring primordium in Epulopiscium. Therefore remains to be elucidated. the offspring must contain at least one copy of the com- In a previous study, we described the earliest stages of plete genome but probably contains more than one copy. endospore formation in M. polyspora and found that a These results support the idea that Epulopiscium har- combination of asymmetric division at both cell poles com- bours a small genome. To account for the huge amount bined with division of engulfed forespores leads to the of DNA in a large mother cell, the unit genome must be formation of multiple dormant offspring (Angert and present in thousands of copies, or portions of the genome Losick, 1998). The ability to produce multiple endospores must be amplified. Our observations differ from previously has become the primary means of reproduction in M. published reports that conclude that mother cell DNA is polyspora and allows the bacteria to survive passage divided into equivalent portions that are partitioned into between host gastrointestinal tracts. The viviparous repro- offspring (Bresler et al., 1998). We believe that our data duction of Epulopiscium bears a remarkable resemblance are more valid for several reasons. For many bacteria, to endospore production by M. polyspora. The common cellular DNA fills the central cytoplasm, while in Epulopi- evolutionary histories of these bacteria and the mecha- scium, DNA is found surrounding the central cytoplasm, nisms involved in these cellular processes suggest a pro-

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 827–835 Intracellular offspring of Epulopiscium 833 gression from a provisional dormancy programme to a 0.2 g KCl; 1.44 g Na2HPO4; 0.24 g KH2PO4 adjusted to novel mode of cellular reproduction. The processes driv- pH 7.4] and allowed to air dry. ing this evolution are still a mystery but may involve the Cells were hybridized as previously described (Angert and Losick, 1998). Rabbit antiserum raised against a fragment of co-evolution of animals that ingest significant amounts of Epulopiscium FtsZ was diluted 1:10 000 in 2% BSA/PBS [2% plant matter or detritus and the efficient acquisition of their (w/v) bovine serum albumin dissolved in PBS]. In immunolo- anaerobic intestinal biota by coprophagy (Clements, calization studies this antiserum specifically hybridizes to 1997). FtsZ rings of close relatives of Epulopiscium including The evolution of any complex developmental pro- Clostridium lentocellum, C. propionicum and M. polyspora gramme relies on the stepwise acquisition of advanta- (Angert and Losick, 1998). Primary antiserum incubations geous processes. In the case of endospore formation, were performed overnight at 4∞C in a humid chamber. Slides were washed with PBS. FITC- or Cy3-labelled anti-rabbit Fc what intermediate stages have lead to the accumulation antibodies (Jackson Laboratories) were prepared as recom- of traits required for the formation of a functional and mended by the manufacturer. The antiserum was diluted resistant endospore? Did an unusual mode of intracellular (1:100 or 1:200 for FITC or Cy3, respectively) in a solution offspring production precede the development of a dor- of 2% BSA/PBS. Cells were simultaneously stained with the mant cell? Although contemporary examples of bacteria DNA-specific dye 4¢, 6-diamidino-2-phenylindole (DAPI), that form multiple live intracellular offspring are most likely 2 mg ml-1. Slides were incubated at room temperature for 2 h the result of incomplete endospore formation, is it possible in a humid chamber and washed with PBS. The cells were mounted in the antifade reagent 0.5% p-phenylenediamine that the formation of multiple live offspring is the ancestral (Sigma) in 20 mM Tris, pH 8.8, 90% glycerol. state?

Experimental procedures Image analysis and processing Epulopiscium collection and fixation Cells were viewed using an Olympus BX61 epifluorescent microscope equipped with low power objectives for scan- Naso tonganus, formerly identified as N. tuberosus (Kuiter ning, as well as 60¥ (N.A. 1.4) UPlanApo and 100¥ UPlan- and Debelius, 2001), were collected by spearfishing from Apo (N.A 1.35) objectives for fluorescent imaging and outer reefs around Lizard Island, Great Barrier Reef, Austra- Nomarski Differential Interference Contrast (DIC) micro- lia. Sections of the gut were removed from the fish and scopy. The microscope is equipped with fluorescence filter intestinal contents were fixed with either 80% ethanol or cold cubes for viewing DAPI, FITC and Cy3. Images were (-20∞C) 80% methanol. Samples were shipped to the labo- acquired using a Cooke SensiCam with a Sony Interline ratory at ambient temperature, but stored at -20∞C upon chip. Image acquisition and constrained iterative deconvolu- arrival. Storage of bacteria in 80% methanol preserves a tion were preformed using SlideBook Software package normal pattern of FtsZ localization in Bacillus subtilis and B. (Intelligent Imaging). For a recent review on the application megaterium cells for at least 6 months (data not shown). of deconvolution and three-dimensional reconstruction to Before immunolocalization experiments paraformaldehyde wide-field microscopy see (Andrews et al., 2002). Final fig- was added to an aliquot of the cells as previously described ures were assembled using Adobe Photoshop version 5.5 (Pedersen et al., 1999). and Canvas 9 (Deneba).

Immunolocalization of FtsZ and staining procedures Acknowledgements Cleaned microscope slides or cover slips were treated with 0.1% poly L-lysine (Sigma), rinsed with distilled water and air- This work was supported by the Jane Coffin Childs Memorial dried. A rectangle was drawn on the glass surface with a PAP Fund for Medical Research and NIH GM18568 to Richard pen (ICN) to form a hydrophobic barrier. The barrier defines Losick. We would like to thank Petra Levin for her insight and a well to contain hybridization solutions but it also serves as many helpful discussions. We are also grateful to Richard a spacer to prevent Epulopiscium cells from being crushed Losick for his enthusiastic support, Howard Choat for his help when the cover slip is placed on a slide. An aliquot of sur- in the field, and Kristen Lee for her technical assistance. geonfish gut contents was placed on the slide surface within the rectangle and allowed to air dry. All subsequent hybridizations and washes were performed on the slide surface. References Epulopiscium cells were permeabilized by incubating the cells in a solution of GTE, pH 6.3 [50 mM glucose; 20 mM Addinall, S.G., Bi, E., and Lutkenhaus, J. (1996) FtsZ ring Tr is-Cl, pH 6.3; 10 mM EDTA] containing mutanolysin formation in fts mutants. J Bacteriol 178: 3877–3884. (Sigma), 500 units ml-1, in a moist chamber at 37∞C for 2 h. Andrews, P.D., Harper, I.S., and Swedlow, J.R. (2002) To 5D This solution was removed and the slide was washed three and beyond: quantitative fluorescence microscopy in the times with GTE, pH 7.6. Cells were then incubated in a solu- postgenomic era. Traffic 3: 29–36. tion of GTE, pH 7.6, containing lysozyme (Sigma), 5 mg Angert, E.R. (1995) Molecular phylogenetic analyses of ml-1, for 2 h at 37∞C. This solution was removed and the slide Epulopiscium-like symbionts. PhD Thesis, Department of was washed 5 times with PBS [containing per litre: 8 g NaCl; Biology, Indiana University, Bloomington, IN, 196 pages.

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