Initiation of intracellular offspring in Epulopiscium

Epulopiscium spp. are the largest heterotrophic bacteria yet described. A distinguishing feature of the Epulopiscium group is their viviparous production of multiple, internal offspring as a means of cellular reproduction. Based on their phylogenetic position, among low G + C Gram-positive endospore-forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation. These observations also raise the possibility that a cell with the capacity to form multiple intracellular offspring was the ancestor of all contemporary endospore-forming bacteria. In an effort to characterize mechanisms common to both processes, we describe the earliest stages of offspring formation in Epulopiscium. First, in anticipation of polar division, some of the mother cell DNA coalesces at the cell poles. FtsZ then localizes in a bipolar pattern and the cell divides. A portion of the pole-associated DNA is trapped within the small cells formed by division at both poles. As development progresses, more pole-associated DNA is apparently packaged into the offspring primordia. These results illustrate three mechanisms, the reorganization of cellular DNA, asymmetric division and DNA packaging, that are common to both endospore formation in Bacillus subtilis and the production of active, intracellular offspring in Epulopiscium. Unlike most endospore formers, Epulopiscium partitions only a small proportion of mother cell DNA into the developing offspring.     

Nocturnal production of endospores in natural populations of epulopiscium-like surgeonfish symbionts

Prior studies have described a morphologically diverse group of intestinal microorganisms associated with surgeonfish. Despite their diversity of form, 16S rRNA gene surveys and fluorescent in situ hybridizations indicate that these bacteria are low-G+C gram-positive bacteria related to Epulopiscium spp. Many of these bacteria exhibit an unusual mode of reproduction, developing multiple offspring intracellularly. Previous reports have suggested that some Epulopiscium-like symbionts produce dormant or phase-bright intracellular offspring. Close relatives of Epulopiscium, such as Metabacterium polyspora and Clostridium lentocellum, are endospore-forming bacteria, which raises the possibility that the phase-bright offspring are endospores. Structural evidence and the presence of dipicolinic acid demonstrate that phase-bright offspring of Epulopiscium-like bacteria are true endospores. In addition, endospores are formed as part of the normal daily life cycle of these bacteria. In the populations studied, mature endospores were seen only at night and the majority of cells in a given population produced one or two endospores per mother cell. Phylogenetic analyses confirmed the close relationship between the endospore-forming surgeonfish symbionts characterized here and previously described Epulopiscium spp. The broad distribution of endospore formation among the Epulopiscium phylogenetic group raises the possibility that sporulation is a characteristic of the group. We speculate that spore formation in Epulopiscium-like symbionts may be important for dispersal and may also enhance survival in the changing conditions of the fish intestinal tract.     

Phylogenetic analysis of Metabacterium polyspora: clues to the evolutionary origin of daughter cell production in Epulopiscium species, the largest bacteria.

It is rare that there are molecular clues to the evolutionary origin of developmental traits. We have encountered an evolutionary juxtaposition that may explain the origin of the unique replicative morphology of Epulopiscium spp., the largest known bacteria, which reproduce by the internal production of multiple live offspring. We report here a 16S rRNA-based phylogenetic analysis of Metabacterium polyspora, a multiple-endospore-forming, uncultivated inhabitant of guinea pig cecum. Cells of M. polyspora were harvested from cecum contents by sedimentation in a Ficoll gradient and lysed. The bacterial 16S rRNA genes of this lysate were amplified by PCR. Sequence analysis of the cloned PCR products revealed two dominant, closely related 16S rRNA types. In situ hybridization of cecum contents with fluorescently labeled oligonucleotides, diagnostic of these two sequences, showed that they represent distinct strains of M. polyspora. Phylogenetic analyses of the sequences showed that M. polyspora is closely related to Epulopiscium spp. On the basis of this result and other correlations, we propose that the process of sporulation was modified in a predecessor of Epulopiscium spp. to produce live offspring instead of quiescent endospores.     

SpoIIE regulates sporulation but does not directly affect solventogenesis in Clostridium acetobutylicum ATCC 824

Using gene expression reporter vectors, we examined the activity of the spoIIE promoter in wild-type and spo0A-deleted strains of Clostridium acetobutylicum ATCC 824. In wild-type cells, the spoIIE promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator spo0A is disrupted, no spoIIE promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress spoIIE and to decrease spoIIE expression via antisense RNA targeted against spoIIE, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of spoIIE. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in spoIIE-downregulated cells are mediated by effects on the cell during sporulation.     

Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis

The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF].     

Characterization of a morphological checkpoint coupling cell-specific transcription to septation in Bacillus subtilis

Early in the process of spore formation in Bacillus subtilis, asymmetric cell division produces a large mother cell and a much smaller prespore. Differentiation of the prespore is initiated by activation of an RNA polymerase sigma factor, sigmaF, specifically in that cell. sigmaF is controlled by a regulatory cascade involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor, SpoIIAA, and a membrane-bound phosphatase, SpoIIE, which converts the inactive, phosphorylated form of SpoIIAA back to the active form. SpoIIE is required for proper asymmetric division and much of the protein is sequestered into the prespore during septation. Importantly, activation of sigmaF is dependent on formation of the asymmetric septum. We have now characterized this morphological checkpoint in detail, using strains affected in cell division and/or spoIIE function. Surprisingly, we found that significant dephosphorylation of SpoIIAA occurred even in the absence of septation. This shows that the SpoIIE phosphatase is at least partially active independent of the morphological event and also that cells can tolerate significant levels of unphosphorylated SpoIIAA without activating sigmaF. We also describe a spoIIE mutant in which the checkpoint is bypassed, probably by an increase in the dephosphorylation of SpoIIAA. Taken together, the results support the idea that sequestration of SpoIIE protein into the prespore plays an important role in the control of sigmaF activation and in coupling this activation to septation.     

An unusual symbiont from the gut of surgeonfishes may be the largest known prokaryote.

Symbionts first reported from the gut of a Red Sea surgeonfish, Acanthurus nigrofuscus (family Acanthuridae), were subsequently described as Epulopiscium fishelsoni. The taxonomic position of this very large (up to 576 microns in length) microorganism has previously been designated in the literature as either uncertain or eukaryotic. We suggest that similar symbionts from Great Barrier Reef surgeonfish may be prokaryotes, which together with E. fishelsoni from the Red Sea may represent the largest known forms of this cell type. Features identifying the symbionts as prokaryotes include the presence of bacterial-type flagella and a bacterial nucleoid and the absence of a nucleus or any other membrane-bound organelle.     

Purification, Kinetic Properties, and Intracellular Concentration of SpoIIE, an Integral Membrane Protein That Regulates Sporulation in Bacillus subtilis

SpoIIE is a bifunctional protein which controls sigmaF activation and formation of the asymmetric septum in sporulating Bacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.     

Food vacuole contents in the ciliate, Balantidium jocularum (Balantididae), a symbiont in the intestine of the surgeonfish, Naso tonganus (Acanthuridae)

During the past 16 years, the ciliate Balantidium jocularum has been collected from the intestines of many specimens of its fish host, Naso tonganus, all collected from the Great Barrier Reef near Lizard Island, Australia. Ciliates for this study of food consumption were isolated in 1988, 1989, 2003, and 2005. Nineteen specimens of B. jocularum were examined in the transmission electron microscope to determine the contents of both food vacuoles and a putative discharging cytoproct vacuole. Food vacuoles contained rod-shaped bacteria, tightly coiled spirilliform bacteria, and one or more euglenid flagellates. In several balantidia of somewhat different form than the type species of B. jocularum, the large bacterium, Epulopiscium fishelsoni, was observed in light microscope protargol preparations. Some putative phagolysosomes retained spirilliform bacteria that were apparently intact, and others contained partially digested flagellates. Food in a single discharging cytoproct vacuole consisted of normal appearing spirilliform bacteria, some other bacteria, and no flagellates. The results argue for non-selective ingestion of food and selective digestion; hence, somewhat inefficient food processing.