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.     

The spoIIE homolog of Epulopiscium sp. type B is expressed early in intracellular offspring development

Epulopiscium sp. type B is an enormous intestinal symbiont of the surgeonfish Naso tonganus. Intracellular offspring production in Epulopiscium shares features with endospore formation. Here, we characterize the spoIIE homolog in Epulopiscium. The timing of spoIIE gene expression and presence of interacting partners suggest that the activation of σ(F) occurs early in Epulopiscium offspring development.     

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.     

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.     

Sporulation of Clostridium acetobutylicum P262 in a Defined Medium

A defined minimal sporulation medium for Clostridium acetobutylicum P262, which produces high levels of solvents, is described. The overall sporulation sequence was similar to that of other endospore-forming bacteria. However, we observed a presporulation stage, during which swollen phase-bright cells which contained large amounts of granulose formed. During sporulation, the initiation of spore coat formation occurred before the onset of cortex formation. Other Clostridium strains tested showed marked variations in ability to grow and sporulate in various minimal media.     

Endospore production of Bacillus spp. for industrial use

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.     

DNA replication during endospore development in Metabacterium polyspora

The guinea pig intestinal symbiont Metabacterium polyspora is an uncultured, endospore-forming member of the Firmicutes. Unlike most endospore-forming bacteria, sporulation is an obligate part of the M. polyspora life cycle when it is associated with a guinea pig. Binary fission is limited to a brief period in its life cycle, if exhibited at all. Instead, M. polyspora relies on the formation of multiple endospores for reproduction. Sporulation is initiated immediately after germination, which leaves little time for the cell to accumulate resources to support spore formation. Using immunolocalization of the nucleotide analogue bromodeoxyuridine (BrdU), we were able to follow replication dynamics in M. polyspora . BrdU was provided to cells within the guinea pig intestinal tract. BrdU was incorporated into DNA located within the forespores throughout development, at all stages prior to spore maturation. Our results suggest that in M. polyspora , DNA replication within the forespore is not suppressed during sporulation as it is in other endospore-forming bacteria. Replication within forespores would allow M. polyspora to maximize its reproductive potential and supply each endospore with at least one complete copy of the genome.     

Ultrastructural evidence for the presence of bacteria, viral-like particles, and mycoplasma-like organisms associated with Giardia spp

Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

Hydrolysis of N'-benzoyl-d-arginine-p-nitroanilide by members of the Haloanaerobiaceae: additional evidence that Haloanaerobium praevalens is related to endospore-forming bacteria

Abstract Three out of the four described halophilic obligately anaerobic bacteria of the family Haloanaerobiaceae hydrolyze d -BAPA (N'-benzoyl- d -arginine-p-nitroanilide), while showing no or little l -BAPA hydrolyzing activity. This property was shown earlier to be characteristic only of non-halophilic Gram-positive endospore-forming bacteria of the genera Bacillus and Clostridium . These results suggest that Haloanaerobium praevalens , which has never been shown to produce endospores, but was shown to be related to the endosphere-forming representatives of the Haloanaerobiaceae on the basis of 16S rRNA nucleotide sequence data, shares other properties characteristics of the endospore-forming bacteria. Neither significant d -BAPA nor l -BAPA hydrolyzing activity was found in Sporohalobacter lortetii .     

Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans

Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts.     

Cell Invasion and Matricide during Photorhabdus luminescens Transmission by Heterorhabditis bacteriophora Nematodes

Many animals and plants have symbiotic relationships with beneficial bacteria. Experimentally tractable models are necessary to understand the processes involved in the selective transmission of symbiotic bacteria. One such model is the transmission of the insect-pathogenic bacterial symbionts Photorhabdus spp. by Heterorhabditis bacteriophora infective juvenile (IJ)-stage nematodes. By observing egg-laying behavior and IJ development, it was determined that IJs develop exclusively via intrauterine hatching and matricide (i.e., endotokia matricida). By transiently exposing nematodes to fluorescently labeled symbionts, it was determined that symbionts infect the maternal intestine as a biofilm and then invade and breach the rectal gland epithelium, becoming available to the IJ offspring developing in the pseudocoelom. Cell- and stage-specific infection occurs again in the pre-IJ pharyngeal intestinal valve cells, which helps symbionts to persist as IJs develop and move to a new host. Synchronous with nematode development are changes in symbiont and host behavior (e.g., adherence versus invasion). Thus, Photorhabdus symbionts are maternally transmitted by an elaborate infectious process involving multiple selective steps in order to achieve symbiont-specific transmission.     

Ultrastructural Evidence for the Presence of Bacteria,Viral-like Particles,and Mycoplasma-like Organisms Associated with Giardia spp.1

ABSTRACT. Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

Endosymbionts of Siboglinum fiordicum and the phylogeny of bacterial endosymbionts in Siboglinidae (Annelida)

Siboglinid worms are a group of gutless marine annelids that are nutritionally dependent upon endosymbiotic bacteria. Four major groups of siboglinids are known-vestimentiferans, moniliferans, Osedax spp. and frenulates. Although endosymbionts of vestimentiferans and Osedax spp. have been previously characterized, little is currently known about endosymbiotic bacteria associated with frenulate and moniliferan siboglinids. This is particularly surprising given that frenulates are the most diverse and widely distributed group of siboglinids. Here, we molecularly characterize endosymbiotic bacteria associated with the frenulate siboglinid Siboglinum fiordicum by using 16S rDNA ribotyping in concert with laser-capture microdissection (LCM). Phylogenetic analysis indicates that at least three major clades of endosymbiotic gamma-proteobacteria associate with siboglinid annelids, with each clade corresponding to a major siboglinid group. S. fiordicum endosymbionts are a group of gamma-proteobacteria that are divergent from bacteria associated with vestimentiferan or Osedax hosts. Interestingly, symbionts of S. fiordicum, from Norway, are most closely related to symbionts of the frenulate Oligobrachia mashikoi from Japan, suggesting that symbionts of frenulates may share common evolutionary history or metabolic features.     

Direct microscopy of bacillus endospore germination in soil microcosms

Antagonistic endospore-forming Bacillus spp. offer a large potential as seed inoculants for control of soil-borne pathogens. In the soil, however, inoculated Bacillus endospores may remain dormant without germination, and plant protection can therefore be inefficient and unpredictable. A method based on direct fluorescence microscopy in soil microcosms was used to determine whether low-cost organic additives incorporated into seed coating material could stimulate endospore germination. Complex organic additives supported a high level of endospore germination of the fungal antagonist Paenibacillus polymyxa CM5-5. Skim milk is a low-cost additive that may be incorporated into seed coating material for efficient induction of Bacillus endospore germination in soil.     

Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores

The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.     

Diet strongly influences the gut microbiota of surgeonfishes

Intestinal tracts are among the most densely populated microbial ecosystems. Gut microbiota and their influence on the host have been well characterized in terrestrial vertebrates but much less so in fish. This is especially true for coral reef fishes, which are among the most abundant groups of vertebrates on earth. Surgeonfishes (family: Acanthuridae) are part of a large and diverse family of reef fish that display a wide range of feeding behaviours, which in turn has a strong impact on the reef ecology. Here, we studied the composition of the gut microbiota of nine surgeonfish and three nonsurgeonfish species from the Red Sea. High‐throughput pyrosequencing results showed that members of the phylum Firmicutes, especially of the genus Epulopiscium, were dominant in the gut microbiota of seven surgeonfishes. Even so, there were large inter‐ and intraspecies differences in the diversity of surgeonfish microbiota. Replicates of the same host species shared only a small number of operational taxonomic units (OTUs), although these accounted for most of the sequences. There was a statistically significant correlation between the phylogeny of the host and their gut microbiota, but the two were not completely congruent. Notably, the gut microbiota of three nonsurgeonfish species clustered with some surgeonfish species. The microbiota of the macro‐ and microalgavores was distinct, while the microbiota of the others (carnivores, omnivores and detritivores) seemed to be transient and dynamic. Despite some anomalies, both host phylogeny and diet were important drivers for the intestinal microbial community structure of surgeonfishes from the Red Sea.     

New Observations on the Ciliate Genus Vestibulongum (Pycnotrichidae): Vestibular Ultrastructure,Macronuclear Endosymbiotic Bacteria,Biogeography, and Evidence for Host Specificity

Two isolates of the pycnotrichid ciliate genus, Vestibulongum, were collected from the host fish, Acanthurus xanthopterus, from two locations in the Southern Pacific Ocean. One was from the Great Barrier Reef (GBR), and a second from Papua New Guinea. These sites are thousands of km from the type locality, off the coast of South Africa. New data were collected from protargol‐stained samples to more fully characterize the general form and light microscopic structures of the ciliate. Specimens from all three sites had a long vestibule, characteristic of most members of the family. Data suggest that specimens from each site are the same genus. The kinetids of the Vestibulongum isolated from the GBR contained the typical components of postciliary, transverse, and nemodesmatal microtubules, and Kd fibrils. Also, two quite different forms of endomacronuclear bacteria were observed and are described. One of those has distinct endospores, which are similar to endospores in nuclear endosymbiotic bacteria in a species of Balantidium from the gut of another species of surgeonfish.     

Identification of β-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils

Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.