The exceptional form and function of the giant bacterium Ca. Epulopiscium viviparus revolves around its sodium motive force.

Epulopiscium spp. are the largest known heterotrophic bacteria; a large cigar-shaped individual is a million times the volume of Escherichia coli. To better understand the metabolic potential and relationship of Epulopiscium sp. type B with its host Naso tonganus, we generated a high-quality draft genome from a population of cells taken from a single fish. We propose the name Candidatus Epulopiscium viviparus to describe populations of this best-characterized Epulopiscium species. Metabolic reconstruction reveals more than 5% of the genome codes for carbohydrate active enzymes, which likely degrade recalcitrant host-diet algal polysaccharides into substrates that may be fermented to acetate, the most abundant short-chain fatty acid in the intestinal tract. Moreover, transcriptome analyses and the concentration of sodium ions in the host intestinal tract suggest that the use of a sodium motive force (SMF) to drive ATP synthesis and flagellar rotation is integral to symbiont metabolism and cellular biology. In natural populations, genes encoding both F-type and V-type ATPases and SMF generation via oxaloacetate decarboxylation are among the most highly expressed, suggesting that ATPases synthesize ATP and balance ion concentrations across the cell membrane. High expression of these and other integral membrane proteins may allow for the growth of its extensive intracellular membrane system. Further, complementary metabolism between microbe and host is implied with the potential provision of nitrogen and B vitamins to reinforce this nutritional symbiosis. The few features shared by all bacterial behemoths include extreme polyploidy, polyphosphate synthesis, and thus far, they have all resisted cultivation in the lab.

Acetobacter pomorum in the Drosophila gut microbiota buffers against host metabolic impacts of dietary preservative formula and batch variation in dietary yeast.

Gut microbiota are fundamentally important for healthy function in animal hosts. Drosophila melanogaster is a powerful system for understanding host-microbiota interactions, with modulation of the microbiota inducing phenotypic changes that are conserved across animal taxa. Qualitative differences in diet, such as preservatives and dietary yeast batch variation, may affect fly health indirectly via microbiota, and may potentially have hitherto uncharacterized effects directly on the fly. These factors are rarely considered, controlled, and are not standardized among laboratories. Here, we show that the microbiota's impact on fly triacylglyceride (TAG) levels-a commonly-measured metabolic index-depends on both preservatives and yeast, and combinatorial interactions among the three variables. In studies of conventional, axenic, and gnotobiotic flies, we found that microbial impacts were apparent only on specific yeast-by-preservative conditions, with TAG levels determined by a tripartite interaction of the three experimental factors. When comparing axenic and conventional flies, we found that preservatives caused more variance in host TAG than microbiota status, and certain yeast-preservative combinations even reversed effects of microbiota on TAG. Preservatives had major effects in axenic flies, suggesting either direct effects on the fly or indirect effects via media. However, Acetobacter pomorum buffers the fly against this effect, despite the preservatives inhibiting growth, indicating that this bacterium benefits the host in the face of mutual environmental toxicity. Our results suggest that antimicrobial preservatives have major impacts on host TAG, and that microbiota modulates host TAG dependent on the combination of the dietary factors of preservative formula and yeast batch. IMPORTANCE Drosophila melanogaster is a premier model for microbiome science, which has greatly enhanced our understanding of the basic biology of host-microbe interactions. However, often overlooked factors such as dietary composition, including yeast batch variability and preservative formula, may confound data interpretation of experiments within the same lab and lead to different findings when comparing between labs. Our study supports this notion; we find that the microbiota does not alter host TAG levels independently. Rather, TAG is modulated by combinatorial effects of microbiota, yeast batch, and preservative formula. Specific preservatives increase TAG even in germ-free flies, showing that a commonplace procedure in fly husbandry alters metabolic physiology. This work serves as a cautionary tale that fly rearing methodology can mask or drive microbiota-dependent metabolic changes and also cause microbiota-independent changes.

Developmental stage influences chromosome segregation patterns and arrangement in the extremely polyploid, giant bacterium Epulopiscium sp. type B.

Few studies have described chromosomal dynamics in bacterial cells with more than two complete chromosome copies or described changes with respect to development in polyploid cells. We examined the arrangement of chromosomal loci in the very large, highly polyploid, uncultivated intestinal symbiont Epulopiscium sp. type B using fluorescent in situ hybridization. We found that in new offspring, chromosome replication origins (oriCs) are arranged in a three-dimensional array throughout the cytoplasm. As development progresses, most oriCs become peripherally located. Siblings within a mother cell have similar numbers of oriCs. When chromosome orientation was assessed in situ by labeling two chromosomal regions, no specific pattern was detected. The Epulopiscium genome codes for many of the conserved positional guide proteins used for chromosome segregation in bacteria. Based on this study, we present a model that conserved chromosomal maintenance proteins, combined with entropic demixing, provide the forces necessary for distributing oriCs. Without the positional regulation afforded by radial confinement, chromosomes are more randomly oriented in Epulopiscium than in most small rod-shaped cells. Furthermore, we suggest that the random orientation of individual chromosomes in large polyploid cells would not hamper reproductive success as it would in smaller cells with more limited genomic resources.

Thiaminase I Provides a Growth Advantage by Salvaging Precursors from Environmental Thiamine and Its Analogs in Burkholderia thailandensis.

Thiamine is essential to life, as it serves as a cofactor for enzymes involved in critical carbon transformations. Many bacteria can synthesize thiamine, while thiamine auxotrophs must obtain it or its precursors from the environment. Thiaminases degrade thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, and thiaminase I specifically has been implicated in thiamine deficiency syndromes in animals. The biological role of this secreted enzyme has been a long-standing mystery. We used the thiaminase I-producing soil bacterium Burkholderia thailandensis as a model to ascertain its function. First, we generated thiamine auxotrophs, which are still able to use exogenous precursors (thiazole and hydroxymethyl pyrimidine), to synthesize thiamine. We found that thiaminase I extended the survival of these strains, when grown in defined media where thiamine was serially diluted out, compared to isogenic strains that could not produce thiaminase I. Thiamine auxotrophs grew better on thiamine precursors than thiamine itself, suggesting thiaminase I functions to convert thiamine to useful precursors. Furthermore, our findings demonstrate that thiaminase I cleaves phosphorylated thiamine and toxic analogs, which releases precursors that can then be used for thiamine synthesis. This study establishes a biological role for this perplexing enzyme and provides additional insight into the complicated nature of thiamine metabolism and how individual bacteria may manipulate the availability of a vital nutrient in the environment.IMPORTANCE The function of thiaminase I has remained a long-standing, unsolved mystery. The enzyme is only known to be produced by a small subset of microorganisms, although thiaminase I activity has been associated with numerous plants and animals, and is implicated in thiamine deficiencies brought on by consumption of organisms containing this enzyme. Genomic and biochemical analyses have shed light on potential roles for the enzyme. Using the genetically amenable thiaminase I-producing soil bacterium Burkholderia thailandensis, we were able to demonstrate that thiaminase I helps salvage precursors from thiamine derivatives in the environment and degrades thiamine to its precursors, which are preferentially used by B. thailandensis auxotrophs. Our study establishes a biological role for this perplexing enzyme and provides insight into the complicated nature of thiamine metabolism. It also establishes B. thailandensis as a robust model system for studying thiamine metabolism.

Epsilon toxin-producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege.

Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin-producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.

Development of a thermostable ß-glucuronidase-based reporter system for monitoring gene expression in hyperthermophiles.

Mesophilic glucuronidases are the most widely used reporters of gene expression in plants, but unsuitable as reporters in (hyper-)thermophiles due their insufficient thermal stability. Here we present the native 66.8 kDa thermostable ß-glucuronidase of Sulfolobus solfataricus. The enzyme activity is characterized in a wide temperature range ideal for, but not limited to, in vivo genetic study of hyperthermophiles. As a proof of concept, we demonstrate its use as a reporter of gene expression in Sulfolobus, by monitoring a promoter fusion created with the ß-glucuronidase coding gene gusB and a copper-responsive promoter.

CopR of Sulfolobus solfataricus represents a novel class of archaeal-specific copper-responsive activators of transcription.

In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)(-1). Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.

Isolation and characterization of arsenic resistant Geobacillus kaustophilus strain from geothermal soils.

A thermophilic, arsenate resistant bacterial strain was isolated from a geothermal field located in the area surrounding Monterotondo (Tuscany, Italy). Based on 16S rRNA gene analysis and recN comparisons the strain was identified as Geobacillus kaustophilus. Cells of the strain, designated A1, were rod-shaped, 2-3 µm long and reacted negatively to Gram staining, despite its taxonomic classification as a Gram positive microorganism. Strain A1 is a thermophilic spore-forming bacterium, and grows optimally at pH 6.5 and 55 °C. An arsenate MIC of 80 mM was determined for strain A1, and the close relative G. kaustophilus DSM 7263(T) showed similar levels of arsenate resistance. These observations were consistent with the presence of arsenic detoxification genes in the genome of G. kaustophilus HTA426. Furthermore, strain A1 growth was not inhibited by 5 mM antimonite and 15 mM arsenite, the highest tested concentrations. This is the first description of arsenic resistance in a Geobacillus strain and supports the hypothesis that members of the genus may have a role in the biogeochemical cycling of arsenic.

Periplasmic binding protein-based magnetic isolation and detection of thiamine in complex biological matrices.

Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine periplasmic binding protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5 nM ranging up to 240 nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2 nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0 nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.

The Drosophila melanogaster Gut Microbiota Provisions Thiamine to Its Host.

The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. We explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, we found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster Likewise, this interplay did not have an impact on egg production. However, we determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, we determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment.IMPORTANCE There has been a long-standing assumption that the microbiota of animals provides their hosts with essential B vitamins; however, there is not a wealth of empirical evidence supporting this idea, especially for vitamin B1 (thiamine). To determine whether this assumption is true, we used Drosophila melanogaster and chemically defined diets with different thiamine concentrations as a model. We found that the microbiota does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet. The power of the Drosophila-microbiota system allowed us to determine that one microbiota member in particular, Acetobacter pomorum, is responsible for the thiamine provisioning. Thereby, our study verifies this long-standing hypothesis. Finally, the methods used in this work are applicable for interrogating the underpinnings of other aspects of the tripartite interaction between diet, host, and microbiota.

Genomic insights into the thiamin metabolism of Paenibacillus thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460.

Paenibacillus thiaminolyticus is the model organism for studying thiaminase I, an enigmatic extracellular enzyme. Originally isolated from the feces of clinical patients suffering from thiamin deficiency, P. thiaminolyticus has been implicated in thiamin deficiencies in humans and other animals due to its ability to produce this thiamin-degrading enzyme. Its close relative, P. apiarius, also produces thiaminase I and was originally isolated from dead honeybee larvae, though it has not been reported to be a honeybee pathogen. We generated draft genomes of the type strains of both species, P. thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460, to deeply explore potential routes of thiamin metabolism. We discovered that the thiaminase I gene is located in a highly conserved operon with thiamin biosynthesis and salvage genes, as well as genes involved in the biosynthesis of the antibiotic bacimethrin. Based on metabolic pathway predictions, P. apiarius NRRL B-23460 has the genomic capacity to synthesize thiamin de novo using a pathway that is rarely seen in bacteria, but P. thiaminolyticus NRRL B-4156 is a thiamin auxotroph. Both genomes encode importers for thiamin and the pyrimidine moiety of thiamin, as well as enzymes to synthesize thiamin from pyrimidine and thiazole.

Host genetic determinants of microbiota-dependent nutrition revealed by genome-wide analysis of Drosophila melanogaster.

Animals bear communities of gut microorganisms with substantial effects on animal nutrition, but the host genetic basis of these effects is unknown. Here we use Drosophila to demonstrate substantial among-genotype variation in the effects of eliminating the gut microbiota on five host nutritional indices (weight, protein, lipid, glucose and glycogen contents); this includes variation in both the magnitude and direction of microbiota-dependent effects. Genome-wide association studies to identify the genetic basis of the microbiota-dependent variation reveal polymorphisms in largely non-overlapping sets of genes associated with variation in the nutritional traits, including strong representation of conserved genes functioning in signalling. Key genes identified by the GWA study are validated by loss-of-function mutations that altered microbiota-dependent nutritional effects. We conclude that the microbiota interacts with the animal at multiple points in the signalling and regulatory networks that determine animal nutrition. These interactions with the microbiota are probably conserved across animals, including humans.

In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors.

Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.