Pre-analytical challenges from adsorptive losses associated with thiamine analysis

Thiamine (vitamin B1) is an essential vitamin serving in its diphosphate form as a cofactor for enzymes in the citric acid cycle and pentose-phosphate pathways. Its concentration reported in the pM and nM range in environmental and clinical analyses prompted our consideration of the components used in pre-analytical processing, including the selection of filters, filter apparatuses, and sample vials. The seemingly innocuous use of glass fiber filters, glass filter flasks, and glass vials, ubiquitous in laboratory analysis of clinical and environmental samples, led to marked thiamine losses. 19.3 nM thiamine was recovered from a 100 nM standard following storage in glass autosampler vials and only 1 nM of thiamine was obtained in the filtrate of a 100 nM thiamine stock passed through a borosilicate glass fiber filter. We further observed a significant shift towards phosphorylated derivatives of thiamine when an equimolar mixture of thiamine, thiamine monophosphate, and thiamine diphosphate was stored in glass (most notably non-silanized glass, where a reduction of 54% of the thiamine peak area was observed) versus polypropylene autosampler vials. The selective losses of thiamine could lead to errors in interpreting the distribution of phosphorylated species in samples. Further, some loss of phosphorylated thiamine derivatives selectively to amber glass vials was observed relative to other glass vials. Our results suggest the use of polymeric filters (including nylon and cellulose acetate) and storage container materials (including polycarbonate and polypropylene) for thiamine handling. Losses to cellulose nitrate and polyethersulfone filters were far less substantial than to glass fiber filters, but were still notable given the low concentrations expected in samples. Thiamine losses were negated when thiamine was stored diluted in trichloroacetic acid or as thiochrome formed in situ, both of which are common practices, but not ubiquitous, in thiamine sample preparation

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

Composition, Diversity, and Origin of the Bacterial Community in Grass Carp Intestine

Gut microbiota has become an integral component of the host, and received increasing attention. However, for many domestic animals, information on the microbiota is insufficient and more effort should be exerted to manage the gastrointestinal bacterial community. Understanding the factors that influence the composition of microbial community in the host alimentary canal is essential to manage or improve the microbial community composition. In the present study, 16S rRNA gene sequence-based comparisons of the bacterial communities in the grass carp (Ctenopharyngodon idellus) intestinal contents and fish culture-associated environments are performed. The results show that the fish intestinal microbiota harbors many cellulose-decomposing bacteria, including sequences related to Anoxybacillus, Leuconostoc, Clostridium, Actinomyces, and Citrobacter. The most abundant bacterial operational taxonomic units (OTUs) in the grass carp intestinal content are those related to feed digestion. In addition, the potential pathogens and probiotics are important members of the intestinal microbiota. Further analyses show that grass carp intestine holds a core microbiota composed of Proteobacteria, Firmicutes, and Actinobacteria. The comparison analyses reveal that the bacterial community in the intestinal contents is most similar to those from the culture water and sediment. However, feed also plays significant influence on the composition of gut microbiota

Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh

Large sulfur bacteria of the genus Achromatium are exceptional among Bacteria and Archaea as they can accumulate high amounts of internal calcite. Although known for more than 100 years, they remain uncultured, and only freshwater populations have been studied so far. Here we investigate a marine population of calcite-accumulating bacteria that is primarily found at the sediment surface of tide pools in a salt marsh, where high sulfide concentrations meet oversaturated oxygen concentrations during the day. Dynamic sulfur cycling by phototrophic sulfide-oxidizing and heterotrophic sulfate-reducing bacteria co-occurring in these sediments creates a highly sulfidic environment that we propose induces behavioral differences in the Achromatium population compared with reported migration patterns in a low-sulfide environment. Fluctuating intracellular calcium/sulfur ratios at different depths and times of day indicate a biochemical reaction of the salt marsh Achromatium to diurnal changes in sedimentary redox conditions. We correlate this calcite dynamic with new evidence regarding its formation/mobilization and suggest general implications as well as a possible biological function of calcite accumulation in large bacteria in the sediment environment that is governed by gradients. Finally, we propose a new taxonomic classification of the salt marsh Achromatium based on their adaptation to a significantly different habitat than their freshwater relatives, as indicated by their differential behavior as well as phylogenetic distance on 16S ribosomal RNA gene level. In future studies, whole-genome characterization and additional ecophysiological factors could further support the distinctive position of salt marsh Achromatium

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

Abstract 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

Microbial circadian clocks: host-microbe interplay in diel cycles

Abstract Background Circadian rhythms, observed across all domains of life, enable organisms to anticipate and prepare for diel changes in environmental conditions. In bacteria, a circadian clock mechanism has only been characterized in cyanobacteria to date. These clocks regulate cyclical patterns of gene expression and metabolism which contribute to the success of cyanobacteria in their natural environments. The potential impact of self-generated circadian rhythms in other bacterial and microbial populations has motivated extensive research to identify novel circadian clocks. Main text Daily oscillations in microbial community composition and function have been observed in ocean ecosystems and in symbioses. These oscillations are influenced by abiotic factors such as light and the availability of nutrients. In the ocean ecosystems and in some marine symbioses, oscillations are largely controlled by light-dark cycles. In gut systems, the influx of nutrients after host feeding drastically alters the composition and function of the gut microbiota. Conversely, the gut microbiota can influence the host circadian rhythm by a variety of mechanisms including through interacting with the host immune system. The intricate and complex relationship between the microbiota and their host makes it challenging to disentangle host behaviors from bacterial circadian rhythms and clock mechanisms that might govern the daily oscillations observed in these microbial populations. Conclusions While the ability to anticipate the cyclical behaviors of their host would likely be enhanced by a self-sustained circadian rhythm, more evidence and further studies are needed to confirm whether host-associated heterotrophic bacteria possess such systems. In addition, the mechanisms by which heterotrophic bacteria might respond to diel cycles in environmental conditions has yet to be uncovered

Comparison of the antimicrobial activity of honey produced by Tetragonisca angustula (Meliponinae) and Apis mellifera from different phytogeographic regions of Costa Rica

The purpose of the present study was to investigate and compare the demonstrated variation in antimicrobial activity of honey produced by introduced A. mellifera and the stingless bee, Tetragonisca angustula, commonly kept in hives in Costa Rica. There was no difference in activity of honey produced by Apis mellifera and T. angustula against the 5 microbes tested. Honey from different phytogeographic regions exhibited differential antimicrobial activity and susceptibility of yeasts to honey of either species was greater than that of bacteria