Biosynthesis Of Vitamin Research Articles

Influence of plant growth regulators on key‐coding genes expression associated with phytochemicals biosynthesis and antioxidant activity in soybean (Glycine max (L.) Merr) sprouts

SummaryThe present study was aimed to investigate the effect of plant growth regulators (PGRs) on phytochemicals biosynthesis and antioxidant activity in soybean sprouts. GmVTC2, GmDHAR and GmGLDH were investigated for the biosynthesis of vitamin C. GmPAL, GmCHS and GmIFS were investigated for the biosynthesis of phenolics, flavonoids and isoflavones. Our results revealed that 6‐benzylaminopurine (6‐BA) treatment significantly affected the expression levels of those major genes, followed by Gibberellin (GA3) and Naphthaleneacetic acid (NAA). Moreover, the highest levels of vitamin C, polyphenolics and antioxidant activity in 6‐BA and GA3 treated groups indicate the synergistic and antagonistic role of growth regulators in the biosynthesis of phytochemicals. Overall, 6‐BA seems the key contributor in genes’ expression involve in the biosynthesis secondary metabolites and their antioxidant activity. Therefore, it could be the most suitable option to enhance nutritional quantity and health benefits of soybean sprouts.

Vitamin D and the gut microbiome: a systematic review of in vivo studies.

Variation in the human microbiome has been linked with a variety of physiological functions, including immune regulation and metabolism and biosynthesis of vitamins, hormones, and neurotransmitters. Evidence for extraskeletal effects of vitamin D has been accruing and it has been suggested that the effect of vitamin D on health is partially mediated through the microbiome. We aimed to critically evaluate the evidence linking vitamin D and the gastrointestinal microbiome. We systematically searched the Embase, Web of Science, PubMed and CINAHL databases, including peer-reviewed publications that reported an association between a measure of vitamin D and the gastrointestinal microbiome in humans or experimental animals. We included 10 mouse and 14 human studies. Mouse studies compared mice fed diets containing different levels of vitamin D (usually high versus low), or vitamin D receptor knockout or Cyp27B1 knockout with wild-type mice. Five mouse studies reported an increase in Bacteroidetes (or taxa within that phylum) in the low vitamin D diet or gene knockout group. Human studies were predominantly observational; all but two of the included studies found some association between vitamin D and the gut microbiome, but the nature of differences observed varied across studies. Despite substantial heterogeneity, we found evidence to support the hypothesis that vitamin D influences the composition of the gastrointestinal microbiome. However, the research is limited, having been conducted either in mice or in mostly small, selected human populations. Future research in larger population-based studies is needed to fully understand the extent to which vitamin D modulates the microbiome.

Invited review: Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility

Various body sites of vertebrates provide stable and nutrient-rich ecosystems for a diverse range of commensal, opportunistic, and pathogenic microorganisms to thrive. The collective genomes of these microbial symbionts (the microbiome) provide host animals with several advantages, including metabolism of indigestible carbohydrates, biosynthesis of vitamins, and modulation of innate and adaptive immune systems. In the context of the bovine udder, however, the relationship between cow and microbes has been traditionally viewed strictly from the perspective of host-pathogen interactions, with intramammary infections by mastitis pathogens triggering inflammatory responses (i.e., mastitis) that are often detrimental to mammary tissues and cow physiology. This traditional view has been challenged by recent metagenomic studies indicating that mammary secretions of clinically healthy quarters can harbor genomic markers of diverse bacterial groups, the vast majority of which have not been associated with mastitis. These observations have given rise to the concept of "commensal mammary microbiota," the ecological properties of which can have important implications for understanding the pathogenesis of mastitis and offer opportunities for development of novel prophylactic or therapeutic products (or both) as alternatives to antimicrobials. Studies conducted to date have suggested that an optimum diversity of mammary microbiota is associated with immune homeostasis, whereas the microbiota of mastitic quarters, or those with a history of mastitis, are considerably less diverse. Whether disruption of the diversity of udder microbiota (dysbiosis) has a role in determining mastitis susceptibility remains unknown. Moreover, little is known about contributions of various biotic and abiotic factors in shaping overall diversity of udder microbiota. This review summarizes current understanding of the microbiota within various niches of the udder and highlights the need to view the microbiota of the teat apex, teat canal, and mammary secretions as interconnected niches of a highly dynamic microbial ecosystem. In addition, host-associated factors, including physiological and anatomical parameters, as well as genetic traits that may affect the udder microbiota are briefly discussed. Finally, current understanding of the effect of antimicrobials on the composition of intramammary microbiota is discussed, highlighting the resilience of udder microbiota to exogenous perturbants.

To B or Not to B: Comparative Genomics Suggests Arsenophonus as a Source of B Vitamins in Whiteflies.

Insect lineages feeding on nutritionally restricted diets such as phloem sap, xylem sap, or blood, were able to diversify by acquiring bacterial species that complement lacking nutrients. These bacteria, considered obligate/primary endosymbionts, share a long evolutionary history with their hosts. In some cases, however, these endosymbionts are not able to fulfill all of their host's nutritional requirements, driving the acquisition of additional symbiotic species. Phloem-feeding members of the insect family Aleyrodidae (whiteflies) established an obligate relationship with Candidatus Portiera aleyrodidarum, which provides its hots with essential amino acids and carotenoids. In addition, many whitefly species harbor additional endosymbionts which may potentially further supplement their host's diet. To test this hypothesis, genomes of several endosymbionts of the whiteflies Aleurodicus dispersus, Aleurodicus floccissimus and Trialeurodes vaporariorum were analyzed. In addition to Portiera, all three species were found to harbor one Arsenophonus and one Wolbachia endosymbiont. A comparative analysis of Arsenophonus genomes revealed that although all three are capable of synthesizing B vitamins and cofactors, such as pyridoxal, riboflavin, or folate, their genomes and phylogenetic relationship vary greatly. Arsenophonus of A. floccissimus and T. vaporariorum belong to the same clade, and display characteristics of facultative endosymbionts, such as large genomes (3 Mb) with thousands of genes and pseudogenes, intermediate GC content, and mobile genetic elements. In contrast, Arsenophonus of A. dispersus belongs to a different lineage and displays the characteristics of a primary endosymbiont—a reduced genome (670 kb) with ~400 genes, 32% GC content, and no mobile genetic elements. However, the presence of 274 pseudogenes suggests that this symbiotic association is more recent than other reported primary endosymbionts of hemipterans. The gene repertoire of Arsenophonus of A. dispersus is completely integrated in the symbiotic consortia, and the biosynthesis of most vitamins occurs in shared pathways with its host. In addition, Wolbachia endosymbionts have also retained the ability to produce riboflavin, flavin adenine dinucleotide, and folate, and may make a nutritional contribution. Taken together, our results show that Arsenophonus hold a pivotal place in whitefly nutrition by their ability to produce B vitamins.

Transcriptional Regulation of Vitamin E Biosynthesis during Germination of Dwarf Fan Palm Seeds.

Vitamin E, a potent antioxidant either presents in the form of tocopherols and/or tocotrienols depending on the plant species, tissue and developmental stage, plays a major role in protecting lipids from oxidation in seeds. Unlike tocopherols, which have a more universal distribution, the occurrence of tocotrienols is limited primarily to monocot seeds. Dwarf fan palm (Chamaerops humilis var. humilis) seeds accumulate tocotrienols in quiescent and dormant seeds, while tocopherols are de novo synthesized during germination. Here, we aimed to elucidate whether tocopherol biosynthesis is regulated at the transcriptional level during germination in this species. We identified and quantified the expression levels of five genes involved in vitamin E biosynthesis, including TYROSINE AMINOTRANSFERASE (ChTAT), HOMOGENTISATE PHYTYLTRANSFERASE (ChHPT), HOMOGENTISATE GERANYLGERANYL TRANSFERASE (ChHGGT), TOCOPHEROL CYCLASE (ChTC) and TOCOPHEROL γ-METHYLTRANSFERASE (Chγ-TMT). Furthermore, we evaluated to what extent variations in the endogenous contents of hormones and hydrogen peroxide (H2O2) correlated with transcriptional regulation. Results showed an increase of ChTAT and ChHPT levels during seed germination, which correlated with an increase of jasmonic acid (JA), gibberellin4 (GA4), and H2O2 contents, while ChHGGT and Chγ-TMT expression levels decreased, thus clearly indicating vitamin E biosynthesis is diverted to tocopherols rather than to tocotrienols. Exogenous application of jasmonic acid increased tocopherol, but not tocotrienol content, thus confirming its regulatory role in vitamin E biosynthesis during seed germination. It is concluded that the biosynthesis of vitamin E is regulated at the transcriptional level during germination in dwarf fan palm seeds, with ChHPT playing a key role in the diversion of the vitamin E pathway towards tocopherols instead of tocotrienols.

Characterization of the Functional Changes in Mouse Gut Microbiome Associated with Increased Akkermansia muciniphila Population Modulated by Dietary Black Raspberries.

Gut microbiome plays an essential role in host health through host–gut microbiota metabolic interactions. Desirable modulation of beneficial gut bacteria, such as Akkermansia muciniphila, can confer health benefits by altering microbiome-related metabolic profiles. The purpose of this study is to examine the effects of a black raspberry-rich diet to reshape the gut microbiome by selectively boosting A. muciniphila population in C57BL/6J mice. Remarkable changes of the mouse gut microbiome were revealed at both compositional and functional levels with an expected increase of A. muciniphila in concert with a profound impact on multiple gut microbiome-related functions, including vitamin biosynthesis, aromatic amino acid metabolism, carbohydrate metabolism, and oxidative stress. These functional alterations in the gut microbiome by an easily accessed freeze-dried black raspberry-supplemented diet may provide novel insights on the improvement of human health via gut microbiome modulation.

Comparative Genomic Analysis and Characterization of Two Salmonella enterica Serovar Enteritidis Isolates From Poultry With Notably Different Survival Abilities in Egg Whites.

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is a globally important foodborne pathogen, and the contaminated chicken eggs are the major source of salmonellosis in humans. Salmonella Enteritidis strains are differentially susceptible to the hostile environment of egg whites. Strains with superior survival ability in egg whites are more likely to contaminate eggs and consequently infect humans. However, the genetic basis for this phenotype is unclear. We characterized two Salmonella Enteritidis strains isolated from chicken meat that had similar genetic backgrounds but large differences in survival ability in egg whites. Although genome comparisons indicated that the gene content and genomic synteny were highly conserved, variations including six insertions or deletions (INDELs) and 70 single nucleotide polymorphisms (SNPs) were observed between the two genomes. Of these, 38 variations including four INDELs and 34 non-synonymous SNPs (nsSNP) were annotated to result in amino acid substitutions or INDELs in coding proteins. These variations were located in 38 genes involved in lysozyme inhibition, vitamin biosynthesis, cell division and DNA damage response, osmotic and oxidative protection, iron-related functions, cell envelope maintenance, amino acid and carbohydrate metabolism, antimicrobial resistance, and type III secretion system. We carried out allelic replacements for two nsSNPs in bioC (biotin synthesis) and pliC (lysozyme inhibition), and two INDELs in ftsK and yqiJ (DNA damage response) by homologous recombination, and these replacements did not alter the bacterial survival ability in egg whites. However, the bacterial survival ability in egg whites was reduced when deletion mutation of the genes bioC and pliC occurred. This study provides initial correlations between observed genotypes and phenotypes and serves as an important caveat for further functional studies.

Using bioreactors to study the effects of drugs on the human microbiota.

The study of complex microbial communities has become a major research focus as mounting evidence suggests the pivotal role microbial communities play in host health and disease. Microbial communities of the gastrointestinal tract, known as the gut microbiota, have been implicated in aiding the host with vitamin biosynthesis, regulation of host energy metabolism, immune system development, and resistance to pathogen invasion. Conversely, disruptions of the gut microbiota have been linked to host morbidity, including the development of inflammatory diseases, metabolic disorders, increased cardiovascular risk, and increased risk of infectious diseases. However, studying the gut microbiota in humans and animals is challenging, as many microorganisms are fastidious with unique nutritional or environmental requirements that are often not met using conventional culture techniques. Bioreactors provide a unique solution to overcome some of the limitations of conventional culture techniques. Bioreactors have been used to propagate and establish complex microbial communities in vitro by recapitulating the physiological conditions found in the GI tract. These systems further our understanding of microbial physiology and facilitate our understanding of the impact of medications and xenobiotics on microbial communities. Here, we review the versatility and breadth of bioreactor systems that are currently available and how they are being used to study faecal and defined microbial communities. Bioreactors provide a unique opportunity to study complex microbial interactions and perturbations in vitro in a controlled environment without confounding biotic and abiotic variables.

Switching to a fibre-rich and low-fat diet increases colonic folate contents among African Americans.

How dietary patterns impact colonic bacterial biosynthesis of vitamins and utilization by humans is poorly understood. Our aim was to investigate whether a reciprocal dietary switch between rural South Africans (traditionally high fibre, low fat) and African Americans (Western diet of low fibre, high fat) affects colonic folate synthesis. Colonic evacuants were obtained from 20 rural South Africans and 20 African Americans consuming their usual diets at baseline. For 2 weeks thereafter, rural South Africans were provided with a Western diet (protein, 27%; fat, 52%; carbohydrate, 20%; and fibre, 8 g/day) and African Americans were provided with a high fibre, low-fat diet (protein, 16%; fat, 17%; carbohydrate, 63%; and fibre, 43 g/day). Colonic evacuants were again collected. No difference between groups at baseline in the folate content of 3-h evacuants was observed. The high-fibre, low-fat diet consumed by African Americans during the intervention produced a 41% increase in mean total folate content compared with baseline values (p = 0.0037). No change was observed in rural South Africans consuming a Western diet. Mean total folate content of colonic evacuants was higher among African Americans at the end of the dietary switch (3107 ± 1811 μg) compared with rural South Africans (2157 ± 1956 μg) (p = 0.0409). In conclusion, consistent with animal studies, switching from a Western diet to one higher in fibre and lower in fat can be expected to result in greater colonic folate content. Future research should confirm that these observations are not transitory and understand the contribution of transit-time to the findings.

Genome Evolution of Bartonellaceae Symbionts of Ants at the Opposite Ends of the Trophic Scale.

Many insects rely on bacterial symbionts to supply essential amino acids and vitamins that are deficient in their diets, but metabolic comparisons of closely related gut bacteria in insects with different dietary preferences have not been performed. Here, we demonstrate that herbivorous ants of the genus Dolichoderus from the Peruvian Amazon host bacteria of the family Bartonellaceae, known for establishing chronic or pathogenic infections in mammals. We detected these bacteria in all studied Dolichoderus species, and found that they reside in the midgut wall, that is, the same location as many previously described nutritional endosymbionts of insects. The genomic analysis of four divergent strains infecting different Dolichoderus species revealed genes encoding pathways for nitrogen recycling and biosynthesis of several vitamins and all essential amino acids. In contrast, several biosynthetic pathways have been lost, whereas genes for the import and conversion of histidine and arginine to glutamine have been retained in the genome of a closely related gut bacterium of the carnivorous ant Harpegnathos saltator. The broad biosynthetic repertoire in Bartonellaceae of herbivorous ants resembled that of gut bacteria of honeybees that likewise feed on carbohydrate-rich diets. Taken together, the broad distribution of Bartonellaceae across Dolichoderus ants, their small genome sizes, the specific location within hosts, and the broad biosynthetic capability suggest that these bacteria are nutritional symbionts in herbivorous ants. The results highlight the important role of the host nutritional biology for the genomic evolution of the gut microbiota—and conversely, the importance of the microbiota for the nutrition of hosts.

Single-Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate-Dependent Catalysis in Vitamin K Biosynthesis.

MenD, or (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase, uses a thiamine diphosphate (ThDP)-dependent tetrahedral Breslow intermediate rather than a canonical enamine for catalysis in the biosynthesis of vitamin K. By real-time monitoring of the cofactor chemical state with circular dichroism spectroscopy, we found that a new post-decarboxylation intermediate was formed from a multistep process that was rate limited by binding of the α-ketoglutarate substrate before it quickly relaxed to the characterized tetrahedral Breslow intermediate. In addition, the chemical steps leading to the reactive post-decarboxylation intermediates were not affected by the electrophilic substrate, isochorismate, whereas release of the product was found to limit the whole catalytic process. Moreover, these intermediates are likely kinetically stabilized owing to the low biological availability of isochorismate under physiological conditions, in contrast to the tight coupling of enamine formation with binding of the electrophilic acceptor in some other ThDP-dependent enzymes. Together with the unusual tetrahedral structure of the intermediates, these findings strongly support a new ThDP-dependent catalytic mode distinct from canonical enamine chemistry.

Vitamin Biosynthesis as an Antifungal Target.

The large increase in the population of immunosuppressed patients, coupled with the limited efficacy of existing antifungals and rising resistance toward them, have dramatically highlighted the need to develop novel drugs for the treatment of invasive fungal infections. An attractive possibility is the identification of possible drug targets within essential fungal metabolic pathways not shared with humans. Here, we review the vitamin biosynthetic pathways (vitamins A–E, K) as candidates for the development of antifungals. We present a set of ranking criteria that identify the vitamin B2 (riboflavin), B5 (pantothenic acid), and B9 (folate) biosynthesis pathways as being particularly rich in new antifungal targets. We propose that recent scientific advances in the fields of drug design and fungal genomics have developed sufficiently to merit a renewed look at these pathways as promising sources for the development of novel classes of antifungals.

Metabolic impact induced by total, water soluble and insoluble components of PM2.5 acute exposure in mice

Fine particulate matter (PM2.5) has been listed as an important environmental risk factor for human health. However, the systemic biological effects on metabolic responses induced by PM2.5 and its components were poorly understood. This study was aimed to evaluate the toxicity of different components of PM2.5 at molecular level via metabolomics approach. In the present study, we adopted a 1H NMR-based metabolomics approach to evaluate metabolic profiles in mice after acute exposure to Total-PM2.5, water soluble components of PM2.5 (WS-PM2.5) and water insoluble components of PM2.5 (WIS-PM2.5). First, we characterized the morphological features and chemical composition of PM2.5. Then, the metabolites changes of serum and urine in mice were systematically analyzed using 800 MHz 1H NMR techniques in combination with multivariate statistical analysis. Total-PM2.5 exposure affected metabolites mainly involved in amino acid metabolism, protein biosynthesis, energy metabolism and metabolism of cofactors and vitamins. WS-PM2.5 exposure influenced lipid metabolism and carbohydrate metabolism. WIS-PM2.5 exposure mainly perturbed amino acid metabolism and energy metabolism. The results suggested that acute exposure to the Total-PM2.5, WS-PM2.5 and WIS-PM2.5 in mice exhibited marked systemic metabolic changes. In addition, the insoluble fraction of PM2.5 contributed greatly to the toxicity of PM2.5.

Metagenomic and metatranscriptomic analysis of the microbial community in Swiss-type Maasdam cheese during ripening

In Swiss-type cheeses, characteristic nut-like and sweet flavor develops during the cheese ripening due to the metabolic activities of cheese microbiota. Temperature changes during warm and cold room ripening, and duration of ripening can significantly change the gene expression of the cheese microbiota, which can affect the flavor formation. In this study, a metagenomic and metatranscriptomic analysis of Swiss-type Maasdam cheese was performed on samples obtained during ripening in the warm and cold rooms. We reconstructed four different bacterial genomes (Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus helveticus, and Propionibacterium freudenreichii subsp. shermanii strain JS) from the Maasdam cheese to near completeness. Based on the DNA and RNA mean coverage, Lc. lactis strongly dominated (~80–90%) within the cheese microbial community. Genome annotation showed the potential for the presence of several flavor forming pathways in these species, such as production of methanethiol, free fatty acids, acetoin, diacetyl, acetate, ethanol, and propionate. Using the metatranscriptomic data, we showed that, with the exception of Lc. lactis, the central metabolism of the microbiota was downregulated during cold room ripening suggesting that fewer flavor compounds such as acetoin and propionate were produced. In contrast, Lc. lactis genes related to the central metabolism, including the vitamin biosynthesis and homolactic fermentation, were upregulated during cold room ripening.

Vitamins Based Novel Target Pathways/Molecules as Possible Emerging Drug Targets for the Management of Tuberculosis.

Tuberculosis (TB) is a deadly infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb). Approximately, 1.8 and 1.3 million people are infected and die, from TB each year as estimated by the World Health Organization. Due to increase in the incidence of drug-resistant strains of Mtb, there is an urgent need to accelerate research which focuses on the development of new drugs with novel mechanism of action that can treat both drugsensitive and resistant TB infections. The purpose of this review study was to describe vitamins as drug target that can be explored to develop new anti tubercular drugs that can treat both drug-sensitive and resistant TB infections. The methodological approaches include literature review which is performed in the databases like PubMed, Web of Science, Scopus, Springer and Science Direct, etc. On the basis of evaluation of literature sources, the review was complied. This review study demonstrated that vitamins biosynthesis pathway could be used in the development of novel drug targets. Further sequencing of the Mtb genome facilitated research in target identification and validation that make possible the discovery of novel anti-TB agent with new mechanisms of action. Several compounds were identified, which target vitamin biosynthesis pathway /enzymes. Some other new targets were also identified and can be explored for the identification of novel structural moiety. Further exploration of these compounds which have been identified to target these vitamins related novel target pathways /molecules could led to the development of antitubercular drug which can be used in the treatment of drug sensitive and resistant TB.

Fermented Food and Non-Communicable Chronic Diseases: A Review.

Fermented foods represent a significant fraction of human diets. Although their impact on health is positively perceived, an objective evaluation is still missing. We have, therefore, reviewed meta-analyses of randomized controlled trials (RCT) investigating the relationship between fermented foods and non-transmissible chronic diseases. Overall, after summarizing 25 prospective studies on dairy products, the association of fermented dairy with cancer was found to be neutral, whereas it was weakly beneficial, though inconsistent, for specific aspects of cardio-metabolic health, in particular stroke and cheese intake. The strongest evidence for a beneficial effect was for yoghurt on risk factors of type 2 diabetes. Although mechanisms explaining this association have not been validated, an increased bioavailability of insulinotropic amino acids and peptides as well as the bacterial biosynthesis of vitamins, in particular vitamin K2, might contribute to this beneficial effect. However, the heterogeneity in the design of the studies and the investigated foods impedes a definitive assessment of these associations. The literature on fermented plants is characterized by a wealth of in vitro data, whose positive results are not corroborated in humans due to the absence of RCTs. Finally, none of the RCTs were specifically designed to address the impact of food fermentation on health. This question should be addressed in future human studies.

ABA participates in the regulation of vitamin C content in the fruit of strawberry using lanthanum nitrate

Abstract By pot experiment method, this study investigated the role of plant hormone abscisic acid (ABA) in the foliar application of 10 μM lanthanum nitrate (La(NO3)3)-regulated the biosynthesis, regeneration and degradation of vitamin C (Vc) and the content of Vc in the fruit of strawberry at different periods of growth and development. The results showed that La(NO3)3 significantly increased the content of Vc by increasingthe activities of recycling enzymes glutathione reductase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and biosynthetic enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH), and decreasing the activities of recycling enzyme APX and degrading enzyme ascorbic acid oxidase (AAO). Compared with La(NO3)3 alone, applications of different ABA biosynthesis inhibitor sodium tungstate (Tu) all markedly decreased the activities of GR, MDHAR and GalLDH, and increased the activities of DHAR and APX, which resulted in the reduction in the content of Vc. Meanwhile, La(NO3)3 induced the production of ABA and Tu reversed the effect of La(NO3)3 on ABA content. Our results suggested that La(NO3)3-induced ABA participated in the regulation of Vc content in the fruit of strawberry.

The biosynthesis and mutual usage of vitamins by marine picoplankton.

The biosynthesis and mutual usage of vitamins by marine picoplankton.

Gut microbiome contributes to impairment of immunity in pulmonary tuberculosis patients by alteration of butyrate and propionate producers.

Tuberculosis (TB) is primarily associated with decline in immune health status. As gut microbiome (GM) is implicated in the regulation of host immunity and metabolism, here we investigate GM alteration in TB patients by 16S rRNA gene and whole-genome shotgun sequencing. The study group constituted of patients with pulmonary TB and their healthy household contacts as controls (HCs). Significant alteration of microbial taxonomic and functional capacity was observed in patients with active TB as compared to the HCs. We observed that Prevotella and Bifidobacterium abundance were associated with HCs, whereas butyrate and propionate-producing bacteria like Faecalibacterium, Roseburia, Eubacterium and Phascolarctobacterium were significantly enriched in TB patients. Functional analysis showed reduced biosynthesis of vitamins and amino acids in favour of enriched metabolism of butyrate and propionate in TB subjects. The TB subjects were also investigated during the course of treatment, to analyse the variation of GM. Although perturbation in microbial composition was still evident after a month's administration of anti-TB drugs, significant changes were observed in metagenome gene pool that pointed towards recovery in functional capacity. Therefore, the findings from this pilot study suggest that microbial dysbiosis may contribute to pathophysiology of TB by enhancing the anti-inflammatory milieu in the host.

RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing.

The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named “RiboFACSeq”. Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis.