The Edible Plant Microbiome represents a diverse genetic reservoir with functional potential in the human host.

Plant microbiomes have been extensively studied for their agricultural relevance on growth promotion and pathogenesis, but little is known about their role as part of the diet when fresh fruits and vegetables are consumed raw. Most studies describing these communities are based on 16S rRNA gene amplicon surveys, limiting our understanding of the taxonomic resolution at the species level and functional capabilities. In this study, we characterized microbes colonizing tomatoes, spinach, brined olives, and dried figs using shotgun metagenomics. We recovered metagenome-assembled genomes of novel lactic acid bacteria from green olives and identified high intra- and inter-specific diversity of Pseudomonas in tomatoes. All samples were colonized by Pseudomonas, consistent with other reports with distinct community structure. Functional characterization showed the presence of enzymes involved in vitamin and short chain fatty acid metabolism and degradation of diverse carbohydrate substrates including plant fibers. The dominant bacterial members were isolated, sequenced, and mapped to its metagenome confirming their identity and indicating the microbiota is culturable. Our results reveal high genetic diversity, previously uncultured genera, and specific functions reflecting a likely plant host association. This study highlights the potential that plant microbes can play when consumed as part of our diet and proposes these as transient contributors to the gut microbiome.

Targeting the Intestinal Microbiota to Prevent Type 2 Diabetes and Enhance the Effect of Metformin on Glycaemia: A Randomised Controlled Pilot Study.

Early treatment may prevent or delay the onset of type 2 diabetes mellitus (T2DM) in individuals who are at high risk. Lifestyle interventions and the hypoglycemic drug metformin have been shown to reduce T2DM incidence. The effectiveness of such interventions may be enhanced by targeting environmental factors such as the intestinal microbiota, which has been proven to predict the response to lifestyle interventions and play a part in mediating the glucose-lowering effects of metformin. Shifts in the intestinal microbiota "towards a more balanced state" may promote glucose homeostasis by regulating short-chain fatty acids' production. This study aimed to investigate the safety and effect of a multi-strain probiotic on glycemic, inflammatory, and permeability markers in adults with prediabetes and early T2DM and to assess whether the probiotic can enhance metformin's effect on glycaemia. A randomised controlled pilot study was conducted in 60 adults with a BMI ≥ 25 kg/m2 and with prediabetes or T2DM (within the previous 12 months). The participants were randomised to a multi-strain probiotic (L. plantarum, L. bulgaricus, L. gasseri, B. breve, B. animalis sbsp. lactis, B. bifidum, S. thermophilus, and S. boulardii) or placebo for 12 weeks. Analyses of the primary outcome (fasting plasma glucose) and secondary outcomes, including, but not limited to, circulating lipopolysaccharide, zonulin, and short chain fatty acids and a metagenomic analysis of the fecal microbiome were performed at baseline and 12 weeks post-intervention. The results showed no significant differences in the primary and secondary outcome measures between the probiotic and placebo group. An analysis of a subgroup of participants taking metformin showed a decrease in fasting plasma glucose, HbA1c, insulin resistance, and zonulin; an increase in plasma butyrate concentrations; and an enrichment of microbial butyrate-producing pathways in the probiotic group but not in the placebo group. Probiotics may act as an adjunctive to metformin by increasing the production of butyrate, which may consequently enhance glucose management.

The impact of skin care products on skin chemistry and microbiome dynamics.

BACKGROUND: Use of skin personal care products on a regular basis is nearly ubiquitous, but their effects on molecular and microbial diversity of the skin are unknown. We evaluated the impact of four beauty products (a facial lotion, a moisturizer, a foot powder, and a deodorant) on 11 volunteers over 9 weeks. RESULTS: Mass spectrometry and 16S rRNA inventories of the skin revealed decreases in chemical as well as in bacterial and archaeal diversity on halting deodorant use. Specific compounds from beauty products used before the study remain detectable with half-lives of 0.5-1.9 weeks. The deodorant and foot powder increased molecular, bacterial, and archaeal diversity, while arm and face lotions had little effect on bacterial and archaeal but increased chemical diversity. Personal care product effects last for weeks and produce highly individualized responses, including alterations in steroid and pheromone levels and in bacterial and archaeal ecosystem structure and dynamics. CONCLUSIONS: These findings may lead to next-generation precision beauty products and therapies for skin disorders.

Predicting proteome allocation, overflow metabolism, and metal requirements in a model acetogen.

The unique capability of acetogens to ferment a broad range of substrates renders them ideal candidates for the biotechnological production of commodity chemicals. In particular the ability to grow with H2:CO2 or syngas (a mixture of H2/CO/CO2) makes these microorganisms ideal chassis for sustainable bioproduction. However, advanced design strategies for acetogens are currently hampered by incomplete knowledge about their physiology and our inability to accurately predict phenotypes. Here we describe the reconstruction of a novel genome-scale model of metabolism and macromolecular synthesis (ME-model) to gain new insights into the biology of the model acetogen Clostridium ljungdahlii. The model represents the first ME-model of a Gram-positive bacterium and captures all major central metabolic, amino acid, nucleotide, lipid, major cofactors, and vitamin synthesis pathways as well as pathways to synthesis RNA and protein molecules necessary to catalyze these reactions, thus significantly broadens the scope and predictability. Use of the model revealed how protein allocation and media composition influence metabolic pathways and energy conservation in acetogens and accurately predicted secretion of multiple fermentation products. Predicting overflow metabolism is of particular interest since it enables new design strategies, e.g. the formation of glycerol, a novel product for C. ljungdahlii, thus broadening the metabolic capability for this model microbe. Furthermore, prediction and experimental validation of changing secretion rates based on different metal availability opens the window into fermentation optimization and provides new knowledge about the proteome utilization and carbon flux in acetogens.

Optimization of carbon and energy utilization through differential translational efficiency.

Control of translation is vital to all species. Here we employ a multi-omics approach to decipher condition-dependent translational regulation in the model acetogen Clostridium ljungdahlii. Integration of data from cells grown autotrophically or heterotrophically revealed that pathways critical to carbon and energy metabolism are under strong translational regulation. Major pathways involved in carbon and energy metabolism are not only differentially transcribed and translated, but their translational efficiencies are differentially elevated in response to resource availability under different growth conditions. We show that translational efficiency is not static and that it changes dynamically in response to mRNA expression levels. mRNAs harboring optimized 5'-untranslated region and coding region features, have higher translational efficiencies and are significantly enriched in genes encoding carbon and energy metabolism. In contrast, mRNAs enriched in housekeeping functions harbor sub-optimal features and have lower translational efficiencies. We propose that regulation of translational efficiency is crucial for effectively controlling resource allocation in energy-deprived microorganisms.

Transcriptome analysis of wild-type and afsS deletion mutant strains identifies synergistic transcriptional regulator of afsS for a high antibiotic-producing strain of Streptomyces coelicolor A3(2).

Most secondary metabolism in Actinobacteria is controlled by multi-layered, gene-regulatory networks. These regulatory mechanisms are not easily identified due to their complexity. As a result, when a strong transcriptional regulator (TR) governs activation of biosynthetic pathways of target antibiotics such as actinorhodin (ACT), additional enhancement of the biosynthesis is difficult in combination with other TRs. To find out any "synergistic transcriptional regulators (sTRs)" that show an additive effect on the major, often strong, transcriptional regulator (mTR), here, we performed a clustering analysis using the transcriptome datasets of an mTR deletion mutant and wild-type strain. In the case of ACT biosynthesis in Streptomyces coelicolor, PhoU (SCO4228) and RsfA (SCO4677) were selected through the clustering analysis, using AfsS (SCO4425) as a model mTR, and experimentally validated their roles as sTRs. Furthermore, through analysis of synergistic effects, we were able to suggest a novel regulation mechanism and formulate a strategy to maximize the synergistic effect. In the case of the double TR mutant strain (ΔrsfA pIBR25::afsS), it was confirmed that the increase of cell mass was the major cause of the synergistic effect. Therefore, the strategy to increase the cell mass of double mutant was further attempted by optimizing the expression of efflux pump, which resulted in 2-fold increase in the cell mass and 24-fold increase in the production of ACT. This result is the highest ACT yield from S. coelicolor ever reported.

Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis.

The microbiome can promote or disrupt human health by influencing both adaptive and innate immune functions. We tested whether bacteria that normally reside on human skin participate in host defense by killing Staphylococcus aureus, a pathogen commonly found in patients with atopic dermatitis (AD) and an important factor that exacerbates this disease. High-throughput screening for antimicrobial activity against S. aureus was performed on isolates of coagulase-negative Staphylococcus (CoNS) collected from the skin of healthy and AD subjects. CoNS strains with antimicrobial activity were common on the normal population but rare on AD subjects. A low frequency of strains with antimicrobial activity correlated with colonization by S. aureus The antimicrobial activity was identified as previously unknown antimicrobial peptides (AMPs) produced by CoNS species including Staphylococcus epidermidis and Staphylococcus hominis These AMPs were strain-specific, highly potent, selectively killed S. aureus, and synergized with the human AMP LL-37. Application of these CoNS strains to mice confirmed their defense function in vivo relative to application of nonactive strains. Strikingly, reintroduction of antimicrobial CoNS strains to human subjects with AD decreased colonization by S. aureus These findings show how commensal skin bacteria protect against pathogens and demonstrate how dysbiosis of the skin microbiome can lead to disease.

The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2).

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development.

Comparative Genomics Reveals the Core and Accessory Genomes of Streptomyces Species.

The development of rapid and efficient genome sequencing methods has enabled us to study the evolutionary background of bacterial genetic information. Here, we present comparative genomic analysis of 17 Streptomyces species, for which the genome has been completely sequenced, using the pan-genome approach. The analysis revealed that 34,592 ortholog clusters constituted the pan-genome of these Streptomyces species, including 2,018 in the core genome, 11,743 in the dispensable genome, and 20,831 in the unique genome. The core genome was converged to a smaller number of genes than reported previously, with 3,096 gene families. Functional enrichment analysis showed that genes involved in transcription were most abundant in the Streptomyces pan-genome. Finally, we investigated core genes for the sigma factors, mycothiol biosynthesis pathway, and secondary metabolism pathways; our data showed that many genes involved in stress response and morphological differentiation were commonly expressed in Streptomyces species. Elucidation of the core genome offers a basis for understanding the functional evolution of Streptomyces species and provides insights into target selection for the construction of industrial strains.

Genome-scale analysis reveals a role for NdgR in the thiol oxidative stress response in Streptomyces coelicolor.

BACKGROUND: NdgR is an IclR-type transcription factor that regulates leucine biosynthesis and other metabolic pathways in Streptomyces coelicolor. Recent study revealed that NdgR is one of the regulatory targets of SigR, an oxidative stress response sigma factor, suggesting that the NdgR plays an important physiological role in response to environmental stresses. Although the regulatory functions of NdgR were partly characterized, determination of its regulon is required for better understanding of the transcriptional regulatory network related with the oxidative stress response. RESULTS: We determined genome-wide binding loci of NdgR by using chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and explored its physiological roles. The ChIP-seq profiles revealed 19 direct binding loci with a 15-bp imperfect palindromic motif, including 34 genes in their transcription units. Most genes in branched-chain amino acid and cysteine biosynthesis pathways were involved in the NdgR regulon. We proved that ndgR is induced by SigR under the thiol oxidation, and that an ndgR mutant strain is sensitive to the thiol oxidizing agent, diamide. Through the expression test of NdgR and the target genes for NdgR under diamide treatment, regulatory motifs were suggested. Interestingly, NdgR constitutes two regulatory motifs, coherent and incoherent feed-forward loops (FFL), in order to control its regulon under the diamide treatment. Using the regulatory motifs, NdgR regulates cysteine biosynthesis in response to thiol oxidative stress, enabling cells to maintain sulfur assimilation with homeostasis under stress conditions. CONCLUSIONS: Our analysis revealed that NdgR is a global transcriptional regulator involved in the regulation of branched-chain amino acids biosynthesis and sulphur assimilation. The identification of the NdgR regulon broadens our knowledge regarding complex regulatory networks governing amino acid biosynthesis in the context of stress responses in S. coelicolor.

Reconstruction of a high-quality metabolic model enables the identification of gene overexpression targets for enhanced antibiotic production in Streptomyces coelicolor A3(2).

Streptomycetes are industrially and pharmaceutically important bacteria that produce a variety of secondary metabolites including antibiotics. Streptomycetes have a complex metabolic network responsible for the production of secondary metabolites and the utilization of organic residues present in soil. In this study, we reconstructed a high-quality metabolic model for Streptomyces coelicolor A3(2), designated iMK1208, in order to understand and engineer the metabolism of this model species. In comparison to iIB711, the previous metabolic model for S. coelicolor, the predictive power of iMK1208 was enhanced by the recent insights that enabled the incorporation of an updated biomass equation, stoichiometric matrix, and energetic parameters. iMK1208 was validated by comparing predictions with the experimental data for growth capability in various growth media. Furthermore, we applied a strain-design algorithm, flux scanning based on enforced objective flux (FSEOF), to iMK1208 for actinorhodin overproduction. FSEOF results identified not only previously known gene overexpression targets such as actII-ORF4 and acetyl-CoA carboxylase, but also novel targets such as branched-chain α-keto acid dehydrogenase (BCDH). We constructed and evaluated the BCDH overexpression mutant, which showed a 52-fold increase in actinorhodin production, validating the prediction power of iMK1208. Hence iMK1208 was shown to be a useful and valuable framework for studying the biotechnologically important Streptomyces species using the principles of systems biology and metabolic engineering.

Identification of the specific electron transfer proteins, ferredoxin, and ferredoxin reductase, for CYP105D7 in Streptomyces avermitilis MA4680.

It was previously proposed that regiospecific hydroxylation of daidzein at 3'-position is mediated by cytochrome P450 hydroxylase (CYP105D7) in the presence of putidaredoxin (CamB) and putidaredoxin reductase (CamA) as electron transfer proteins from Pseudomonas putida. The genome sequence of Streptomyces avermitilis MA4680 revealed 33 P450 (CYPs) with 6 ferredoxin reductases (Fprs) and 9 ferredoxins (Fdxs) as their putative electron transfer partner proteins. To identify right endogenous electron transfer proteins for CYP105D7 activity, in vitro reconstitution, gene disruption, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mRNA expression profile analysis were examined. The most effective electron transfer proteins for CYP105D7 appear to be FdxH (SAV7470), which is located downstream to CYP105D7 as a cluster, and FprD (SAV5675). Throughout our overall analysis, we proposed that the primary electron transfer pathway for CYP105D7 follows as such NAD(P)H→FdxH→FprD→CYP105D7.

NdgR, a common transcriptional activator for methionine and leucine biosynthesis in Streptomyces coelicolor.

We show here that NdgR, a known transcriptional activator of isopropylmalate dehydratase in actinomycetes, may have other targets in the cell. An in-frame deletion mutant of ndgR showed unexpectedly poor growth in defined minimal medium even in the presence of leucine. To our surprise, it was supplementation of cysteine and methionine that corrected the growth. Based on this, we propose that NdgR induces cysteine-methionine biosynthesis. Direct involvement of NdgR in the very last steps of methionine synthesis with methionine synthase (metH) and 5,10-methylenetetrahydrofolate reductase (metF) was examined. From a pulldown assay, it was seen that NdgR was enriched from crude cell lysates with a strong affinity to metH and metF upstream sequences. Direct physical interaction of NdgR with these targets was further examined with a gel mobility shift assay. ndgR, leuC, metH, and metF were inducible in M145 cells upon nutrient downshift from rich to minimal medium but were not induced in the ndgR knockout mutant. Taking these observations together, NdgR-dependent metH-metF expression would account for the abnormal growth phenotype of the ndgR mutant although there may be additional NdgR-dependent genes in the Cys-Met metabolic pathways. As the first transcriptional factor reported for regulating Cys-Met metabolism in Streptomyces, NdgR links two disparate amino acid families, branched-chain amino acids (BCAAs) and sulfur amino acids, at the transcriptional level. Considering that Cys-Met metabolism is connected to mycothiol and one-carbon metabolism, NdgR may have broad physiological impacts.

A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.

Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor.

γ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

A novel function of Streptomyces integration host factor (sIHF) in the control of antibiotic production and sporulation in Streptomyces coelicolor.

Bacterial integration host factors (IHFs) play important roles in site-specific recombination, DNA replication, transcription, genome organization and bacterial pathogenesis. In Streptomyces coelicolor, there are three putative IHFs: SCO1480, SCO2950 and SCO5556. SCO1480 or Streptomyces IHF (sIHF) was previously identified as a transcription factor that binds to the promoter region of redD, the pathway-specific regulatory gene for the undecylprodigiosin biosynthetic gene cluster. Here we show that production of the pigmented antibiotics actinorhodin and undecylprodigiosin is strongly enhanced in sihf null mutants, while sporulation was strongly inhibited, with an on average 25% increase in spore size. Furthermore, the sihf mutant spores showed strongly reduced viability, with high sensitivity to heat and live/dead staining revealing a high proportion of empty spores, while enhanced expression of sIHF increased viability. This suggests a major role for sIHF in controlling viability, perhaps via the control of DNA replication and/or segregation. Proteomic analysis of the sihf null mutant identified several differentially expressed transcriptional regulators, indicating that sIHF may have an extensive response regulon. These data surprisingly reveal that a basic architectural element conserved in many actinobacteria such as mycobacteria, corynebacteria, streptomycetes and rhodococci may act as a global regulator of secondary metabolism and cell development.

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor.

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor∆manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor∆manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor∆manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.

Loss of phosphomannomutase activity enhances actinorhodin production in Streptomyces coelicolor.

Phosphomannomutase (ManB), whose main function is the conversion of mannose-6-phosphate to mannose-1-phosphate, is involved in biosynthesis of GDP-mannose for numerous processes such as synthesis of structural carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins in prokaryotes and eukaryotes. ManB isolated from Streptomyces coelicolor was shown to have both phosphomannomutase and phosphoglucomutase activities. Deletion of manB in S. coelicolor caused a dramatic increase in actinorhodin (ACT) production in the low-glucose Difco nutrient (DN) medium, whereas the wild-type strain did not produce ACT on this medium. Experiments involving complementation of the manB deletion showed that increased ACT production in DN media was due to blockage of phosphomannomutase activity rather than phosphoglucomutase activity. This result therefore provides useful information for the design of strategies that enhance antibiotic production through the control of carbon flux.

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of Streptomyces coelicolor.

In order to identify the regulators involved in antibiotic production or time-specific cellular events, the messenger ribonucleic acid (mRNA) expression data of the two gene clusters, actinorhodin (ACT) and undecylprodigiosin (RED) biosynthetic genes, were clustered with known mRNA expression data of regulators from S. coelicolor using a filtering method based on standard deviation and clustering analysis. The result identified five regulators including two well-known regulators namely, SCO3579 (WlbA) and SCO6722 (SsgD). Using overexpression and deletion of the regulator genes, we were able to identify two regulators, i.e., SCO0608 and SCO6808, playing roles as repressors in antibiotics production and sporulation. This approach can be easily applied to mapping out new regulators related to any interesting target gene clusters showing characteristic expression patterns. The result can also be used to provide insightful information on the selection rules among a large number of regulators.