Culture-Independent Metagenomic Surveillance of Commercially Available Probiotics with High-Throughput Next-Generation Sequencing.

Millions of people consume dietary supplements either following a doctor's recommendation or at their own discretion to improve their overall health and well-being. This is a rapidly growing trend, with an associated and expanding manufacturing industry to meet the demand for new health-related products. In this study, we examined the contents and microbial viability of several popular probiotic products on the United States market. Culture-independent methods are proving ideal for fast and efficient analysis of foodborne pathogens and their associated microbial communities but may also be relevant for analyzing probiotics containing mixed microbial constituents. These products were subjected to next-generation whole-genome sequencing and analyzed by a custom in-house-developed k-mer counting method to validate manufacturer label information. In addition, the batch variability of respective products was examined to determine if any changes in their formulations and/or the manufacturing process occurred. Overall, the products we tested adhered to the ingredient claims and lot-to-lot differences were minimal. However, there were a few discrepancies in the naming of closely related Lactobacillus and Bifidobacterium species, whereas one product contained an apparent Enterococcus contaminant in two of its three lots. With the microbial contents of the products identified, we used traditional PCR and colony counting methods to comparatively assess our results and verify the viability of the microbes in these products with regard to the labeling claims. Of all the supplements examined, only one was found to be inaccurate in viability. Our use of next-generation sequencing as an analytical tool clearly demonstrated its utility for quickly analyzing commercially available products containing multiple microbes to ensure consumer safety. IMPORTANCE The rapidly growing supplement industry operates without a formal premarket approval process. Consumers rely on product labels to be accurate and true. Those products containing live microbials report both identity and viability on most product labels. This study used next-generation sequencing technology as an analytical tool in conjunction with classic culture methods to examine the validity of the labels on supplement products containing live microbials found in the United States marketplace. Our results show the importance of testing these products for identity, viability, and potential contaminants, as well as introduce a new culture-independent diagnostic approach for testing these products. Podcast: A podcast concerning this article is available.

The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

To better understand the energetics of accurate DNA replication, we directly measured ΔG(o) for the incorporation of a nucleotide into elongating dsDNA in solution (ΔG(o)(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔG(o)(incorporation) = 5.2 ± 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that ΔΔG(o)(incorporation) alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids; they may only have had to take advantage of the inherently more favorable ΔG(o) for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.

B family DNA polymerases asymmetrically recognize pyrimidines and purines.

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.

Interaction of human DNA polymerase alpha and DNA polymerase I from Bacillus stearothermophilus with hypoxanthine and 8-oxoguanine nucleotides.

To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase alpha (pol alpha), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol alpha strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N(6), or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N(6) from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base pair and that pol alpha uses N3 of a purine dNTP to block this incorporation. Pol alpha also polymerized 8-oxo-dGTP across from a templating A, and removing N(6) from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N(6), and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base pairs by pol alpha, and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N(6), or N7 greatly inhibits incorporation. N(6) and, to a lesser extent, N1 help drive hypoxanthine:adenine base-pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed.

Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.

We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N(2), N-3, and N(6) of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects. Second, it dramatically increased the rate of misincorporation of the resulting dNTP analogues, with alterations in either N-1 or N(6) having the most severe impacts. Adding N(2) to dNTPs containing the bases adenine and purine increased the degree of polymerization opposite T but also tremendously increased the degree of misincorporation opposite A, C, and G. Thus, BF uses N-1, N(2), N-3, and N(6) of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation. Comparing how BF discriminates between right and wrong dNTPs with both B family polymerases and low-fidelity polymerases indicates that BF has chosen a unique solution vis-a-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.

Role of the 2-amino group of purines during dNTP polymerization by human DNA polymerase alpha.

We used a series of dNTP analogues in conjunction with templates containing modified bases to elucidate the role that N(2) of a purine plays during dNTP polymerization by human DNA polymerase alpha. Removing N(2) from dGTP had small effects during correct incorporation opposite C but specifically increased misincorporation opposite A. Adding N(2) to dATP and related analogues had small and variable effects on the efficiency of polymerization opposite T. However, the presence of N(2) greatly enhanced polymerization of these dATP analogues opposite a template C. The ability of N(2) to enhance polymerization opposite C likely results from formation of a hydrogen bond between the purine N(2) and pyrimidine O(2). Even in those cases where formation of a wobble base pair, tautomerization, and/or protonation of the base pair between the incoming dNTP and template base cannot occur (e.g., 2-pyridone.purine (or purine analogue) base pairs), N(2) enhanced formation of the base pair. Importantly, N(2) had similar effects on dNTP polymerization both when added to the incoming purine dNTP and when added to the template base being replicated. The mechanistic implications of these results regarding how pol alpha discriminates between right and wrong dNTPs are discussed.

Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli.

Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were T transversions.

Synthesis, DNA polymerase incorporation, and enzymatic phosphate hydrolysis of formamidopyrimidine nucleoside triphosphates.

The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.

Excision of formamidopyrimidine lesions by endonucleases III and VIII is not a major DNA repair pathway in Escherichia coli.

Proper maintenance of the genome is of great importance. Consequently, damaged nucleotides are repaired through redundant pathways. We considered whether the genome is protected from formamidopyrimidine nucleosides (Fapy*dA, Fapy*dG) via a pathway distinct from the Escherichia coli guanine oxidation system. The formamidopyrimidines are produced in significant quantities in DNA as a result of oxidative stress and are efficiently excised by formamidopyrimidine DNA glycosylase. Previous reports suggest that the formamidopyrimidine nucleosides are substrates for endonucleases III and VIII, enzymes that are typically associated with pyrimidine lesion repair in E.coli. We investigated the possibility that Endo III and/or Endo VIII play a role in formamidopyrimidine nucleoside repair by examining Fapy*dA and Fapy*dG excision opposite all four native 2'-deoxyribonucleotides. Endo VIII excises both lesions more efficiently than does Endo III, but the enzymes exhibit similar selectivity with respect to their action on duplexes containing the formamidopyrimidines opposite native deoxyribonucleotides. Fapy*dA is removed more rapidly than Fapy*dG, and duplexes containing purine nucleotides opposite the lesions are superior substrates compared with those containing formamidopyrimidine-pyrimidine base pairs. This dependence upon opposing nucleotide indicates that Endo III and Endo VIII do not serve as back up enzymes to formamidopyrimidine DNA glycosylase in the repair of formamidopyrimidines. When considered in conjunction with cellular studies [J. O. Blaisdell, Z. Hatahet and S. S. Wallace (1999) J. Bacteriol., 181, 6396-6402], these results also suggest that Endo III and Endo VIII do not protect E.coli against possible mutations attributable to formamidopyrimidine lesions.

Synthesis of oligonucleotides containing Fapy.dG (N(6)-(2-deoxy-alpha,beta-D-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) using a 5'-dimethoxytrityl dinucleotide phosphoramidite.

Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine (11a) or 5'-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS.

Probing the configurations of formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA using endonuclease IV.

The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of oxidative stress. These lesions readily epimerize in water, an unusual property for nucleosides. The equilibrium mixture slightly favors the beta-anomer, but the configurational status in DNA is unknown. The ability of endonuclease IV (Endo IV) to efficiently incise alpha-deoxyadenosine was used as a tool to determine the configuration of Fapy.dA and Fapy.dG in DNA. Endo IV incision of the C-nucleoside analogues of Fapy.dA was used to establish selectivity for the alpha-anomer. Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0 +/- 7.5 nM, k(cat) = 0.58 +/- 0.21 min(-1)), but the beta-isomer is a poor substrate. Fapy.dA incision is considerably slower than that of alpha-C-Fapy.dA, and does not proceed to completion. Endo IV incision of Fapy.dA proceeds further upon rehybridization, suggesting that the lesion reequilibrates and that the enzyme preferentially cleaves duplex DNA containing alpha-Fapy.dA. The extent of Fapy.dA incision suggests that the lesion exists predominantly ( approximately 90%) as the beta-anomer in DNA. Endo IV incises Fapy.dG to less than 5% under comparable reaction conditions, suggesting that the lesion exists almost exclusively as its beta-anomer in DNA.