Deuterium-depletion has no significant impact on the mutation rate of Escherichia coli, invalidating the Double D-Bridge Hypothesis

Deuterium (D), the second most abundant isotope of hydrogen is present in natural waters at an approximate concentration of 145-155 ppm (cca. 1.5E-4 atom/atom). D is known to influence various biological processes due to its physical and chemical properties, which significantly differ from those of hydrogen. For example, increasing D-concentration to >1000-fold above its natural abundance has been shown to increase the frequency of genetic mutations in several species. The Double D-Bridge Hypothesis, formulated with the intent of explaining the mechanism of D-mutagenicity is based on the calculation that the theoretical frequency of A:T base pairs that comprise two D-bridges instead of H-bridges is 2E-8, which is equal to the mutation rate of certain species. To experimentally challenge this hypothesis, and to infer the mutagenicity of D present at natural concentrations, we investigated the effect of a nearly 100-fold reduction of D concentration on the bacterial mutation rate. Using fluctuation tests, we measured the mutation rate of three Escherichia coli genes (cycA, ackA and galK) in media containing D at either <2 ppm or 150 ppm concentrations. Out of 15 pair-wise fluctuation analyses, nine indicated a significant decrease, while three marked the significant increase of the mutation/culture value upon D-depletion. Overall, growth in D-depleted minimal medium led to a geometric mean of 0.663-fold (95% confidence interval: 0.483-0.911) change in the mutation rate. This falls nowhere near the expected 10,000-fold reduction, indicating that the Double D-Bridge Hypothesis does not hold. In addition, the combined results did not display a statistically significant change in the mutation/culture value, the mutation rate or the mutant frequency upon D-depletion. The potential mutagenic effect of D present at natural concentrations on E. coli is therefore below the limit of detection using the indicated methods.


Introduction 45
Hydrogen (H or 1 H) and deuterium (D or  melanogaster [17] and mammalian cell lines [18] are all inhibited in growth or reproduction. 68 intoxication has not been studied for ethical reasons, but a 100-fold elevation of cellular D 69 concentration for 6 weeks led to only mild and transient symptoms [21,22]. 70 The mutagenic effect of deuterium has also been in the focus of multiple investigations for 71 several species. The presence of high (50-99.8 %) D 2 O concentration was shown to elevate 72 mutation frequencies in bacteriophage T4 [12], in various bacteria including Escherichia coli 73 [13], Salmonella typhimurium [23], Proteus mirabilis [24] and Streptococcus pneumoniae 74 [25], and in Oryza sativa [16]. The observable changes in mutation frequencies were often 75 strain and locus-dependent, but on average meant an order of magnitude of increase. Two 76 investigations reported no mutagenic effect of D 2 O in D. melanogaster [26] and E. coli [27], 77 respectively, which may have resulted from the low number of parallels used. A study also 78 proved that deuterium-oxide enhanced the mutation rate induced by gamma rays in cultured 79 mouse leukemia cells [28]. More recently, the in vitro error-promoting effect of 99 % D 2 O 80 was demonstrated for Taq DNA polymerase, increasing the frequency of mutations within 81 PCR products approx. nine fold [29]. 82 Among the numerous explanations regarding the mutagenic effect of D, perhaps the most 83 interesting one is what we refer to as the Double D-Bridge Hypothesis [30]. The basis of this 84 explanation is the fact that the frequency of A:T base pairs harboring two D-bridges instead 85 of two H-bridges (i.e. 1.5 x 10 -4 squared) is approximately equal to the mutation rate (~2 x 86 10 -8 mutations/nucleotide/generation) calculated for humans and Caenorhabditis elegans 87 [30]. This model implicitly assumes that the presence of D is the only cause of single 88 nucleotide exchange events, and that practically each such double D-bridge leads to a point 89 mutation. If this were the case, a 2-order of magnitude decrease in the labile D-content of 90 DNA should yield a 4-order of magnitude decrease in the mutation rate, considering that the 91 frequency of double D-bridges is proportional to the square of D-abundance.

92
Apart from mutagenicity-tests, the past decade has brought about an increasing amount of 93 data indicating that the D content of natural waters may have a significant effect on biological 94 processes, e.g. concerning brain function [31], cellular aging [32] and tumor growth [33].

95
Although the mutagenic effect of D 2 O seems to be convincingly demonstrated at >1000-fold 96 increased concentrations, no study has yet investigated the potential effect of D on the rate of 97 mutations at its natural abundance.

98
In this work, we measure the effect of reducing the D content of the culture medium on the 99 mutation rate of E. coli for two reasons: to test the Double D-Bridge Hypothesis, and to 100 assess the contribution of natural D concentrations to the spontaneous mutagenesis of 101 bacterial cells. We report a minor decrease in mutation rate upon D-depletion, not meeting 102 most of our criteria for statistical significance, which stands far from the 10,000-fold (=100 103 squared) reduction expected based on the Double D-Bridge Hypothesis.    The instrument uses off-axis integrated cavity output spectroscopy to measure absolute 136 abundances of 2 HHO via laser absorption [35]. Isotopic ratios are reported as δD value with 137 respect to the international VSMOW standard in permill according to the following formula:  galU_SpF and galU_SpR, which carry homology arms specific for galU (S1 Table). The 154 PCR product was electroporated into E. coli MG1655 carrying the arabinose-induced pKD46 155 plasmid (GenBank ID: AY048746.1). Recombinants were selected on LB+Sp plates.

156
Colonies were screened by colony-PCR using primers galU_D and SmFw. One positive  valid experiments can be found in S2_Table, marked as plain and bold text, respectively.

194
Raw data (colony counts) corresponding to all experiments are available in S1_File.

237
In this study, our main goal was to compare the mutation rates of bacterial cultures grown in  Table). Among the two remaining assays, a 23.2% increase was detected upon D-

407
When analyzing individual fluctuation test-pairs, a method more informative than t-tests is to 408 seek non-overlapping 95% confidence intervals, for this type of analysis takes the magnitude 409 of the effect into account as well [39,55]. Only three of the 15 valid fluctuation test-pairs 410 yielded non-overlapping CI95 ranges (Fig 2), which means that in 12 cases, we cannot  examples of mutation-inhibition, certain fruit juices were classified as anti-mutagens based 477 on their capability to reduce UV-induced mutant frequency 2-2.5 fold, measured using the 478 Rif R assay [61]. What is common in all the papers cited above is that only those changes in 479 mutation rate or mutation frequency are considered relevant which are greater than two-fold.

480
Exceptions to this rule are rare, the only ones we found from other authors was a 1.