NCT02788071.

Background Metagenomic sequencing is a well-established tool in the modern biosciences. While it promises unparalleled insights into the genetic content of the biological samples studied, conclusions drawn are at risk from biases inherent to the DNA sequencing methods, including inaccurate abundance estimates as a function of genomic guanine-cytosine (GC) contents. Results We explored such GC biases across many commonly used platforms in experiments sequencing multiple genomes (with mean GC contents ranging from 28.9% to 62.4%) and metagenomes. GC bias profiles varied among different library preparation protocols and sequencing platforms. We found that our workflows using MiSeq and NextSeq were hindered by major GC biases, with problems becoming increasingly severe outside the 45–65% GC range, leading to a falsely low coverage in GC-rich and especially GC-poor sequences, where genomic windows with 30% GC content had >10-fold less coverage than windows close to 50% GC content. We also showed that GC content correlates tightly with coverage biases. The PacBio and HiSeq platforms also evidenced similar profiles of GC biases to each other, which were distinct from those seen in the MiSeq and NextSeq workflows. The Oxford Nanopore workflow was not afflicted by GC bias. Conclusions These findings indicate potential sources of difficulty, arising from GC biases, in genome sequencing that could be pre-emptively addressed with methodological optimizations provided that the GC biases inherent to the relevant workflow are understood. Furthermore, it is recommended that a more critical approach be taken in quantitative abundance estimates in metagenomic studies. In the future, metagenomic studies should take steps to account for the effects of GC bias before drawing conclusions, or they should use a demonstrably unbiased workflow.

Mobilization of insoluble soil inorganic phosphate by plant beneficial rhizobacteria is a trait of key importance to the development of microbial biofertilizers. In this study, the ability of several Pseudomonas spp. to solubilize Ca3 (PO4 )2 was compared. While all Pseudomonas spp. were found to facilitate a decrease in pH and solubilize inorganic phosphate by the production of extracellular organic acids, strains varied by producing either gluconic or 2-ketogluconic acid. Furthermore, comparison between the Pseudomonas spp. of the genes involved in oxidative glucose metabolism revealed variations in genomic organization. To further investigate the genetic mechanisms involved in inorganic phosphate solubilization by Pseudomonas spp., a transposon mutant library of P. fluorescens F113 was screened for mutants with reduced Ca3 (PO4 )2 solubilization ability. Mutations in the gcd and pqqE genes greatly reduced the solubilization ability, whereas mutations in the pqqB gene only moderately reduced this ability. The combination of biochemical analysis and genomic comparisons revealed that alterations in the pqq biosynthetic genes, and the presence/absence of the gluconate dehydrogenase (gad) gene, fundamentally affect phosphate solublization in strains of P. fluorescens.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.