Adaptive Laboratory Evolution Restores Solvent Tolerance in Plasmid-Cured Pseudomonas putida S12: a Molecular Analysis.

Pseudomonas putida S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, arpR, that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit ß', a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FoF1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in P. putida S12 lacking its megaplasmid.IMPORTANCE Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

Long term treatment with metformin in patients with type 2 diabetes and risk of vitamin B-12 deficiency: randomised placebo controlled trial.

OBJECTIVES: To study the effects of metformin on the incidence of vitamin B-12 deficiency (<150 pmol/l), low concentrations of vitamin B-12 (150-220 pmol/l), and folate and homocysteine concentrations in patients with type 2 diabetes receiving treatment with insulin. DESIGN: Multicentre randomised placebo controlled trial. SETTING: Outpatient clinics of three non-academic hospitals in the Netherlands. PARTICIPANTS: 390 patients with type 2 diabetes receiving treatment with insulin. INTERVENTION: 850 mg metformin or placebo three times a day for 4.3 years. MAIN OUTCOME MEASURES: Percentage change in vitamin B-12, folate, and homocysteine concentrations from baseline at 4, 17, 30, 43, and 52 months. RESULTS: Compared with placebo, metformin treatment was associated with a mean decrease in vitamin B-12 concentration of -19% (95% confidence interval -24% to -14%; P<0.001) and in folate concentration of -5% (95% CI -10% to -0.4%; P=0.033), and an increase in homocysteine concentration of 5% (95% CI -1% to 11%; P=0.091). After adjustment for body mass index and smoking, no significant effect of metformin on folate concentrations was found. The absolute risk of vitamin B-12 deficiency (<150 pmol/l) at study end was 7.2 percentage points higher in the metformin group than in the placebo group (95% CI 2.3 to 12.1; P=0.004), with a number needed to harm of 13.8 per 4.3 years (95% CI 43.5 to 8.3). The absolute risk of low vitamin B-12 concentration (150-220 pmol/l) at study end was 11.2 percentage points higher in the metformin group (95% CI 4.6 to 17.9; P=0.001), with a number needed to harm of 8.9 per 4.3 years (95% CI 21.7 to 5.6). Patients with vitamin B-12 deficiency at study end had a mean homocysteine level of 23.7 micromol/l (95% CI 18.8 to 30.0 micromol/l), compared with a mean homocysteine level of 18.1 micromol/l (95% CI 16.7 to 19.6 micromol/l; P=0.003) for patients with a low vitamin B-12 concentration and 14.9 micromol/l (95% CI 14.3 to 15.5 micromol/l; P<0.001 compared with vitamin B-12 deficiency; P=0.005 compared with low vitamin B-12) for patients with a normal vitamin B-12 concentration (>220 pmol/l). CONCLUSIONS: Long term treatment with metformin increases the risk of vitamin B-12 deficiency, which results in raised homocysteine concentrations. Vitamin B-12 deficiency is preventable; therefore, our findings suggest that regular measurement of vitamin B-12 concentrations during long term metformin treatment should be strongly considered. Trial registration Clinicaltrials.gov NCT00375388.