Design of a Whole-Cell Biosensor Based on Bacillus subtilis Spores and the Green Fluorescent Protein To Monitor Arsenic

ABSTRACT A green fluorescent protein (GFP)-based whole-cell biosensor (WCB-GFP) for monitoring arsenic (As) was developed in Bacillus subtilis. To this end, we designed a reporter gene fusion carrying the gfpmut3a gene under the control of the promoter/operator region of the arsenic operon (Pars::gfpmut3a) in the extrachromosomal plasmid pAD123. This construct was transformed into B. subtilis 168, and the resultant strain was used as a whole-cell biosensor (BsWCB-GFP) for the detection of As. The BsWCB-GFP was specifically activated by inorganic As(III) and As(V), but not by dimethylarsinic acid [DMA(V)], and exhibited high tolerance to the noxious effects of arsenic. Accordingly, after 12 h exposure, B. subtilis cells carrying the Pars::gfpmut3a fusion exhibited 50 and 90% lethal doses (LD50 and LD90) to As(III) of 0.89 mM and As 1.71 mM, respectively. Notably, dormant spores from the BsWCB-GFP were able to report the presence of As(III) in a concentration range from 0.1 to 1,000 μM 4 h after the onset of germination. In summary, the specificity and high sensitivity for As, as well as its ability to proliferate under concentrations of the metal that are considered toxic in water and soil, makes the B. subtilis biosensor developed here a potentially important tool for monitoring environmental samples contaminated with this pollutant. IMPORTANCE Arsenic (As) contamination of groundwater is associated with serious worldwide health risks. Detection of this pollutant at concentrations that are established as permissible for water consumption by WHO is a matter of significant interest. Here, we report the generation of a whole-cell biosensor for As detection in the Gram-positive spore former B. subtilis. This biosensor reports the presence of inorganic As, activating the expression of the green fluorescent protein (GFP) under the control of the promoter/operator of the ars operon. The biosensor can proliferate under concentrations of As(III) that are considered toxic in water and soil and detect this ion at concentrations as low as 0.1 μM. Of note, spores of the Pars-GFP biosensor exhibited the ability to detect As(III) following germination and outgrowth. Therefore, this novel tool has the potential to be directly applied to monitor As contamination in environmental samples

Polymorphisms in DNA Repair Genes (APEX1, XPD, XRCC1 and XRCC3) and Risk of Preeclampsia in a Mexican Mestizo Population

Variations in genes involved in DNA repair systems have been proposed as risk factors for the development of preeclampsia (PE). We conducted a case-control study to investigate the association of Human apurinic/apyrimidinic (AP) endonuclease (APEX1) Asp148Glu (rs1130409), Xeroderma Pigmentosum group D (XPD) Lys751Gln (rs13181), X-ray repair cross-complementing group 1 (XRCC) Arg399Gln (rs25487) and X-ray repair cross-complementing group 3 (XRCC3) Thr241Met (rs861539) polymorphisms with PE in a Mexican population. Samples of 202 cases and 350 controls were genotyped using RTPCR. Association analyses based on a χ2 test and binary logistic regression were performed to determine the odds ratio (OR) and a 95% confidence interval (95% CI) for each polymorphism. The allelic frequencies of APEX1 Asp148Glu polymorphism showed statistical significant differences between preeclamptic and normal women (p = 0.036). Although neither of the polymorphisms proved to be a risk factor for the disease, the APEX1 Asp148Glu polymorphism showed a tendency of association (OR: 1.74, 95% CI = 0.96–3.14) and a significant trend (p for trend = 0.048). A subgroup analyses revealed differences in the allelic frequencies of APEX1 Asp148Glu polymorphism between women with mild preeclampsia and severe preeclampsia (p = 0.035). In conclusion, our results reveal no association between XPD Lys751Gln, XRCC Arg399Gln and XRCC3 Thr241Met polymorphisms and the risk of PE in a Mexican mestizo population; however, the results in the APEX1 Asp148Glu polymorphism suggest the need for future studies using a larger sample size

Hypocholesterolemia is an independent risk factor for depression disorder and suicide attempt in Northern Mexican population

Abstract Background Cholesterol has been associated as a risk factor for cardiovascular disease. Recently, however, there is growing evidence about crucial requirement of neuron membrane cholesterol in the organization and function of the 5-HT1A serotonin receptor. For this, low cholesterol level has been reported to be associated with depression and suicidality. However there have been inconsistent reports about this finding and the exact relationship between these factors remains controversial. Therefore, we investigated the link between serum cholesterol and its fractions with depression disorder and suicide attempt in 467 adult subjects in Mexican mestizo population. Methods Plasma levels of total cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol (LDL-c) were determined in 261 MDD patients meeting the DSM-5 criteria for major depressive disorder (MDD), 59 of whom had undergone an episode of suicide attempt, and 206 healthy controls. Results A significant decrease in total cholesterol, LDL-cholesterol, VLDL-cholesterol and triglyceride serum levels was observed in the groups of MDD patients and suicide attempt compared to those without suicidal behavior (p < 0.05). After adjusting for covariates, lower cholesterol levels were significantly associated with MDD (OR 4.229 CI 95% 2.555 – 7.000, p<.001) and suicide attempt (OR 5.540 CI 95% 2.825 – 10.866, p<.001) Conclusions These results support the hypothesis that lower levels of cholesterol are associated with mood disorders like MDD and suicidal behavior. More mechanistic studies are needed to further explain this association

YwqL (EndoV), ExoA and PolA act in a novel alternative excision pathway to repair deaminated DNA bases in Bacillus subtilis.

DNA deamination generates base transitions and apurinic/apyrimidinic (AP)-sites which are potentially genotoxic and cytotoxic. In Bacillus subtilis uracil can be removed from DNA by the uracil DNA-glycosylase through the base excision repair pathway. Genetic evidence suggests that B. subtilis YwqL, a homolog of Endonuclease-V (EndoV), acts on a wider spectrum of deaminated bases but the factors that complete this pathway have remained elusive. Here, we report that a purified His6-YwqL (hereafter BsEndoV) protein had in vitro endonuclease activity against double-stranded DNAs containing a single uracil (U), hypoxanthine (Hx), xanthine (X) or an AP site. Interestingly, while BsEndoV catalyzed a single strand break at the second phosphodiester bond towards the 3'-end of the U and AP lesions, there was an additional cleavage of the phosphodiester bond preceding the Hx and X lesions. Remarkably, the repair event initiated by BsEndoV on Hx and X, was completed by a recombinant B. subtilis His6-DNA polymerase A (BsPolA), but not on BsEndoV-processed U and AP lesions. For the latter lesions a second excision event performed by a recombinant B. subtilis His6-ExoA (BsExoA) was necessary before completion of their repair by BsPolA. These results suggest the existence of a novel alternative excision repair pathway in B. subtilis that counteracts the genotoxic effects of base deamination. The presence of this novel pathway in vivo in B. subtilis was also supported by analysis of effects of single or multiple deletions of exoA, endoV and polA on spontaneous mutations in growing cells, and the sensitivity of growing wild-type and mutant cells to a DNA deaminating agent