Using photocatalyzed-peroxonization to disinfect and denature genetic material of bacterial plasmids present in hospital wastewater.

The literature reports the presence of multiresistant microorganisms in wastewater discharged from municipal and hospital wastewater treatment plants (WWTPs). This has led to questions concerning the disinfection efficiency of the treatments applied. Thus, this study aimed to assess the efficiency of different chemical oxidation methods to disinfect and to degrade bacterial plasmids present in hospital wastewaters, to avoid the dispersion of antibiotic resistance genes in the environment. The methods tested were UV254nm alone or associated with an Ag or Ti-photocatalyst in photo-peroxonization (UV254 nm/H2O2/O3/Ag2O/Ag2CO3@PU or UV254 nm/H2O2/O3/TiO2@PU) under different pH conditions (4, 7, and 10). The application of plasmid DNA electrophoresis to hospital wastewater treated using an advanced oxidation process (AOP) achieved the total structural denaturation of microorganism plasmids at the three pH values tested. Also, UV254 nm alone was partially efficient in the disinfection of hospital wastewater. AOPs performed with the two functionalized catalysts resulted in 100% disinfection after 10 min at the three pH values tested. No intact plasmids were observed after 20 min of treatment with photocatalysis. This study could contribute to the development and improvement of wastewater treatment aimed at mitigating the spread of multiresistant microorganisms in the environment.

Genetic material present in hospital wastewaters: Evaluation of the efficiency of DNA denaturation by ozonolysis and ozonolysis/sonolysis treatments.

Hospital wastewater treatments must ensure that all genetic material is destroyed, since nuclear and extra-nuclear DNA can show antimicrobial resistance and contain recombinant genes, which promote vertical and/or horizontal gene transfer, amplifying the current problem of the emergence of antibiotic-resistant microorganisms. In this study, we investigated whether ozonolysis or ozonolysis/sonolysis in combination can denature genetic material, i.e., destroy the integrity of DNA molecules, present in hospital wastewaters. To achieve this goal, hospital wastewaters were treated by ozonolysis or ozonolysis/sonolysis in combination (at 70 and 100 W L(-1)) and both raw and treated wastewaters were analyzed in terms of disinfection and DNA denaturation efficiency quantified by viable cell counts and by agarose gel electrophoresis. In the ozonolysis treatment, the agarose gel electrophoresis technique showed that the ozone-treated samples contained DNA molecules, while combined ozonolysis/sonolysis destroyed the DNA in a power density-dependent manner (64% at 70 W L(-1) and 81% at 100 W L(-1)). Care must be taken by environmental managers to distinguish disinfection processes from DNA denaturation processes, since these two terms are not synonymous.

The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.

The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.

Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte â-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2). Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH) and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp) and modE2 (372 bp) genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae.