The faulty SOS response of Pseudomonas putida KT2440 stems from an inefficient RecA-LexA interplay.

Despite its environmental robustness Pseudomonas putida strain KT2440 is very sensitive to DNA damage and displays poor homologous recombination efficiencies. To gain an insight into this deficiency isogenic ∆recA and ∆lexA1 derivatives of prophage-free strain P. putida EM173 were generated and responses of the recA and lexA1 promoters to DNA damage tested with GFP reporter technology. Basal expression of recA and lexA1 of P. putida were high in the absence of DNA damage and only moderately induced by norfloxacin. A similar behaviour was observed when equivalent GFP fusions to the recA and lexA promoters of E. coli were placed in P. putida EM173. In contrast, all SOS promoters were subject to strong repression in E. coli, which was released only when cells were treated with the antibiotic. Replacement of P. putida's native LexA1 and RecA by E. coli homologues did not improve the responsiveness of the indigenous functions to DNA damage. Taken together, it seems that P. putida fails to mount a strong SOS response due to the inefficacy of the crucial RecA-LexA interplay largely tractable to the weakness of the corresponding promoters and the inability of the repressor to shut them down entirely in the absence of DNA damage.

Evolving metabolism of 2,4-dinitrotoluene triggers SOS-independent diversification of host cells.

The molecular mechanisms behind the mutagenic effect of reactive oxygen species (ROS) released by defective metabolization of xenobiotic 2,4-dinitrotoluene (DNT) by a still-evolving degradation pathway were studied. To this end, the genes required for biodegradation of DNT from Burkholderia cepacia R34 were implanted in Escherichia coli and the effect of catabolizing the nitroaromatic compound monitored with stress-related markers and reporters. Such a proxy of the naturally-occurring scenario faithfully recreated the known accumulation of ROS caused by faulty metabolism of DNT and the ensuing onset of an intense mutagenesis regime. While ROS triggered an oxidative stress response, neither homologous recombination was stimulated nor the recA promoter activity increased during DNT catabolism. Analysis of single-nucleotide changes occurring in rpoB during DNT degradation suggested a relaxation of DNA replication fidelity rather than direct damage to DNA. Mutants frequencies were determined in strains defective in either converting DNA damage into mutagenesis or mediating inhibition of mismatch repair through a general stress response. The results revealed that the mutagenic effect of ROS was largely SOS-independent and stemmed instead from stress-induced changes of rpoS functionality. Evolution of novel metabolic properties thus resembles the way sublethal antibiotic concentrations stimulate the appearance of novel resistance genes.

Comparison of proliferation and osteogenic differentiation potential of bovine adipose tissue and bone marrow derived stem cells.

Bone marrow derived stem cells (BMSC) are the most utilized cell type in the field of bone regeneration. Although BMSC are both safe and efficacious, the search for alternative sources for stem cells continues. We investigated bovine BMSC and adipose tissue derived mesenchymal stem cells (ATSC) using immunofluorescence and PCR. We further compared the osteogenic differentiation potentials of both sources of stem cells. We assessed alkaline phosphatase (ALP) enzyme levels and calcium deposition in differentiating cells at days 7, 14 and 21 to compare the osteogenic differentiation capability of both cell types. We found that ATSC expressed significantly higher ALP levels compared to BMSC throughout osteogenic differentiation. Calcium deposition was greater in ATSC than BMSC at days 7 and 14. By the end of day 21, BMSC produced greater calcium deposition. We found that ATSC undergo osteogenic differentiation more rapidly than BMSC, but BMSC provide greater mineralization over longer periods.

Mutations in the translation initiation region of the pac gene resulting in increased levels of activity of penicillin G acylase.

Penicillin G acylase (PA) is an important enzyme used in the industrial production of b-lactam antibiotics. In this study, the effects of mutations in the translation initiation region of the Escherichia coli pac gene, encoding periplasmic PA, were examined. Several mutations led to increased amounts of PA activity, including those that lengthened the spacer region between the ribosome binding site and the ATG start codon, and those with altered codons on positions +2 and +4 relative to the start codon. These results indicated that the wild-type sequence of the pac gene does not provide maximum expression levels and that the strategies applied in this study can be used to improve production of PA in E. coli. Unexpectedly, our study also suggested that translocation of PA was, in contrast to earlier reports, shown not to require the Twin-arginine translocation pathway for transport into the periplasm.

Nicotiana tabacum-associated bioengineered Pseudomonas putida can enhance rhizoremediation of soil containing 2,4-dinitrotoluene.

Rhizoremediation processes are based on plant-bacteria interactions and can be effectively used for cleaning many pollutants from the environment to overcome the constraints of individual phytoremediation. Here, 1 mM and 1.5 mM concentrations of 2,4-dinitrotoluene (2,4-DNT) degrading Pseudomonas putida (P. putida) strain KT.DNT and various growth stages of Nicotiana tabacum (N. tabacum) were initially assayed in in vitro tissue culture system and the best conditions for the association of plant-rhizobacterium were ascertained to remediation of the soil contaminated with 2,4-DNT. 5-days old N. tabacum plants inoculated with 2 × 106 cfu/mL bacterial inoculum for 3 weeks were preferred for rhizoremediation experiments as they showed a nearly threefold increase in the fresh and dry biomass in comparison to noninoculated ones. When these seedlings were planted either alone or together with P. putida KT2440 or P. putida KT.DNT in soils contaminated with 1 mM and 1.5 mM of 2,4-DNT, the maximum degradation rate of 98% and ~ 93% were determined at the end of 14 days by KT.DNT inoculated tobacco plants. Our results indicate that it would be advantageous to use the 2,4-DNT-degrading bacterium inoculated with N. tabacum plants to accelerate and enhance the cleanup of soil contaminated with 2,4-DNT.

Molecular characterization of bovine amniotic fluid derived stem cells with an underlying focus on their comparative neuronal potential at different passages.

BACKGROUND: The excellence in the field of stem cell therapy demands alternative and more convenient stem cells for potential applications. Researchers have opted for least invasive and broadly multipotent cells with minimum ethical concerns. Bovine amniotic fluid derived mesenchymal stem cells (BAF-MSCs) due to their ease of collection and owing similar gestational length to that of human could be presumed as an attractive large animal model for biomedical and biotechnology research. METHODS: Bovine amniotic fluid derived stem cells were isolated from abattoir based samples and characterized for epithelial, neuronal, mesenchymal and pluripotent markers by qPCR and immunofluorescence studies at P1, P3, P5 and P7 alongside population doubling time, growth curve and multilineage differentiation studies. RESULTS: The cells were explored for unique expression of Sox2, which was observed to be up regulated with increase in passage number and Nestin was found to be downregulated during further passaging of mesenchymal cells in this study. The cells also co-expressed Oct ¾ at initial passages which diminished within further passages. Evidence regarding diversity and heterogeneity in different cell population in amniotic fluid was recorded by positive expression of epithelial cell markers like pan Cytokeratin and p63 during early passages. The study suggested that cells with higher expression of Sox2 generated comparatively larger neurospheres with comparative strong expression of Sox2 and Nestin by immunofluorescence staining and qPCR analysis. Besides BAF-MSCs derived neurospheres were also shown to express pro-neuronal markers like ß-III Tubulin, GAP43 and ASCL-1. CONCLUSIONS: This study explores and characterizes BAF-MSCs for their multipotent and neurogenic potentials and their use for clinical applications, though more detailed studies are needed to determine the exact pathways linked with neurogenic capacities of these cells and their morphological assessments at different gestational ages in bovines. The knowledge from the bovine model after detailed studies, proven safety and efficacy could also be used to understand substitutive strategies to investigate MSCs physiology at different trimesters and potential application of these cells for human and veterinary regenerative medicine provided the animal ethics are carefully monitored.

Biotransformation of 2,4-dinitrotoluene by the beneficial association of engineered Pseudomonas putida with Arabidopsis thaliana.

2,4-dinitrotoluene (2,4-DNT) is a priority environmental xenobiotic pollutant which has toxic, mutagenic, and carcinogenic properties. Thus, its biodegradation by applying recent approaches such as taking advantage of plant-bacteria interactions is crucial. In this work, the genes from Burkholderia sp. R34, necessary for 2,4-DNT degradation, were integrated into wild-type Pseudomonas putida (P. putida) KT2440 genome, and this strain, named KT.DNT, was inoculated to soil in in vitro conditions. To estimate the disappearance of 2,4-DNT in contaminated soil, samples were taken from different time intervals, extracted and analyzed using high-performance liquid chromatography (HPLC). Biotransformation of 2,4-DNT increased gradually and the degradation in soil after 14-days of treatment with the bacterium was found to be the 97.1%, indicating that the engineered strain could be a remarkable candidate for in situ bioremediation of 2,4-DNT-contaminated sites. In addition, in vitro interaction of this bacterium with a model plant, Arabidopsis thaliana (A. thaliana), enhanced lateral root and root hair formation together with dry root weight. Moreover, the initial 2,4-DNT concentration was decreased to 68% within 2 h with the plant-associated KT.DNT in liquid culture. Hence, the usage of this bacterium with plants could also be a promising application for the 2,4-DNT biotransformation.

The Metabolic Redox Regime of Pseudomonas putida Tunes Its Evolvability toward Novel Xenobiotic Substrates.

During evolution of biodegradation pathways for xenobiotic compounds involving Rieske nonheme iron oxygenases, the transition toward novel substrates is frequently associated with faulty reactions. Such events release reactive oxygen species (ROS), which are endowed with high mutagenic potential. In this study, we evaluated how the operation of the background metabolic network by an environmental bacterium may either foster or curtail the still-evolving pathway for 2,4-dinitrotoluene (2,4-DNT) catabolism. To this end, the genetically tractable strain Pseudomonas putida EM173 was implanted with the whole genetic complement necessary for the complete biodegradation of 2,4-DNT (recruited from the environmental isolate Burkholderia sp. R34). By using reporter technology and direct measurements of ROS formation, we observed that the engineered P. putida strain experienced oxidative stress when catabolizing the nitroaromatic substrate. However, the formation of ROS was neither translated into significant activation of the SOS response to DNA damage nor did it result in a mutagenic regime (unlike what has been observed in Burkholderia sp. R34, the original host of the pathway). To inspect whether the tolerance of P. putida to oxidative challenges could be traced to its characteristic reductive redox regime, we artificially altered the NAD(P)H pool by means of a water-forming, NADH-specific oxidase. Under the resulting low-NAD(P)H status, catabolism of 2,4-DNT triggered a conspicuous mutagenic and genomic diversification scenario. These results indicate that the background biochemical network of environmental bacteria ultimately determines the evolvability of metabolic pathways. Moreover, the data explain the efficacy of some bacteria (e.g., pseudomonads) to host and evolve with new catabolic routes.IMPORTANCE Some environmental bacteria evolve with new capacities for the aerobic biodegradation of chemical pollutants by adapting preexisting redox reactions to novel compounds. The process typically starts by cooption of enzymes from an available route to act on the chemical structure of the substrate-to-be. The critical bottleneck is generally the first biochemical step, and most of the selective pressure operates on reshaping the initial reaction. The interim uncoupling of the novel substrate to preexisting Rieske nonheme iron oxygenases usually results in formation of highly mutagenic ROS. In this work, we demonstrate that the background metabolic regime of the bacterium that hosts an evolving catabolic pathway (e.g., biodegradation of the xenobiotic 2,4-DNT) determines whether the cells either adopt a genetic diversification regime or a robust ROS-tolerant status. Furthermore, our results offer new perspectives to the rational design of efficient whole-cell biocatalysts, which are pursued in contemporary metabolic engineering.