ABSTRACT
Marine environments are a reservoir of relevant information on dangerous contaminants such as hydrocarbons, as well as microbial communities with probable degradation skills. However, to access microbial diversity, it is necessary to obtain high-quality DNA. An inexpensive, reliable, and effective metagenomic DNA (mgDNA) extraction protocol from marine sediments contaminated with petroleum hydrocarbons was established in this study from modifications to Zhou's protocol. The optimization included pretreatment of sediment with saline solutions for the removal of contaminants, a second precipitation and enzymatic degradation of RNA, followed by purification of mgDNA extracted by electroelution. The results obtained indicated that the modifications applied to 12 sediments with total petroleum hydrocarbon (TPH) concentrations from 22.6-174.3 (µg/g dry sediment) yielded 20.3-321.3 ng/µL mgDNA with A260/A280 and A260/A230 ratios of 1.75 ± 0.08 and 1.19 ± 0.22, respectively. The 16S rRNA amplification confirmed the purity of the mgDNA. The suitability of this mgDNA extraction protocol lies in the fact that all chemical solutions utilized are common in all molecular biology laboratories, and the use of dialysis membrane does not require any sophisticated or expensive equipment, only an electrophoretic chamber.
Subject(s)
Geologic Sediments/microbiology , Metagenome , Petroleum Pollution/adverse effects , Seawater/microbiology , Chemical Fractionation/methods , Geologic Sediments/chemistry , Hydrocarbons/chemistry , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/standards , Seawater/chemistryABSTRACT
Most dramatic examples of actin reorganization have been described during host-microbe interactions. Plasticity of actin is, in part, due to posttranslational modifications such as phosphorylation or ubiquitylation. Here, we show for the first time that actins found in root nodules of Phaseolus vulgaris are modified transiently during nodule development by monoubiquitylation. This finding was extended to root nodules of other legumes and to other plants infected with mycorrhiza or plant pathogens such as members of the genera Pseudomonas and Phytophthora. However, neither viral infections nor diverse stressful conditions (heat shock, wounding, or osmotic stress) induced this response. Additionally, this phenomenon was mimicked by the addition of a yeast elicitor or H2O2 to Phaseolus vulgaris suspension culture cells. This modification seems to provide increased stability of the microfilaments to proteolytic degradation and seems to be found in fractions in which the actin cytoskeleton is associated with membranes. All together, these data suggest that actin monoubiquitylation may be considered an effector mechanism of a general plant response against microbes.
