RESUMO
Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.
RESUMO
Macrolide antibiotics are common contaminants in the aquatic environment. They are toxic to a wide range of primary producers, inhibiting the algal growth and further hindering the delivery of several ecosystem services. Yet the molecular mechanisms of macrolides in algae remain undetermined. The objectives of this study were therefore to: 1. evaluate whether macrolides at the environmentally relevant level inhibit the growth of algae; and 2. test the hypothesis that macrolides bind to ribosome and inhibit protein translocation in algae, as it does in bacteria. In this study, transcriptomic analysis was applied to elucidate the toxicological mechanism in a model green alga Raphidocelis subcapitata treated with 5 and 90 µg L-1 of a typical macrolide roxithromycin (ROX). While exposure to ROX at 5 µg L-1 for 7 days did not affect algal growth and the transciptome, ROX at 90 µg L-1 resulted in 45% growth inhibition and 2306 (983 up- and 1323 down-regulated) DEGs, which were primarily enriched in the metabolism of energy, lipid, vitamins, and DNA replication and repair pathways. Nevertheless, genes involved in pathways in relation to translation and protein translocation and processing were dysregulated. Surprisingly, we found that genes involved in the base excision repair process were mostly repressed, suggesting that ROX may be genotoxic and cause DNA damage in R. subcapitata. Taken together, ROX was unlikely to pose a threat to green algae in the environment and the mode of action of macrolides in bacteria may not be directly extrapolated to green algae.