Polyhydroxyalkanoate Production by Caenibius tardaugens from Steroidal Endocrine Disruptors.

The α-proteobacterium Caenibius tardaugens can use estrogens and androgens as the sole carbon source. These compounds are steroidal endocrine disruptors that are found contaminating soil and aquatic ecosystems. Here, we show that C. tardaugens, which has been considered as a valuable biocatalyst for aerobic steroidal hormone decontamination, is also able to produce polyhydroxyalkanoates (PHA), biodegradable and biocompatible polyesters of increasing biotechnological interest as a sustainable alternative to classical oil-derived polymers. Steroid catabolism yields a significant amount of propionyl-CoA that is metabolically directed towards PHA production through condensation into 3-ketovaleryl-CoA, rendering a PHA rich in 3-hydroxyvalerate. To the best of our knowledge, this is the first report where PHAs are produced from steroids as carbon sources.

Identification of the EdcR Estrogen-Dependent Repressor in Caenibius tardaugens NBRC 16725: Construction of a Cellular Estradiol Biosensor.

In this work, Caenibius tardaugens NBRC 16725 (strain ARI-1) (formerly Novosphingobium tardaugens) was isolated due to its capacity to mineralize estrogenic endocrine disruptors. Its genome encodes the edc genes cluster responsible for the degradation of 17ß-estradiol, consisting of two putative operons (OpA and OpB) encoding the enzymes of the upper degradation pathway. Inside the edc cluster, we identified the edcR gene encoding a TetR-like protein. Genetic studies carried out with C. tardaugens mutants demonstrated that EdcR represses the promoters that control the expression of the two operons. These genetic analyses have also shown that 17ß-estradiol and estrone, the second intermediate of the degradation pathway, are the true effectors of EdcR. This regulatory system has been heterologously expressed in Escherichia coli, foreseeing its use to detect estrogens in environmental samples. Genome comparisons have identified a similar regulatory system in the edc cluster of Altererythrobacter estronivorus MHB5, suggesting that this regulatory arrangement has been horizontally transferred to other bacteria.

Enhancing the Rice Seedlings Growth Promotion Abilities of Azoarcus sp. CIB by Heterologous Expression of ACC Deaminase to Improve Performance of Plants Exposed to Cadmium Stress.

Environmental pollutants can generate stress in plants causing increased ethylene production that leads to the inhibition of plant growth. Ethylene production by the stressed plant may be lowered by Plant Growth-Promoting Bacteria (PGPB) that metabolizes the immediate precursor of ethylene 1-aminocyclopropane-1-carboxylate (ACC). Thus, engineering PGPB with ACC deaminase activity can be a promising alternative to mitigate the harmful effects of pollutants and thus enhance plant production. Here we show that the aromatics-degrading and metal-resistant Azoarcus sp. CIB behaves as a PGP-bacterium when colonizing rice as an endophyte, showing a 30% increment in plant weight compared to non-inoculated plants. The cloning and expression of an acdS gene led to a recombinant strain, Azoarcus sp. CIB (pSEVA237acdS), possessing significant ACC deaminase activity (6716 nmol mg-1 h-1), constituting the first PGPB of the Rhodocyclaceae family equipped with this PGP trait. The recombinant CIB strain acquired the ability to protect inoculated rice plants from the stress induced by cadmium (Cd) exposure and to increase the Cd concentration in rice seedlings. The observed decrease of the levels of reactive oxygen species levels in rice roots confirms such a protective effect. The broad-host-range pSEVA237acdS plasmid paves the way to engineer PGPB with ACC deaminase activity to improve the growth of plants that might face stress conditions.

Unraveling the 17ß-Estradiol Degradation Pathway in Novosphingobium tardaugens NBRC 16725.

We have analyzed the catabolism of estrogens in Novosphingobium tardaugens NBRC 16725, which is able to use endocrine disruptors such as 17ß-estradiol, estrone, and estriol as sole carbon and energy sources. A transcriptomic analysis enabled the identification of a cluster of catabolic genes (edc cluster) organized in two divergent operons that are involved in estrogen degradation. We have developed genetic tools for this estrogen-degrading bacterium, allowing us to delete by site-directed mutagenesis some of the genes of the edc cluster and complement them by using expression plasmids to better characterize their precise role in the estrogen catabolism. Based on these results, a catabolic pathway is proposed. The first enzyme of the pathway (17ß-hydroxysteroid dehydrogenase) used to transform 17ß-estradiol into estrone is encoded out of the cluster. A CYP450 encoded by the edcA gene performs the second metabolic step, i.e., the 4-hydroxylation of estrone in this strain. The edcB gene encodes a 4-hydroxyestrone-4,5-dioxygenase that opens ring A after 4-hydroxylation. The initial steps of the catabolism of estrogens and cholate proceed through different pathways. However, the degradation of estrogens converges with the degradation of testosterone in the final steps of the lower catabolic pathway used to degrade the common intermediate 3aα-H-4α(3'-propanoate)7a-ß-methylhexahydro-1,5-indanedione (HIP). The TonB-dependent receptor protein EdcT appears to be involved in estrogen uptake, being the first time that this kind of proteins has been involved in steroid transport.

Testosterone Degradative Pathway of Novosphingobium tardaugens.

In this work, we have shown that Novosphingobium tardaugens NBRC 16725 (strain ARI-1), a bacterial strain that was isolated due to its capacity to mineralize the estrogenic endocrine compound 17ß-estradiol, is also able to mineralize testosterone, the androgenic endocrine compound. Using in silico analysis, we predicted a new putative steroid degradation (SD) gene cluster in strain ARI-1, which resembles genes involved in testosterone degradation in Comamonas testosteroni and other testosterone degrading bacteria like Actinobacteria (like Rhodococcus and Mycobacteria genera) although with significant differences in gene organization. A whole transcriptomic analysis of N. tardaugens revealed that testosterone produces a limited induction of the genes of the SD cluster that show a high basal expression in its absence. The 3ß/17ß-hydroxysteroid dehydrogenase involved in the first metabolic step of testosterone degradation was identified by using genetic and biochemical approaches. The construction of knockout mutant strains in the genes of the SD cluster together with in silico analyses suggests the existence of gene redundancy in the genome of N. tardaugens. This work will expand the knowledge about the metabolic pathways and biotransformation capabilities of a Gram-negative bacterium that could become a new model system in the bacterial steroid degradation field.