Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

Tailoring modifications in labrenzin synthesis: a-la-carte production of pathway intermediates.

Pederin-family polyketides today constitute a group of more than 30 molecules being produced as natural products by different microorganisms across multitude of ecological niches. They are mostly known for their extreme cytotoxic activity and the decades of long exploration as potential antitumor drugs. The difference in their potency and biological activity lies in the tailoring modifications of the core molecule. Despite the isolation of many pederin-like molecules until the date, only marine bacterium Labrenzia sp. PHM005 was reported as a cultivable producer and able to be genetically modified. Here, we study the role of tailoring enzymes from the lab gene cluster responsible for methylation and hydroxylation of labrenzin core molecule. We managed to produce a spectrum of differently tailored labrenzin analogs for the development of future drugs. This work constitutes one-step forward in understanding the biosynthesis of pederin-family polyketides and provides the tools to modify and overproduce these anticancer drugs in a-la-carte manner in Labrenzia sp. PHM005, but also in other producers in the future.

In vivo production of pederin by labrenzin pathway expansion.

Pederin is a potent polyketide toxin that causes severe skin lesions in humans after contact with insects of genus Paederus. Due to its potent anticancer activities, pederin family compounds have raised the interest of pharmaceutical industry. Despite the extensive studies on the cluster of biosynthetic genes responsible for the production of pederin, it has not yet been possible to isolate and cultivate its bacterial endosymbiont producer. However, the marine bacterium Labrenzia sp. PHM005 was recently reported to produce labrenzin, the closest pederin analog. By cloning a synthetic pedO gene encoding one of the three O-methyltraferase of the pederin cluster into Labrenzia sp. PHM005 we have been able to produce pederin for the first time by fermentation in the new recombinant strain.

Integrating greenhouse gas capture and C1 biotechnology: a key challenge for circular economy.

State of the art on the valorisation of C1 carbon sources obtained either from natural or anthropogenic origins as a key challenge for the circular economy.

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.

Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors.

Glutarimide-containing polyketides are known as potent antitumoral and antimetastatic agents. The associated gene clusters have only been identified in a few Streptomyces producers and Burkholderia gladioli symbiont. The new glutarimide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologues in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutarimide-like and pederin-like pathways, as well as the functionality of their synthetic products.

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.

Genome of Labrenzia sp. PHM005 Reveals a Complete and Active Trans-AT PKS Gene Cluster for the Biosynthesis of Labrenzin.

The complete genome of the strain Labrenzia sp. PHM005, a free-living producer of a pederin analog 18-O-demethyl pederin, hereinafter labrenzin, has been sequenced. This strain contains two replicons comprising a circular chromosome of 6,167,349 bp and a circular plasmid (named p1BIR) of 19,450 bp. A putative gene cluster responsible for the synthesis of labrenzin (lab cluster) has been identified showing that it encodes a trans-AT mixed type PKS/NRPS biosynthetic pathway that is responsible for the synthesis of pederin and possibly an onnamide analog. The putative boundaries of the lab gene cluster were determined by genetic comparisons with other related strains, suggesting that the cluster consists of a 79-kb region comprising 3 genes encoding multidomain hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) proteins (PKS4, PKS/NRPS13, and PKS/NRPS15), and 16 auxiliary enzymes. Transcriptomic analyses suggest that all the genes of the cluster are expressed in our culture conditions (i.e., in minimal medium in the absence of any specific inducer) at detectable levels. We have developed genetic tools to facilitate the manipulation of this strain and the functional characterization of the cluster genes. We have created a site-directed mutant unable to produce pederin, demonstrating experimentally for the first time the role of the cluster in the synthesis of pederin. This work paves the way to unravel the clues of the biosynthesis of pederin family compounds and opens the door to modify and overproduce these anticancer drugs for industrial and pharmaceutical purposes.

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.

Unravelling the pleiotropic role of the MceG ATPase in Mycobacterium smegmatis.

The Mce systems are complex ABC transporters that are encoded by different numbers of homologous operons in Actinobacteria. While the four Mce systems of Mycobacterium tuberculosis are all energized by a single ATPase, MceG, each system appears to import different fatty acids or sterols. To explore if this behaviour can be extended to saprophytic mycobacteria, whose more complex genomes encode more Mce systems, we have identified and characterized the MceG orthologue of Mycobacterium smegmatis. This bacterium relies on MceG to energize its six Mce systems that contribute to a variety of cellular functions including sterol uptake and cell envelope maintenance. In the absence of MceG, M. smegmatis was not able to utilize cholesterol or phytosterols as carbon sources implying that this ATPase is necessary to energize the Mce4-sterol transport system. Other phenotypic alterations observed in the ΔMceG mutant, such as cell envelope modifications, suggest a pleiotropic functionality of the Mce systems that are particularly important for stress responses. Several ΔMceG phenotypes were recapitulated in a strain lacking only the unique C-terminal region of MceG, suggesting an important functional or regulatory function for this domain.

Gas phase reactions of unsaturated esters with Cl atoms.

BACKGROUND, AIM, AND SCOPE: Acrylate and methacrylate esters are alpha,beta-unsaturated esters that contain vinyl groups directly attached to the carbonyl carbon (CH(2)=CHCOO- and CH(2)=CCH(3)COO-, respectively) and are widely used in the polymer plastic and resin production. Rate coefficients for Cl reactions for most of the unsaturated esters have not been previously determined, and a good understanding is needed of all the atmospheric oxidation processes of these compounds in order to determine lifetimes in the atmosphere and to evaluate the impact of these reactions on the formation of photo-oxidants and therefore on health and environment. MATERIALS AND METHODS: The relative rate technique has been used to obtain rate coefficients for the reactions between the Cl atom and a series of unsaturated esters. The experiments have been carried out in a static Teflon reactor at room temperature and atmospheric pressure (N(2) as bath gas) using gas chromatography with flame ionization detection as detection system. RESULTS: The following rate coefficients are obtained (in cubic meter per molecule per second): methyl acrylate + Cl = 1.71 +/- 0.13 x 10(-10); methyl methacrylate + Cl = 2.30 +/- 0.18 x 10(-10); ethyl acrylate + Cl = 1.82 +/- 0.13 x 10(-10); ethyl methacrylate + Cl = 2.71 +/- 0.21 x 10(-10); butyl acrylate + Cl = 2.94 +/- 0.23 x 10(-10); butyl methacrylate + Cl = 3.83 +/- 0.30 x 10(-10); methyl 3-methyl acrylate + Cl = 2.21 +/- 0.17 x 10(-10); and methyl 3,3-dimethyl acrylate + Cl = 3.58 +/- 0.28 x 10(-10). DISCUSSION: Rate coefficients calculated for Cl reactions are around one order of magnitude higher than OH ones. The effect in the reactivity of increased substitution at the carbon-carbon double bond is analyzed and also the effect of the identity of the alkyl group R in the -C(O)OR. Atmospheric lifetimes of the compounds against the attack by the major oxidants are estimated and the atmospheric implications are discussed. CONCLUSIONS: The dominant atmospheric loss process for acrylate esters is clearly their daytime reaction with the hydroxyl radical. However, in coastal areas and in the marine boundary layer and in some industrial zones, Cl-atom-initiated degradation of the unsaturated esters considered here can be a significant if not dominant homogeneous loss process. RECOMMENDATIONS AND PERSPECTIVES: Product analysis should be necessary in order to evaluate the real environmental impact of these reactions. OH and ozone reactions of most of the considered compounds have already been studied and products determined, but kinetic and products information for NO(3) radical reactions is especially scarce.