Detection by metagenomic functional analysis and improvement by experimental evolution of β-lactams resistance genes present in oil contaminated soils.

The spread of antibiotic resistance genes has become a global health concern identified by the World Health Organization as one of the greatest threats to health. Many of antimicrobial resistance determinants found in bacterial pathogens originate from environmental bacteria, so identifying the genes that confer resistance to antibiotics in different habitats is mandatory to better understand resistance mechanisms. Soil is one of the most diverse environments considered reservoir of antimicrobial resistance genes.

Characterization of a dszEABC operon providing fast growth on dibenzothiophene and construction of broad-host-range biodesulfurization catalysts.

A new operon for biodesulfurization (BDS) of dibenzothiophene and derivatives has been isolated from a metagenomic library made from oil-contaminated soil, by selecting growth of E. coli on DBT as the sulfur source. This operon is similar to a dszEABC operon also isolated by metagenomic functional screening but exhibited substantial differences: (i) the new fosmid provides much faster growth on DBT; (ii) associated dszEABC genes can be expressed without the need of heterologous expression from the vector promoter; and (iii) monooxygenases encoded in the fosmid cannot oxidize indole to produce indigo.

The Regulatory Hierarchy Following Signal Integration by the CbrAB Two-Component System: Diversity of Responses and Functions.

CbrAB is a two-component system, unique to bacteria of the family , capable of integrating signals and involved in a multitude of physiological processes that allow bacterial adaptation to a wide variety of varying environmental conditions. This regulatory system provides a great metabolic versatility that results in excellent adaptability and metabolic optimization. The two-component system (TCS) CbrA-CbrB is on top of a hierarchical regulatory cascade and interacts with other regulatory systems at different levels, resulting in a robust output.

The functional differences between paralogous regulators define the control of the general stress response in Sphingopyxis granuli TFA.

Sphingopyxis granuli TFA is a contaminant degrading alphaproteobacterium that responds to adverse conditions by inducing the general stress response (GSR), an adaptive response that controls the transcription of a variety of genes to overcome adverse conditions. The core GSR regulators (the response regulator PhyR, the anti-σ factor NepR and the σ factor EcfG) are duplicated in TFA, being PhyR1 and PhyR2, NepR1 and NepR2 and EcfG1 and EcfG2. Based on multiple genetic, phenotypical and biochemical evidences including in vitro transcription assays, we have assigned distinct functional features to each paralogue and assessed their contribution to the GSR regulation, dictating its timing and the intensity.

Extracytoplasmic Function σ Factors as Tools for Coordinating Stress Responses.

The ability of bacterial core RNA polymerase (RNAP) to interact with different σ factors, thereby forming a variety of holoenzymes with different specificities, represents a powerful tool to coordinately reprogram gene expression. Extracytoplasmic function σ factors (ECFs), which are the largest and most diverse family of alternative σ factors, frequently participate in stress responses. The classification of ECFs in 157 different groups according to their phylogenetic relationships and genomic context has revealed their diversity.

Identification of two fnr genes and characterisation of their role in the anaerobic switch in Sphingopyxis granuli strain TFA.

Sphingopyxis granuli strain TFA is able to grow on the organic solvent tetralin as the only carbon and energy source. The aerobic catabolic pathway for tetralin, the genes involved and their regulation have been fully characterised. Unlike most of the bacteria belonging to the sphingomonads group, this strain is able to grow in anoxic conditions by respiring nitrate, though not nitrite, as the alternative electron acceptor.

Isolation and genomic characterization of the ibuprofen-degrading bacterium Sphingomonas strain MPO218.

The presence of pharmaceutical compounds in waters and soils is of particular concern because these compounds can be biologically active, even at environmental concentrations. Most pharmaceutical contaminants result from inefficient removal of these compounds during wastewater treatment. Although microorganisms able to biodegrade pharmaceuticals compounds have been described, the isolation and characterization of new bacterial strains capable of degrading drugs remain important to improve the removal of this pollutant.

Understanding the metabolism of the tetralin degrader Sphingopyxis granuli strain TFA through genome-scale metabolic modelling.

Sphingopyxis granuli strain TFA is an α-proteobacterium that belongs to the sphingomonads, a group of bacteria well-known for its degradative capabilities and oligotrophic metabolism. Strain TFA is the only bacterium in which the mineralisation of the aromatic pollutant tetralin has been completely characterized at biochemical, genetic, and regulatory levels and the first Sphingopyxis characterised as facultative anaerobe. Here we report additional metabolic features of this α-proteobacterium using metabolic modelling and the functional integration of genomic and transcriptomic data.

Special Issue: Genetics of Biodegradation and Bioremediation.

Many different biodegradation pathways, both aerobic and anaerobic, have already been characterised, and the phylogenetic relationships among catabolic genes within the different types of pathways have been studied. However, new biodegradation activities and their coding genes are continuously being reported, including those involved in the catabolism of emerging contaminants or those generally regarded as non-biodegradable. Gene regulation is also an important issue for the efficient biodegradation of contaminants.

Two paralogous EcfG σ factors hierarchically orchestrate the activation of the General Stress Response in Sphingopyxis granuli TFA.

Under ever-changing environmental conditions, the General Stress Response (GSR) represents a lifesaver for bacteria in order to withstand hostile situations. In α-proteobacteria, the EcfG-type extracytoplasmic function (ECF) σ factors are the key activators of this response at the transcriptional level. In this work, we address the hierarchical function of the ECF σ factor paralogs EcfG1 and EcfG2 in triggering the GSR in Sphingopyxis granuli TFA and describe the role of EcfG2 as global switch of this response.

Identification of a complete dibenzothiophene biodesulfurization operon and its regulator by functional metagenomics.

Functional screening for aromatic ring oxygenases of an oil contaminated soil metagenome identified 25 different clones bearing monooxygenases coding genes. One fosmid bore an operon containing four tightly linked genes coding for a complete dibenzothiophene biodesulfurization pathway, which included the predicted monooxygenases DszC and DszA, the desulfinase DszB, and an FMN-oxidoreductase designated DszE. The dszEABC operon provided Escherichia coli with the ability to use dibenzothiophene as the only sulfur source.

Unraveling the role of the CbrA histidine kinase in the signal transduction of the CbrAB two-component system in Pseudomonas putida.

The histidine kinase CbrA of the CbrAB two-component system of Pseudomonas putida is a key element to recognise the activating signal and mediate auto- and trans-phosphorylation of the response element CbrB. CbrA is encoded by the gene cbrA which is located downstream of a putative open reading frame we have named cbrX. We describe the role of the CbrX product in the expression of CbrA and show there is translational coupling of the genes.

Biodegradation of Tetralin: Genomics, Gene Function and Regulation.

Tetralin (1,2,3,4-tetrahydonaphthalene) is a recalcitrant compound that consists of an aromatic and an alicyclic ring. It is found in crude oils, produced industrially from naphthalene or anthracene, and widely used as an organic solvent. Its toxicity is due to the alteration of biological membranes by its hydrophobic character and to the formation of toxic hydroperoxides.

The response of Sphingopyxis granuli strain TFA to the hostile anoxic condition.

Sphingomonads comprises a group of interesting aerobic bacteria because of their ubiquity and metabolic capability of degrading many recalcitrant contaminants. The tetralin-degrader Sphingopyxis granuli strain TFA has been recently reported as able to anaerobically grow using nitrate as the alternative electron acceptor and so far is the only bacterium with this ability within the sphingomonads group. To understand how strain TFA thrives under anoxic conditions, a differential transcriptomic analysis while growing under aerobic or anoxic conditions was performed.

Development of an inducible lytic system for functional metagenomic screening.

Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamper the technique. Here we have used an inducible autolysis system that lyses bacteria thus releasing its content in both, liquid and solid cultures, in response to anhydrotetracycline.

The CbrB Regulon: Promoter dissection reveals novel insights into the CbrAB expression network in Pseudomonas putida.

CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P.

SuhB, a small non-coding RNA involved in catabolite repression of tetralin degradation genes in Sphingopyxis granuli strain TFA.

Global dRNA-seq analysis of transcription start sites combined with in silico annotation using Infernal software revealed the expression of 91 putative non-coding sRNA in Sphingopyxis granuli TFA cells grown on different carbon sources. Excluding housekeeping sRNAs, only one additional sRNA, which belongs to the Rfam SuhB family (RF00519), was detected by Infernal but with an incorrect size according to the experimental results. SuhB is highly conserved across the Sphingopyxis genus.

Molecular Methods to Analyze the Effect of Proteins Expressed by Salmonella During Its Intracellular Stage.

Salmonella is probably the intracellular pathogen most extensively studied. Once inside the cell, this bacterium produces different proteins involved in the infection process known as effectors that translocate through its own secretion systems to the eukaryotic cytosol exerting diverse effects on the cell. Additionally, Salmonella can be engineered to include a protein expression system that, upon the addition of an inducer molecule, can produce heterologous proteins at a specific time during the course of the infection.

Engineering Salmonella as intracellular factory for effective killing of tumour cells.

Salmonella have many desirable properties as antitumour-agent due to its ability to proliferate inside tumours and induce tumour regression. Additionally, this bacterium can be genetically engineered to deliver therapeutic proteins intratumourally. The main limitation of this approach is the efficient release of therapeutic molecules from intratumoural bacteria.

Development of Genetic Tools for the Manipulation of the Planctomycetes.

Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system.

Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzT Promoter.

Unlabelled: PatzT is an internal promoter of the atzRSTUVW operon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzT is σ(N) dependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzT promoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC.

Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters.

Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together.

Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain.