Pseudomonas taetrolens ULE-PH5 and Pseudomonas sp. ULE-PH6 Isolated from the Hop Rhizosphere Increase Phosphate Assimilation by the Plant.

Most of the phosphorus incorporated into agricultural soils through the use of fertilizers precipitates in the form of insoluble salts that are incapable of being used by plants. This insoluble phosphorus present in large quantities in soil forms the well-known "phosphorus legacy". The solubilization of this "phosphorus legacy" has become a goal of great agronomic importance, and the use of phosphate-solubilizing bacteria would be a useful tool for this purpose. In this work, we have isolated and characterized phosphate-solubilizing bacteria from the rhizosphere of hop plants. Two particular strains, Pseudomonas taetrolens ULE-PH5 and Pseudomonas sp. ULE-PH6, were selected as plant growth-promoting rhizobacteria due to their high phosphate solubilization capability in both plate and liquid culture assays and other interesting traits, including auxin and siderophore production, phytate degradation, and acidic and alkaline phosphatase production. These strains were able to significantly increase phosphate uptake and accumulation of phosphorus in the aerial part (stems, petioles, and leaves) of hop plants, as determined by greenhouse trials. These strains are promising candidates to produce biofertilizers specifically to increase phosphate adsorption by hop plants.

Albocycline Is the Main Bioactive Antifungal Compound Produced by Streptomyces sp. OR6 against Verticillium dahliae.

Verticillium wilt is a soil-borne fungal disease that affects olive trees (Olea europaea) and poses a serious threat to their cultivation. The causal agent of this disease is Verticillium dahliae, a pathogen that is difficult to control with conventional methods. Therefore, there is a need to explore alternative strategies for the management of Verticillium wilt. In this study, we aimed to isolate and characterize actinobacteria from the rhizosphere of olive trees that could act as potential biocontrol agents against V. dahliae. We selected a Streptomyces sp. OR6 strain based on its in vitro antifungal activity and its ability to suppress the pathogen growth in soil samples. We identified the main active compound produced by this strain as albocycline, a macrolide polyketide with known antibacterial properties and some antifungal activity. Albocycline was able to efficiently suppress the germination of conidiospores. To our knowledge, this is the first report of albocycline as an effective agent against V. dahliae. Our results suggest that Streptomyces sp. OR6, or other albocycline-producing strains, could be used as a promising tool for the biological control of Verticillium wilt.

Molecular Identification and Acid Stress Response of an Acidithiobacillus thiooxidans Strain Isolated from Rio Tinto (Spain).

Acidithiobacillus thiooxidans is of paramount importance in the development of biomining technologies. Being widely recognized as an extreme acidophile, extensive research has been dedicated to understanding its significant role in the extraction of several ores in recent years. However, there still exist significant molecular uncertainties surrounding this species. This study focuses on developing a taxonomic assignment method based on the sequencing of the 16S-5S rRNA cluster, along with a qPCR-based technology enabling precise growth determination. Additionally, an approach to understanding its response to acid stress is explored through RT-PCR and MALDI-TOF analysis. Our findings indicate that when subjected to pH levels below 1, the cell inhibits central (carbon fixation and metabolism) and energy (sulfur metabolism) metabolism, as well as chaperone synthesis, suggesting a potential cellular collapse. Nevertheless, the secretion of ammonia is enhanced to raise the environmental pH, while fatty acid synthesis is upregulated to reinforce the cell membrane.

Changes in the Microbial Composition of the Rhizosphere of Hop Plants Affected by Verticillium Wilt Caused by Verticillium nonalfalfae.

Verticillium wilt is a devastating disease affecting many crops, including hops. This study aims to describe fungal and bacterial populations associated with bulk and rhizosphere soils in a hop field cultivated in Slovenia with the Celeia variety, which is highly susceptible to Verticillium nonalfalfae. As both healthy and diseased plants coexist in the same field, we focused this study on the detection of putative differences in the microbial communities associated with the two types of plants. Bacterial communities were characterized by sequencing the V4 region of the 16S rRNA gene, whereas sequencing of the ITS2 region was performed for fungal communities. The bacterial community was dominated by phyla Proteobacteria, Acidobacteriota, Bacteroidota, Actinobacteriota, Planctomycetota, Chloroflexi, Gemmatimonadota, and Verrucomicrobiota, which are typically found in crop soils throughout the world. At a fungal level, Fusarium sp. was the dominant taxon in both bulk and rhizosphere soils. Verticillium sp. levels were very low in all samples analyzed and could only be detected by qPCR in the rhizosphere of diseased plants. The rhizosphere of diseased plants underwent important changes with respect to the rhizosphere of healthy plants where significant increases in potentially beneficial fungi such as the basidiomycetes Ceratobasidium sp. and Mycena sp., the zygomycete Mortierella sp., and a member of Glomeralles were observed. However, the rhizosphere of diseased plants experienced a decrease in pathogenic basidiomycetes that can affect the root system, such as Thanatephorus cucumeris (the teleomorph of Rhizoctonia solani) and Calyptella sp.

Biodegradation of Pine Processionary Caterpillar Silk Is Mediated by Elastase- and Subtilisin-like Proteases.

Pine processionary caterpillar nests are made from raw silk. Fibroin protein is the main component of silk which, in the case of pine processionary caterpillar, has some unusual properties such as a higher resistance to chemical hydrolysis. Isolation of microorganisms naturally present in silk nests led to identification of Bacillus licheniformis and Pseudomonas aeruginosa strains that in a defined minimal medium were able to carry out extensive silk biodegradation. A LasB elastase-like protein from P. aeruginosa was shown to be involved in silk biodegradation. A recombinant form of this protein expressed in Escherichia coli and purified by affinity chromatography was able to efficiently degrade silk in an in vitro assay. However, silk biodegradation by B. licheniformis strain was mediated by a SubC subtilisin-like protease. Homologous expression of a subtilisin Carlsberg encoding gene (subC) allowed faster degradation compared to the biodegradation kinetics of a wildtype B. licheniformis strain. This work led to the identification of new enzymes involved in biodegradation of silk materials, a finding which could lead to possible applications for controlling this pest and perhaps have importance from sanitary and biotechnological points of view.

The Grapevine Microbiome to the Rescue: Implications for the Biocontrol of Trunk Diseases.

Grapevine trunk diseases (GTDs) are one of the most devastating pathologies that threaten the survival and profitability of vineyards around the world. Progressive banning of chemical pesticides and their withdrawal from the market has increased interest in the development of effective biocontrol agents (BCAs) for GTD treatment. In recent years, considerable progress has been made regarding the characterization of the grapevine microbiome, including the aerial part microbiome (flowers, berries and leaves), the wood microbiome, the root environment and vineyard soil microbiomes. In this work, we review these advances especially in relation to the etiology and the understanding of the composition of microbial populations in plants affected by GTDs. We also discuss how the grapevine microbiome is becoming a source for the isolation and characterization of new, more promising BCAs that, in the near future, could become effective tools for controlling these pathologies.

Using Rhizosphere Phosphate Solubilizing Bacteria to Improve Barley (Hordeum vulgare) Plant Productivity.

On average less than 1% of the total phosphorous present in soils is available to plants, making phosphorous one of the most limiting macronutrients for crop productivity worldwide. The aim of this work was to isolate and select phosphate solubilizing bacteria (PSB) from the barley rhizosphere, which has other growth promoting traits and can increase crop productivity. A total of 104 different bacterial isolates were extracted from the barley plant rhizosphere. In this case, 64 strains were able to solubilize phosphate in agar plates. The 24 strains exhibiting the highest solubilizing index belonged to 16 different species, of which 7 isolates were discarded since they were identified as putative phytopathogens. The remaining nine strains were tested for their ability to solubilize phosphate in liquid medium and in pot trials performed in a greenhouse. Several of the isolated strains (Advenella mimigardefordensis, Bacillus cereus, Bacillus megaterium and Burkholderia fungorum) were able to significantly improve levels of assimilated phosphate, dry weight of ears and total starch accumulated on ears compared to non-inoculated plants. Since these strains were able to increase the growth and productivity of barley crops, they could be potentially used as microbial inoculants (biofertilizers).

Advances in the control of phytopathogenic fungi that infect crops through their root system.

Productivity and economic sustainability of many herbaceous and woody crops are seriously threatened by numerous phytopathogenic fungi. While symptoms associated with phytopathogenic fungal infections of aerial parts (leaves, stems and fruits) are easily observable and therefore recognizable, allowing rapid or preventive action to control this type of infection, the effects produced by soil-borne fungi that infect plants through their root system are more difficult to detect. The fact that these fungi initiate infection and damage underground implies that the first symptoms are not as easily noticeable, and therefore both crop yield and plant survival are frequently severely compromised by the time the infection is found. In this paper we will review and discuss recent insights into plant-microbiota interactions in the root system crucial to understanding the beginning of the infectious process. We will also review different methods for diminishing and controlling the infection rate by phytopathogenic fungi penetrating through the root system including both the traditional use of biocontrol agents such as antifungal compounds as well as some new strategies that could be used because of their effective application, such as nanoparticles, virus-based nanopesticides, or inoculation of plant material with selected endophytes. We will also review the possibility of modeling and influencing the composition of the microbial population in the rhizosphere environment as a strategy for nudging the plant-microbiome interactions toward enhanced beneficial outcomes for the plant, such as controlling the infectious process.

Necrotic and Cytolytic Activity on Grapevine Leaves Produced by Nep1-Like Proteins of Diplodia seriata.

Many phytopathogenic fungi produce necrosis and ethylene inducing peptide 1 (Nep1-like proteins or NLP) that trigger leaf necrosis and the activation of defense mechanisms. These proteins have been widely studied in plant pathogens as Moniliophthora perniciosa or Botrytis cinerea between others, but little is known about their biological roles in grapevine trunk pathogens. Advances in the sequencing of genomes of several fungi involved in grapevine trunk diseases have revealed that these proteins are present in several copies in their genomes. The aim of this project was to analyze the presence of genes encoding NLP proteins in the Diplodia seriata genome and to characterize their putative role as virulence factors associated to grapevine trunk diseases. In this study, we characterized four NLPs from Diplodia seriata. All proteins showed highly similar amino acid sequences and contained the characteristic peptide motifs of NLPs. DserNEPs slightly reduced the viability of Vitis vinifera L. cell cultures. The cytolytic activity from DserNEP1 was stronger than that from DserNEP2, even at low concentrations. Purified DserNEPs also produced necrosis in leaves when they were inoculated into micropropagules of V. vinifera L. This is the first record of Nep1-like proteins from a fungus associated with grapevine trunk diseases and also from a member of the Botryosphaeriaceae family.

Developing tools for evaluating inoculation methods of biocontrol Streptomyces sp. strains into grapevine plants.

The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents.

Use of Endophytic and Rhizosphere Actinobacteria from Grapevine Plants To Reduce Nursery Fungal Graft Infections That Lead to Young Grapevine Decline.

Endophytic and rhizosphere actinobacteria isolated from the root system of 1-year-old grafted Vitis vinifera plants were evaluated for their activities against fungi that cause grapevine trunk diseases. A total of 58 endophytic and 94 rhizosphere isolates were tested. Based on an in vitro bioassay, 15.5% of the endophytic isolates and 30.8% of the rhizosphere isolates exhibited antifungal activity against the fungal pathogen Diplodia seriata, whereas 13.8% of the endophytic isolates and 16.0% of the rhizosphere isolates showed antifungal activity against Dactylonectria macrodidyma (formerly Ilyonectria macrodidyma). The strains which showed the greatest in vitro efficacy against both pathogens were further analyzed for their ability to inhibit the growth of Phaeomoniella chlamydospora and Phaeoacremonium minimum (formerly Phaeoacremonium aleophilum). Based on their antifungal activity, three rhizosphere isolates and three endophytic isolates were applied on grafts in an open-root field nursery in a 3-year trial. The field trial led to the identification of one endophytic strain, Streptomyces sp. VV/E1, and two rhizosphere isolates, Streptomyces sp. VV/R1 and Streptomyces sp. VV/R4, which significantly reduced the infection rates produced by the fungal pathogens Dactylonectria sp., Ilyonectria sp., P. chlamydospora, and P. minimum, all of which cause young grapevine decline. The VV/R1 and VV/R4 isolates also significantly reduced the mortality level of grafted plants in the nursery. This study shows that certain actinobacteria could represent a promising new tool for controlling fungal trunk pathogens that infect grapevine plants through the root system in nurseries.IMPORTANCE Grapevine trunk diseases are a major threat to the wine and grape industry worldwide. They cause a significant reduction in yields as well as in grape quality, and they can even cause plant death. Trunk diseases are caused by fungal pathogens that enter through pruning wounds and/or the root system. Although different strategies have recently been developed to protect pruning wounds using antifungal compounds (natural or synthetic) or biocontrol agents, no tools are yet available for controlling soil pathogens that infect plants through their root system. This study shows that different actinobacterial isolates, when applied to grafts in a nursery, can significantly reduce the infection rate caused by fungal pathogens that enter through the root system. This is a new, promising, and green alternative for preventing the decline of young grapevines in nurseries and vineyards.

Effectiveness of Natural Antifungal Compounds in Controlling Infection by Grapevine Trunk Disease Pathogens through Pruning Wounds.

Grapevine trunk fungal pathogens, such as Diplodia seriata and Phaeomoniella chlamydospora, can infect plants through pruning wounds. They cause grapevine trunk diseases and are involved in grapevine decline. Accordingly, the protection of pruning wounds is crucial for the management of grapevine trunk diseases. The efficacy of different natural antifungals in inhibiting the growth of several fungi causing grapevine trunk diseases was evaluated in vitro. The fungi showing greater in vitro efficacy were tested on autoclaved grape wood assays against D. seriata and P. chlamydospora. Based on results from these assays, chitosan oligosaccharide, vanillin, and garlic extract were selected for further evaluation on pruning wounds inoculated with D. seriata and P. chlamydospora in field trials. A significant decrease in plant mortality was observed after 2 years of growth in the plants treated with the different natural antifungals compared to the mortality rate observed in infected plants that were not treated with antifungals. Also, the infection rate for the inoculated pathogens was significantly reduced in plants treated with the selected natural antifungals. Therefore, natural antifungals represent a promising alternative for disease control and could provide significant economic benefits for the grape-growing industry.

Sensory and chemical characterisation of the aroma of Prieto Picudo rosé wines: the differential role of autochthonous yeast strains on aroma profiles.

This study evaluates the specific impact of isolated yeast strains on the aromatic profile of fermented musts from Prieto Picudo, an autochthonous Castilla y León (Spain) red grape variety with an increasing demand in the local marketplace. For this purpose, the aroma profiles of wines elaborated from Prieto Picudo grapes have been studied by sensory analysis, gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS), with the aim of determining the potential of each strain to generate distinctive varietal and fermentation-derived aromatic compounds. The results have shown that the yeast strain exerts a critical influence on the levels of some fermentative (linear and branched ethyl esters, fatty acids, ethyl phenylacetate) and varietal compounds (4-mercapto-4-methyl-2-pentanone, 3-mercaptohexylacetate, ß-damascenone), thus inducing a deep influence on the final aroma of the wine. Combination of both sensory and chemical data arises as a major tool to monitor the different patterns of aroma release and formation from selected yeast strains during the winemaking process.

Destruction of chloroanisoles by using a hydrogen peroxide activated method and its application to remove chloroanisoles from cork stoppers.

A chemical method for the efficient destruction of 2,4,6-trichloroanisole (TCA) and pentachloroanisole (PCA) in aqueous solutions by using hydrogen peroxide as an oxidant catalyzed by molybdate ions in alkaline conditions was developed. Under optimal conditions, more than 80.0% TCA and 75.8% PCA were degraded within the first 60 min of reaction. Chloroanisoles destruction was followed by a concomitant release of up to 2.9 chloride ions per TCA molecule and 4.6 chloride ions per PCA molecule, indicating an almost complete dehalogenation of chloroanisoles. This method was modified to be adapted to chloroanisoles removal from the surface of cork materials including natural cork stoppers (86.0% decrease in releasable TCA content), agglomerated corks (78.2%), and granulated cork (51.3%). This method has proved to be efficient and inexpensive with practical application in the cork industry to lower TCA levels in cork materials.

Cytoplasmic- and extracellular-proteome analysis of Diplodia seriata: a phytopathogenic fungus involved in grapevine decline.

BACKGROUND: The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome. RESULTS: Intracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism. CONCLUSIONS: This study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity process. In fact, several of the identified proteins have been reported as pathogenicity factors in other phytopathogenic fungi. Moreover, this proteomic analysis supposes a useful basis for deepening into D. seriata knowledge and will contribute to the development of the molecular biology of this fungal strain as it has been demonstrated by cloning the gene Prx1 encoding mitochondrial peroxiredoxin-1 of D. seriata (the first gene to be cloned in this microorganism; data not shown).

Characterization of a novel 2,4,6-trichlorophenol-inducible gene encoding chlorophenol O-methyltransferase from Trichoderma longibrachiatum responsible for the formation of chloroanisoles and detoxification of chlorophenols.

De novo sequencing of eight internal peptides of purified chlorophenol O-methyltransferase, or CMT1 (before named as CPOMT), from Trichoderma longibrachiatum was performed by MALDI-TOF/TOF and ESI-IT. A novel gene (cmt1) encoding CMT1 was cloned by using a PCR approach based on the amino acid sequence of two internal peptides. The gene (1637 bp) encoded a protein of 468 amino acids with a deduced molecular mass of 52.4 kDa, and a theoretical isoelectric point of 5.93. This gene contains four introns, whose location was confirmed by comparison of cDNA and chromosomal sequences. The expression of cmt1 gene was induced at transcriptional level by exposure of fungal mycelia to 2,4,6-trichlorophenol (2,4,6-TCP). Putative homologous genes were detected in many different fungal strains, including other Trichoderma species. Partial silencing of cmt1 gene resulted in a 48.9% (+/-5.2) decrease of CMT1 activity levels in a T. longibrachiatum At37 transformant strain by comparison with the wild type, whereas a decrease of up to 53.0% was observed in the levels of 2,4,6-TCA produced in liquid cultures. Efficient expression of cmt1 gene in Escherichia coli unequivocally confirmed that it encodes a CMT1 enzyme.

Two overlapping antiparallel genes encoding the iron regulator DmdR1 and the Adm proteins control siderophore [correction of sedephore] and antibiotic biosynthesis in Streptomyces coelicolor A3(2).

The dmdR1 gene of Streptomyces coelicolor encodes an important regulator of iron metabolism. An antiparallel gene (adm) homologous to a development-regulated gene of Streptomyces aureofaciens has been found to overlap with dmdR1. Both proteins DmdR1 and Adm are formed in solid and liquid cultures of S. coelicolor A3(2). The purpose of this study was to assess possible interaction between the products of these two antiparallel genes. Two mutants with stop codons resulting in arrested translation of either DmdR1 or Adm were obtained by gene replacement and compared with a deletion mutant (DeltadmdR1/adm) that was defective in both genes. The deletion mutant was unable to form either protein, did not sporulate and lacked desferrioxamine, actinorhodin and undecylprodigiosin biosynthesis; biosynthesis of these compounds was recovered by complementation with dmdR1/adm genes. The mutant in which formation of Adm protein was arrested showed normal levels of DmdR1, lacked Adm and over-produced the antibiotics undecylprodigiosin and actinorhodin (in MS medium), suggesting that Adm plays an important role in secondary metabolism. The mutant in which DmdR1 formation was arrested synthesized desferrioxamines in a constitutive (deregulated) manner, and produced relatively normal levels of antibiotics. In conclusion, our results suggest that there is a fine interplay of expression of these antiparallel genes, as observed for other genes that encode lethal proteins such as the toxin/antitoxin systems. The Adm protein seems to have a major effect on the control of secondary metabolism, and its formation is probably tightly controlled, as expected for a key regulator.

Biodegradation of 2,4,6-TCA by the white-rot fungus Phlebia radiata is initiated by a phase I (O-demethylation)-phase II (O-conjugation) reactions system: implications for the chlorine cycle.

Thirteen species of white-rot fungi tested have been shown to efficiently biodegrade 1 mM 2,4,6-trichloroanisole (2,4,6-TCA) in liquid cultures. The maximum biodegradation rate (94.5% in 10-day incubations) was exhibited by a Phlebia radiata strain. The enzymes of the ligninolytic complex, laccase, lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) were not able to transform 2,4,6-TCA in in vitro reactions, indicating that the ligninolytic complex was not involved in the initial attack to 2,4,6-TCA. Instead, the first biodegradative steps were carried out by a phase I and phase II reactions system. Phase I reaction consisted on a O-demethylation catalysed by a microsomal cytochrome P-450 monooxygenase to produce 2,4,6-trichlorophenol (2,4,6-TCP). Later, in a phase II reaction catalysed by a microsomal UDP-glucosyltransferase, 2,4,6-TCP was detoxified by O-conjugation with D-glucose to produce 2,4,6-TCP-1-O-d-glucoside (TCPG). This compound accumulated in culture supernatants, reaching its maximum concentration between 48 and 72 h of growth. TCPG levels decreased constantly by the end of fermentation, indicating that it was subsequently metabolized. A catalase activity was able to break in vitro the glycosidic link to produce 2,4,6-TCP, whereas ligninolytic enzymes did not have a significant effect on the biotransformation of that compound. Once formed, 2,4,6-TCP was further degraded as detected by a concomitant release of 2.6 mol of chloride ions by 1 mol of initial 2,4,6-TCA, indicating that this compound underwent almost a complete dehalogenation and biodegradation. It was concluded that P. radiata combines two different degradative mechanisms in order to biodegrade 2,4,6-TCA. The significance of the capability of white-rot fungi to O-demethylate chloroanisoles for the global chlorine cycle is discussed.

Environmental significance of O-demethylation of chloroanisoles by soil bacterial isolates as a mechanism that improves the overall biodegradation of chlorophenols.

The biodegradation rate of chlorophenols in the environment seems to be limited by a competitive mechanism of O-methylation which produces chloroanisoles with a high potential of being bioconcentrated in living organisms. In this work we report for the first time the isolation of three soil bacterial strains able to efficiently degrade 2,4,6-trichloroanisole (2,4,6-TCA). These strains were identified as Xanthomonas retroflexus INBB4, Pseudomonas putida INBP1 and Acinetobacter radioresistens INBS1. In these isolates 2,4,6-TCA was efficiently metabolized in a minimal medium containing methanol and 2,4,6-TCA as the only carbon sources, with a concomitant release of 3 mol of chloride ion from 1 mol of 2,4,6-TCA, indicating complete dehalogenation of 2,4,6-TCA. 2,4,6-trichlorophenol (2,4,6-TCP) was identified as a degradative intermediate, indicating that 2,4,6-TCA underwent O-demethylation as the first step in the biodegradation process. 2,4,6-TCP was further transformed into 2,6-dichloro-para-hydroquinone (2,6-DCHQ) and subsequently mineralized. The degradation of chloroanisoles could improve the overall biodegradation of chlorophenols in the environment, because those chlorophenols previously biomethylated might also be later biodegraded. Xanthomonas retroflexus INBB4 has two O-demethylation systems: one is an oxygenase-type demethylase, and the other is a tetrahydrofolate (THF)-dependent O-demethylase. On the contrary O-demethylation of 2,4,6-TCA in P. putida INBP1 is just catalysed by an oxygenase-type NADH/NADPH-dependent O-demethylase, whereas in A. radioresistens INBS1 a THF-dependent O-demethylase activity was detected.

Transcriptional regulation of the desferrioxamine gene cluster of Streptomyces coelicolor is mediated by binding of DmdR1 to an iron box in the promoter of the desA gene.

Streptomyces coelicolor and Streptomyces pilosus produce desferrioxamine siderophores which are encoded by the desABCD gene cluster. S. pilosus is used for the production of desferrioxamine B which is utilized in human medicine. We report the deletion of the desA gene encoding a lysine decarboxylase in Streptomyces coelicolor A3(2). The DeltadesA mutant was able to grow on lysine as the only carbon and nitrogen source but its desferrioxamine production was blocked, confirming that the L-lysine decarboxylase encoded by desA is a dedicated enzyme committing L-lysine to desferrioxamine biosynthesis. Production of desferrioxamine was restored by complementation with the whole wild-type desABCD cluster, but not by desA alone, because of a polar effect of the desA gene replacement on expression of the downstream des genes. The transcription pattern of the desABCD cluster in S. coelicolor showed that all four genes were coordinately induced under conditions of iron deprivation. The transcription start point of the desA gene was identified by primer extension analysis at a thymine located 62 nucleotides upstream of the translation start codon. The -10 region of the desA promoter overlaps the 19-nucleotide palindromic iron box sequence known to be involved in iron regulation in Streptomyces. Binding of DmdR1 divalent metal-dependent regulatory protein to the desA promoter region of both S. coelicolor and S. pilosus was shown using electrophoretic mobility-shift assays, validating the conclusion that iron regulation of the desABCD cluster is mediated by the regulatory protein DmdR1. We conclude that the genes involved in desferrioxamine production are under transcriptional control exerted by the DmdR1 regulator in the presence of iron and are expressed under conditions of iron limitation.