Juan José R. Coque

Characterization of the cmcH genes of Nocardia lactamdurans and streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis

Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC.

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

SummaryThe cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a Mr of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a Mr of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.

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 ΔdesA 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.

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, β-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.

Functional analysis of two divalent metal‐dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants

In Gram‐positive bacteria, the expression of iron‐regulated genes is mediated by a class of divalent metal‐dependent regulatory (DmdR) proteins. We cloned and characterized two dmdR genes of Streptomyces coelicolor that were located in two different nonoverlapping cosmids. Functional analysis of dmdR1 and dmdR2 was performed by deletion of each copy. Deletion of dmdR1 resulted in the derepression of at least eight proteins and in the repression of three others, as shown by 2D proteome analysis. These 11 proteins were characterized by MALDI‐TOF peptide mass fingerprinting. The proteins that show an increased level in the mutant correspond to a DNA‐binding hemoprotein, iron‐metabolism proteins and several divalent metal‐regulated enzymes. The levels of two other proteins – a superoxide dismutase and a specific glutamatic dehydrogenase – were found to decrease in this mutant. Complementation of the dmdR1‐deletion mutant with the wild‐type dmdR1 allele restored the normal proteome profile. By contrast, deletion of dmdR2 did not affect significantly the protein profile of S. coelicolor. One of the proteins (P1, a phosphatidylethanolamine‐binding protein), overexpressed in the dmdR1‐deleted mutant, is encoded by ORF3 located immediately upstream of dmdR2; expression of both ORF3 and dmdR2 is negatively controlled by DmdR1. Western blot analysis confirmed that dmdR2 is only expressed when dmdR1 is disrupted. Species of Streptomyces have evolved an elaborated regulatory mechanism mediated by the DmdR proteins to control the expression of divalent metal‐regulated genes.

Overexpression of the Nocardia lactamduransα-Aminoadipyl-Cysteinyl-Valine Synthetase in Streptomyces lividans

Formation of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (Aad-Cys-Val) is catalyzed by a multienzyme peptide synthetase encoded by the pcbAB gene in producers of beta-lactam antibiotics. The pcbAB gene of Nocardia lactamdurans was overexpressed in Streptomyces lividans giving a high Aad-Cys-Val synthetase activity. The synthetase was purified 2785-fold to near homogeneity showing a molecular mass of 430 kDa by SDS/PAGE. The protein was identified in the gels with antibodies to Aad-Cys-Val synthetase and by the formation of aminoacyl-synthetase thioester complex with [14C]valine. The purified synthetase used alpha-aminoadipic acid or its lactam 6-oxopiperidine 2-carboxylic acid but was unable to use piperideine 6-carboxylic acid or pipecolic acid as substrates to form Aad-Cys-Val. L-Cystathionine, (2-amino-2-carboxyethyl)-L-homocysteine, was used as substrate and formed Aad-Cys-Val with the same efficiency as L-cysteine. The product of the reaction eluted with authentic Aad-Cys-Val. The synthetase preparation was able to hydrolyze L-cystathionine by a pyridoxal-phosphate-independent mechanism which is not inhibited by propargylglycine, to form Aad-Cys-Val.

Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements an Escherichia coli mutant defective in the ATPase activity of SecA

The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes. It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins. All the previously described conserved motifs of SecA proteins were conserved in the S. lividans protein. The secA gene of S. lividans restored sensitivity to sodium azide in E. coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein. However, it did not complement the temperature-sensitive mutation in E. coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature. secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.

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

ABSTRACT 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.

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 (ΔdmdR1/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.

Using endophytic and rhizospheric actinobacteria from grapevine plants to reduce fungal graft infections in nurseries that lead to young grapevine decline

ABSTRACT 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.