Marta V. Mendes

A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis

BACKGROUND Polyene macrolides are a class of large macrocyclic polyketides that interact with membrane sterols, having antibiotic activity against fungi but not bacteria. Their rings include a chromophore of 3-7 conjugated double bonds which constitute the distinct polyene structure. Pimaricin is an archetype polyene, important in the food industry as a preservative to prevent mould contamination of foods, produced by Streptomyces natalensis. We set out to clone, sequence and analyse the gene cluster responsible for the biosynthesis of this tetraene. RESULTS A large cluster of 16 open reading frames spanning 84985 bp of the S. natalensis genome has been sequenced and found to encode 13 homologous sets of enzyme activities (modules) of a polyketide synthase (PKS) distributed within five giant multienzyme proteins (PIMS0-PIMS4). The total of 60 constituent active sites, 25 of them on a single enzyme (PIMS2), make this an exceptional multienzyme system. Eleven additional genes appear to govern modification of the polyketide-derived framework and export. Disruption of the genes encoding the PKS abolished pimaricin production. CONCLUSIONS The overall architecture of the PKS gene cluster responsible for the biosynthesis of the 26-membered polyene macrolide pimaricin has been determined. Eleven additional tailoring genes have been cloned and analysed. The availability of the PKS cluster will facilitate the generation of designer pimaricins by combinatorial biosynthesis approaches. This work represents the extensive description of a second polyene macrolide biosynthetic gene cluster after the one for the antifungal nystatin.

Engineered biosynthesis of novel polyenes: a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis

BACKGROUND The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups. Therefore, the identification of novel macrolide monooxygenases with activities toward alternative substrates, particularly epoxidases, is a fundamental aspect of the growing field of combinatorial biosynthesis. The specific alteration of these activities should constitute a further source of novel analogues. We investigated this possibility by directed inactivation of one of the P450s belonging to the biosynthetic gene cluster of an archetype polyene, pimaricin. RESULTS A recombinant mutant of the pimaricin-producing actinomycete Streptomyces natalensis produced a novel pimaricin derivative, 4,5-deepoxypimaricin, as a major product. This biologically active product resulted from the phage-mediated targeted disruption of the gene pimD, which encodes the cytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification. CONCLUSIONS We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides.

DNA signature-based approaches for bacterial detection and identification.

During the late eighties, environmental microbiologists realized the potential of the polymerase chain reaction (PCR) for the design of innovative approaches to study microbial communities or to detect and identify microorganisms in diverse and complex environments. In contrast to long-established methods of cultivation-based microbial identification, PCR-based techniques allow for the identification of microorganisms regardless of their culturability. A large number of reports have been published that describe PCR-inspired methods, frequently complemented by sequencing or hybridization profiling, to infer taxonomic and clonal microbial diversity or to detect and identify microorganisms using taxa-specific genomic markers. Typing methods have been particularly useful for microbial ecology-driven studies; however, they are not suitable for diagnostic purposes, such as the detection of specific species, strains or clones. Recently, comprehensive reviews have been written describing the panoply of typing methods available and describing their advantages and limitations; however, molecular approaches for bacterial detection and identification were either not considered or only vaguely discussed. This review focuses on DNA-based methods for bacterial detection and identification, highlighting strategies for selecting taxa-specific loci and emphasizing the molecular techniques and emerging technological solutions for increasing the detection specificity and sensitivity. The massive and increasing number of available bacterial sequences in databases, together with already employed bioinformatics tools, hold promise of more reliable, fast and cost-effective methods for bacterial identification in a wide range of samples in coming years. This tendency will foster the validation and certification of these methods and their routine implementation by certified diagnostic laboratories.

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.

A walk into the LuxR regulators of Actinobacteria: phylogenomic distribution and functional diversity.

LuxR regulators are a widely studied group of bacterial helix-turn-helix (HTH) transcription factors involved in the regulation of many genes coding for important traits at an ecological and medical level. This regulatory family is particularly known by their involvement in quorum-sensing (QS) mechanisms, i.e., in the bacterial ability to communicate through the synthesis and binding of molecular signals. However, these studies have been mainly focused on Gram-negative organisms, and the presence of LuxR regulators in the Gram-positive Actinobacteria phylum is still poorly explored. In this manuscript, the presence of LuxR regulators among Actinobacteria was assayed using a domain-based strategy. A total of 991 proteins having one LuxR domain were identified in 53 genome-sequenced actinobacterial species, of which 59% had an additional domain. In most cases (53%) this domain was REC (receiver domain), suggesting that LuxR regulators in Actinobacteria may either function as single transcription factors or as part of two-component systems. The frequency, distribution and evolutionary stability of each of these sub-families of regulators was analyzed and contextualized regarding the ecological niche occupied by each organism. The results show that the presence of extra-domains in the LuxR-regulators was likely driven by a general need to physically uncouple the signal sensing from the signal transduction. Moreover, the total frequency of LuxR regulators was shown to be dependent on genetic, metabolic and ecological variables. Finally, the functional annotation of the LuxR regulators revealed that the bacterial ecological niche has biased the specialization of these proteins. In the case of pathogens, our results suggest that LuxR regulators can be involved in virulence and are therefore promising targets for future studies in the health-related biotechnology field.

Crosstalk between ROS homeostasis and secondary metabolism in S. natalensis ATCC 27448: modulation of pimaricin production by intracellular ROS.

Streptomyces secondary metabolism is strongly affected by oxygen availability. The increased culture aeration enhances pimaricin production in S. natalensis, however the excess of O2 consumption can lead to an intracellular ROS imbalance that is harmful to the cell. The adaptive physiological response of S. natalensis upon the addition of exogenous H2O2 suggested that the modulation of the intracellular ROS levels, through the activation of the H2O2 inducible catalase during the late exponential growth phase, can alter the production of pimaricin. With the construction of defective mutants on the H2O2 related enzymes SodF, AhpCD and KatA1, an effective and enduring modulation of intracellular ROS was achieved. Characterization of the knock-out strains revealed different behaviours regarding pimaricin production: whilst the superoxide dismutase defective mutant presented low levels of pimaricin production compared to the wild-type, the mutants defective on the H2O2-detoxifying enzymes displayed a pimaricin overproducer phenotype. Using physiological and molecular approaches we report a crosstalk between oxidative stress and secondary metabolism regulatory networks. Our results reveal that the redox-based regulation network triggered by an imbalance of the intracellular ROS homeostasis is also able to modulate the biosynthesis of pimaricin in S. natalensis.

Quercetin Protects Saccharomyces cerevisiae against Oxidative Stress by Inducing Trehalose Biosynthesis and the Cell Wall Integrity Pathway

Background Quercetin is a naturally occurring flavonol with antioxidant, anticancer and anti-ageing properties. In this study we aimed to identify genes differentially expressed in yeast cells treated with quercetin and its role in oxidative stress protection. Methods A microarray analysis was performed to characterize changes in the transcriptome and the expression of selected genes was validated by RT-qPCR. Biological processes significantly affected were identified by using the FUNSPEC software and their relevance in H2O2 resistance induced by quercetin was assessed. Results Genes associated with RNA metabolism and ribosome biogenesis were down regulated in cells treated with quercetin, whereas genes associated with carbohydrate metabolism, endocytosis and vacuolar proteolysis were up regulated. The induction of genes related to the metabolism of energy reserves, leading to the accumulation of the stress protectant disaccharide trehalose, and the activation of the cell wall integrity pathway play a key role in oxidative stress resistance induced by quercetin. Conclusions These results suggest that quercetin may act as a modulator of cell signaling pathways related to carbohydrate metabolism and cell integrity to exert its protective effects against oxidative stress.

A novel approach for the identification of bacterial taxa-specific molecular markers

Aims:  To develop and establish a methodology for an oriented and fast identification of species taxa‐specific molecular markers useful for the identification of micro‐organisms.

Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

Background Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. Methodology In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. Significance This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with Novel Markers and Using a Dot Blot Platform Coupled with Automatic Data Analysis

ABSTRACT Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNA-based methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads.