Fernando Tavares

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.

Draft Genome Sequence of Frankia sp. Strain CN3, an Atypical, Noninfective (Nod - ) Ineffective (Fix - ) Isolate from Coriaria nepalensis

ABSTRACT We report here the genome sequence of Frankia sp. strain CN3, which was isolated from Coriaria nepalensis. This genome sequence is the first from the fourth lineage of Frankia, strains of which are unable to reinfect actinorhizal plants. At 10 Mb, it represents the largest Frankia genome sequenced to date.

Draft Genome sequence of Frankia sp. Strain QA3, a nitrogen-fixing actinobacterium isolated from the root nodule of Alnus nitida

ABSTRACT Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a high-quality draft genome sequence for Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated from root nodules of Alnus nitida.

A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia

A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris-HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5+/-7.44 microg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50+/-0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.

A phylogenomic analysis of bacterial helix-turn-helix transcription factors.

Perception by each individual organism of its environments parameters is a key factor for survival. In a constantly changing environment, the ability to assess nutrient sources and potentially stressful situations constitutes the main basis for ecological adaptability. Transcription regulators are key decision-making proteins that mediate the communication between environmental conditions and DNA transcription through a multifaceted network. The parallel study of these regulators across microbial organisms adapted to contrasting biotopes constitutes an unexplored approach to understand the evolution of genome plasticity and cell function. We present here a reassessment of bacterial helix-turn-helix regulator diversity in different organisms from a multidisciplinary perspective, on the interface that links metabolism, ecology and phylogeny, further sustained by a statistically based approach. The present revision brought to light evidence of patterns among families of regulators, suggesting that multiple selective forces modulate the number and kind of regulators present in a given genome. Besides being an important step towards understanding the adaptive traits that influence the microbial responses to the varying environment on the very first and most prevalent line of reaction, the transcription of DNA, this approach is a promising tool to extract biological trends from genomic databases.

On the nature of fur evolution: A phylogenetic approach in Actinobacteria

BackgroundAn understanding of the evolution of global transcription regulators is essential for comprehending the complex networks of cellular metabolism that have developed among related organisms. The fur gene encodes one of those regulators – the ferric uptake regulator Fur – widely distributed among bacteria and known to regulate different genes committed to varied metabolic pathways. On the other hand, members of the Actinobacteria comprise an ecologically diverse group of bacteria able to inhabit various natural environments, and for which relatively little is currently understood concerning transcriptional regulation.ResultsBLAST analyses revealed the presence of more than one fur homologue in most members of the Actinobacteria whose genomes have been fully sequenced. We propose a model to explain the evolutionary history of fur within this well-known bacterial phylum: the postulated scenario includes one duplication event from a primitive regulator, which probably had a broad range of co-factors and DNA-binding sites. This duplication predated the appearance of the last common ancestor of the Actinobacteria, while six other duplications occurred later within specific groups of organisms, particularly in two genera: Frankia and Streptomyces. The resulting paralogues maintained main biochemical properties, but became specialised for regulating specific functions, coordinating different metal ions and binding to unique DNA sequences. The presence of syntenic regions surrounding the different fur orthologues supports the proposed model, as do the evolutionary distances and topology of phylogenetic trees built using both Neighbor-Joining and Maximum-Likelihood methods.ConclusionThe proposed fur evolutionary model, which includes one general duplication and two in-genus duplications followed by divergence and specialization, explains the presence and diversity of fur genes within the Actinobacteria. Although a few rare horizontal gene transfer events have been reported, the model is consistent with the view of gene duplication as a main force of microbial genomes evolution. The parallel study of Fur phylogeny across diverse organisms offers a solid base to guide functional studies and allows the comparison between response mechanisms in relation with the surrounding environment. The survey of regulators among related genomes provides a relevant tool for understanding the evolution of one of the first lines of cellular adaptability, control of DNA transcription.

Increasing Awareness about Antibiotic Use and Resistance: A Hands-On Project for High School Students

Background Health-promoting education is essential to foster an informed society able to make decisions about socio-scientific issues based on scientifically sustained criteria. Antibiotic resistance is currently a major public health issue. Considering that irrational antibiotic use has been associated with the development and widespread of antibiotic resistant bacteria, educational interventions to promote prudent antibiotic consumption are required. Methodology/Principal Findings This study focuses on the outcomes of an interventional program implemented at the University of Porto, Portugal, to promote awareness about antibiotic resistance at high school levels (15–17 year old). The project Microbiology recipes: antibiotics à la carte articulates a set of wet and dry lab activities designed to promote the participants’ understanding of concepts and processes underlying antibiotics’ production and activity, such as the notion of mechanisms of action of antibiotics. Following a mix-method approach based on a pre−/post design, the effectiveness of this project was assessed by gathering data from surveys, direct observation and analysis of artifacts of 42 high school students (aged 15 and 16 years). The results indicate that the participants developed a more comprehensive picture of antibiotic resistance. The project was shown to promote more sophisticated conceptualizations of bacteria and antibiotics, increased awareness about the perils of antibiotic resistance, and enhanced consciousness towards measures that can be undertaken to mitigate the problem. The participants regarded their experiences as enjoyable and useful, and believed that the project contributed to improve their understanding and raise their interest about the issues discussed. Furthermore, there were also improvements in their procedural skills concerning the laboratory techniques performed. Conclusions/Significance This study evidences the possibility of increasing high school students’ awareness about the consequences of antibiotic resistance and the importance of judicious antibiotic use. The findings inform about the educational benefits of incorporating hands-on activities in science education programs.

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.