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.

The blackspot seabream, Pagellus bogaraveo, is a sparid fish of great economic importance in the northeast Atlantic. The main aim of this work was to assess the infection levels and diversity of anisakid nematodes parasitizing P. bogaraveo from Portuguese waters. The anisakid larvae were identified by polymerase chain reaction-restriction fragment length polymorphism analysis and ten different patterns were observed, four of which were not previously reported in the literature. Moreover, several species were detected for the first time in this host: Anisakis simplex × Anisakis pegreffii hybrids, Anisakis ziphidarum, Anisakis typica, Anisakis physeteris, as well as three undescribed anisakids Anisakis sp. PB-2009, Anisakis sp. PB-2010, and Contracaecum sp. PB-2010. The ITS1-5.8S-ITS2 region was sequenced and analyzed phylogenetically, revealing that our anisakids were distributed by the two distinct clades reported previously, corresponding to the two recognized larval morphotypes. Moreover, a group of organisms, including our specimens from Madeira and the previously reported Anisakis sp. HC-2005, cluster together and seem to belong to clade I. A certain degree of intraspecific diversity was also detected. Samples from mainland waters had the highest infection levels and were dominated by A. pegreffii. Madeira had the highest diversity overall, dominated by Anisakis sp. PB-2010. Fish from the Azores had the lowest infection levels, and the species with the highest relative abundance was A. physeteris. The anisakid nematode communities were relatively similar in mainland waters but very distinct in both the Azores and Madeira islands, suggesting the existence of at least three different stocks of P. bogaraveo in the northeast Atlantic.

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes).The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments.The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N2-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.

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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.

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70–0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

The existing data support Portugal as the western European country with the highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1-infected individuals followed at the reference hospital of the province of Minho, Portugal, at which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%) and the C (14.5%), F1 (7.3%), and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with the A1 and F1 subtypes increased over the years compared with those with B, G, or C subtypes or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of the B and G subtypes were found. However, transmission clusters of non-B and non-G viruses among native individuals were also found, with the dates of the most recent common ancestor estimated to be in the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and are mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

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