Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure.

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

Pseudomonas aeruginosa strains belonging to phylogroup 3 frequently exhibit an atypical quorum sensing response: the case of MAZ105, a tomato rhizosphere isolate.

Pseudomonas aeruginosa is a widespread γ-proteobacterium and an important opportunistic pathogen. The genetically diverse P. aeruginosa phylogroup 3 strains are characterized by producing the pore-forming ExlA toxin and by their lack of a type III secretion system. However, like all strains of this species, they produce several virulence-associated traits, such as elastase, rhamnolipids and pyocyanin, which are regulated by quorum sensing (QS). The P. aeruginosa QS response comprises three systems (Las, Rhl and Pqs, respectively) that hierarchically regulate these virulence factors. The Pqs QS system is composed of the PqsR transcriptional factor, which, coupled with the alkyl-quinolones HHQ or PQS, activates the transcription of the pqsABCDE operon. The products of the first four genes of this operon produce HHQ, which is then converted to PQS by PqsH, while PqsE forms a complex with RhlR and stabilizes it. In this study we report that mutations affecting the Pqs system are particularly common in phylogroup 3 strains. To better understand QS in phylogroup 3 strains we studied strain MAZ105 isolated from tomato rhizosphere and showed that it contains mutations in the central QS transcriptional regulator, LasR, and in the gene encoding the PqsA enzyme involved in the synthesis of PQS. However, it can still produce QS-regulated virulence factors and is virulent in Galleria mellonella and mildly pathogenic in the mouse abscess/necrosis model; our results show that this may be due to the expression of pqsE from a different PqsR-independent promoter than the pqsA promoter. Our results indicate that using anti-virulence therapy based on targeting the PQS system will not be effective against infections by P. aeruginosa phylogroup 3 strains.

PqsR-independent quorum-sensing response of Pseudomonas aeruginosa ATCC 9027 outlier-strain reveals new insights on the PqsE effect on RhlR activity.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its ΔlasR-derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain.IMPORTANCEPseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections.

Testing the Two-Step Model of Plant Root Microbiome Acquisition Under Multiple Plant Species and Soil Sources.

The two-step model for plant root microbiomes considers soil as the primary microbial source. Active selection of the plant's bacterial inhabitants results in a biodiversity decrease toward roots. We collected sixteen samples of in situ ruderal plant roots and their soils and used these soils as the main microbial input for single genotype tomatoes grown in a greenhouse. Our main goal was to test the soil influence in the structuring of rhizosphere microbiomes, minimizing environmental variability, while testing multiple plant species. We massively sequenced the 16S rRNA and shotgun metagenomes of the soils, in situ plants, and tomato roots. We identified a total of 271,940 bacterial operational taxonomic units (OTUs) within the soils, rhizosphere and endospheric microbiomes. We annotated by homology a total of 411,432 (13.07%) of the metagenome predicted proteins. Tomato roots did follow the two-step model with lower α-diversity than soil, while ruderal plants did not. Surprisingly, ruderal plants are probably working as a microenvironmental oasis providing moisture and plant-derived nutrients, supporting larger α-diversity. Ruderal plants and their soils are closer according to their microbiome community composition than tomato and its soil, based on OTUs and protein comparisons. We expected that tomato ß-diversity clustered together with their soil, if it is the main rhizosphere microbiome structuring factor. However, tomato microbiome ß-diversity was associated with plant genotype in most samples (81.2%), also supported by a larger set of enriched proteins in tomato rhizosphere than soil or ruderals. The most abundant bacteria found in soils was the Actinobacteria Solirubrobacter soli, ruderals were dominated by the Proteobacteria Sphingomonas sp. URGHD0057, and tomato mainly by the Bacteroidetes Ohtaekwangia koreensis, Flavobacterium terrae, Niastella vici, and Chryseolinea serpens. We calculated a metagenomic tomato root core of 51 bacterial genera and 2,762 proteins, which could be the basis for microbiome-oriented plant breeding programs. We attributed a larger diversity in ruderal plants roots exudates as an effect of the moisture and nutrient acting as a microbial harbor. The tomato and ruderal metagenomic differences are probably due to plant domestication trade-offs, impacting plant-bacteria interactions.

The outlier Pseudomonas aeruginosa strain ATCC 9027 harbors a defective LasR quorum-sensing transcriptional regulator.

Pseudomonas aeruginosa infections represent an important health problem that has been recognized by the World Health Organization as a research priority. A complex regulatory network called the quorum sensing (QS) regulates several P. aeruginosa virulence-related traits, including production of elastase, rhamnolipids and pyocyanin. The avirulent P. aeruginosa strain ATCC 9027 belongs to clade 3, which is the more distant phylogroup in relationship to the other four clades of this species. This strain does not produce QS-regulated virulence factors such as elastase and rhamnolipids when cultured in rich LB medium. We report here that ATCC 9027 harbors a defective LasR protein, presumably due to the presence of an aspartic acid in position 196 instead of a glutamic acid which is the amino acid present in this position in functional LasR proteins of the type strains PAO1 (clade 1) and PA7 (also belonging to clade 3), among others. In addition, we report that ATCC 9027 and PA7 strains present differences compared to the PAO1 strain in lasB which encodes elastase, and in the rhlR regulatory sequences that modify las-boxes, and that these mutations have a little effect in the expression of these genes by a functional LasR transcriptional regulator.

Virulence factors regulation by the quorum-sensing and Rsm systems in the marine strain Pseudomonas aeruginosa ID4365, a natural mutant in lasR.

Pseudomonas aeruginosa is an opportunistic human pathogen that is able to produce several virulence factors such as pyocyanin, rhamnolipids and elastase. In the clinical reference strain PAO1, synthesis of these virulence factors is regulated transcriptionally by quorum sensing (QS) and post-transcriptionally by the Rsm system. Herein, we investigated the role of these systems in the control of the pyocyanin, rhamnolipids and elastase production in the marine strain ID4365. We found that this strain carries a nonsense mutation in lasR that makes it a natural mutant in the Las QS system. However, its QS response is still functional with the Rhl system activating virulence factors synthesis. We found that the Rsm system affects virulence factors production, since overexpression of RsmA reduces pyocyanin production whereas RsmY overexpression increases its synthesis. Unexpectedly, and in contrast to the type strain PAO1, inactivation of rsmA increases pyocyanin but reduces elastase and rhamnolipids production by a reduction of RhlR levels. Thus, QS and Rsm systems are involved in regulating virulence factors production, but this regulation is different to the PAO1 strain even though their genomes are highly conserved. It is likely that these differences are related to the different ecological niches in which these strains lived.

Evolution of bacteria seen through their essential genes: the case of Pseudomonas aeruginosa and Azotobacter vinelandii.

Pseudomonas aeruginosa is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its pathogenicity-related traits belongs to its core-genome while both environmental and clinical isolates are part of the same population with a highly conserved genomic sequence. Unexpectedly, considering the high level of sequence identity and homologue gene number shared between different P. aeruginosa isolates, the presence of specific essential genes of the two type strains PAO1 and PA14 has been reported to be highly variable. Here we report the detailed bioinformatics analysis of the essential genes of P. aeruginosa PAO1 and PA14 that have been previously experimentally identified and show that the reported gene variability was owed to sequencing and annotation inconsistencies, but that in fact they are highly conserved. This bioinformatics analysis led us to the definition of 348 P. aeruginosa general essential genes. In addition we show that 342 of these 348 essential genes are conserved in Azotobacter vinelandii, a nitrogen-fixing, cyst-forming, soil bacterium. These results support the hypothesis of A. vinelandii having a polyphyletic origin with a Pseudomonads genomic backbone, and are a challenge to the accepted theory of bacterial evolution.

A Novel Two-Component System, Encoded by the sco5282/sco5283 Genes, Affects Streptomyces coelicolor Morphology in Liquid Culture.

Streptomyces are mycelial bacteria adapted to grow in soil. They have become important producers of biomolecules with medical applications, but their growth in industrial fermenters is challenged by their peculiar morphology in liquid culture: the hyphae tend to clump and grow as large pellets, which are oxygen- and nutrient-limited, grow slowly and present diminished protein production. Here, by implementing an experimental evolution strategy, a S. coelicolor strain, 2L12, with dispersed morphology and reduced pellet size in liquid culture and no defects in either differentiation or secondary metabolism was selected. Genome sequencing revealed a single amino acid substitution in a sensor kinase, Sco5282, of unknown function to be responsible for the morphological changes. Moreover, genetic and biochemical scrutiny identified Sco5283 as the cognate response regulator and demonstrated that the acquired mutation activates this two-component system. Finally, transcriptomic analysis of the mutant strain revealed changes in expression of genes involved in central processes such as glycolysis, gluconeogenesis, stress-signaling pathways, proteins secretion and cell envelope metabolism. Thus a novel two-component system is proposed to play a key role in the control of Streptomyces extracellular metabolism.

Role of ß-oxidation and de novo fatty acid synthesis in the production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa.

Pseudomonas aeruginosa are ubiquitous γ-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the ß-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(R)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and ß-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (R)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through ß-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (S)-3-hydroxyacyl-CoA).

Inactivation of the quorum-sensing transcriptional regulators LasR or RhlR does not suppress the expression of virulence factors and the virulence of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an environmental bacterium but is also an opportunistic pathogen. The aim of this work is to evaluate the contribution of P. aeruginosa LasR and RhlR transcriptional regulators of the quorum-sensing response (QSR) to the production of virulence factors, and to its virulence in a mouse abscess model. The QSR is a complex regulatory network that modulates the expression of several virulence factors, including elastase, pyocyanin and rhamnolipids. LasR, when complexed with the auto-inducer 3-oxo-dodecanoyl lactone (3O-C12-HSL), produced by LasI, is at the top of the QSR regulatory cascade since it activates transcription of some genes encoding virulence factors (such as the gene coding for elastase, lasB) and also transcription of both rhlR and rhlI, encoding the synthase of the auto-inducer butanoyl-homoserine lactone (C4-HSL). In turn RhlR, coupled with C4-HSL, activates the transcription of genes encoding for the enzymes involved in pyocyanin and rhamnolipid production. Several efforts have been made to obtain inhibitors of LasR activity that would suppress the QSR. However, these attempts have used chemical compounds that might not be specific for LasR inactivation. In this work we show that individual inactivation of either lasR or rhlR did not block the QSR, nor did it impair P. aeruginosa virulence, and that even a lasR rhlR double mutant still presented residual virulence, even lacking the production of virulence factors. These results show that the inhibition of either lasR or rhlR is not a straightforward approach to blocking P. aeruginosa virulence, due to the great complexity of the QSR.

5p and 3p Strands of miR-34 Family Members Have Differential Effects in Cell Proliferation, Migration, and Invasion in Cervical Cancer Cells.

The micro RNA (miR)-34 family is composed of 5p and 3p strands of miR-34a, miR-34b, and miR-34c. The 5p strand's expression and function is studied in cervical cancer. The 3p strand's function and regulation remain to be elucidated. To study the function of the passenger strands of miR-34 family members, we overexpressed 5p and 3p strands using a synthetic miRNA in cervical cell lines. Cell proliferation was evaluated using crystal violet. Migration and invasion were tested using transwell assays, Western blot, and zymography. Possible specific targets and cell signaling were investigated for each strand. We found that miR-34a-5p inhibited proliferation, migration, and cell invasion accompanied by matrix metalloproteinase 9 (MMP9) activity and microtubule-associated protein 2 (MAP2) protein reduction. We also found that miR-34b-5p and miR-34c-5p inhibit proliferation and migration, but not invasion. In contrast, miR-34c-5p inhibits MMP9 activity and MAP2 protein, while miR-34b-5p has no effect on these genes. Furthermore, miR-34a-3p and miR-34b-3p inhibit proliferation and migration, but not invasion, despite the later reducing MMP2 activity, while miR-34c-3p inhibit proliferation, migration, and cell invasion accompanied by MMP9 activity and MAP2 protein inhibition. The difference in cellular processes, MMP2 and MMP9 activity, and MAP2 protein inhibition by miR-34 family members suggests the participation of other regulated genes. This study provides insights into the roles of passenger strands (strand*) of the miR-34 family in cervical cancer.

Corrigendum: Variability of Bacterial Essential Genes Among Closely Related Bacteria: The Case of Escherichia coli.

[This corrects the article DOI: 10.3389/fmicb.2018.01059.].

Overproduction of rhamnolipids in Pseudomonas aeruginosa PA14 by redirection of the carbon flux from polyhydroxyalkanoate synthesis and overexpression of the rhlAB-R operon.

OBJECTIVE: To construct Pseudomonas aeruginosa PA14 derivatives that overproduce rhamnolipids (RL) by blocking the synthesis of the carbon-storage polymer polyhydroxyalkanoates (PHA) and by overexpressing the rhlAB-R operon that encodes for enzymes of RL synthesis and the RhlR transcriptional regulator. RESULTS: In contrast to previous results showing that overexpression of rhlAB-R genes in two P. aeruginosa strains (PAO1 and ATCC 9027) is sufficient to overproduce RL, we show that a PA14 derivative overexpressing the rhlAB-R operon did not increase the synthesis of these biosurfactants. In addition, PA14 mutants deficient in PHA production did not overproduce RL either. However, if the rhlAB-R genes were expressed in a mutant that is completely impaired in PHA synthesis, a significant increase in RL production was observed (59%). These results show that RL production in PA14 is limited both by the availability of fatty acid precursors and by the levels of the RhlA and RhlB enzymes that are involved in the synthesis of mono-RL. CONCLUSIONS: The limitation of RL production by P. aeruginosa PA14 is multifactorial and diverse from the results obtained with other strains. Thus, the factors that limit RL production are particular to each P. aeruginosa strain, so strain-specific strategies should be developed to increase their production.

The Escherichia coli bcsB gene is a conditional essential gene in the context of functional cellulose synthesis.

The understanding of why a gene is essential for a bacterium has implications in different research areas, such as bacterial evolution, synthetic biology and biotechnology, making the identification of essential genes a very active research field. Bacterial essential genes have been defined, among other criteria, by the inability to obtain viable mutants in such genes. In the case of Escherichia coli this approach led to the construction of the Keio collection of single-gene knockout mutants that contains deletions of all the open reading frames present in the genome with the exception of 303 genes that were found to be essential for the growth of this bacterium. One of the genes that was identified as essential is bcsB, which is involved in synthesis of extracellular cellulose. However, the reason for the essential nature of BcsB for E. coli viability has not been determined. In this work we show that bcsB is essential only in strains that have a functional capacity to synthesize cellulose, presumably due to the activity of BcsB in the translocation of this polymer across the periplasm. Thus, we propose that bcsB is a conditionally essential gene in E. coli.

Variability of Bacterial Essential Genes Among Closely Related Bacteria: The Case of Escherichia coli.

The definition of bacterial essential genes has been widely pursued using different approaches. Their study has impacted several fields of research such as synthetic biology, the construction of bacteria with minimal chromosomes, the search for new antibiotic targets, or the design of strains with biotechnological applications. Bacterial genomes are mosaics that only share a small subset of gene-sequences (core genome) even among members of the same species. It has been reported that the presence of essential genes is highly variable between closely related bacteria and even among members of the same species, due to the phenomenon known as "non-orthologous gene displacement" that refers to the coding for an essential function by genes with no sequence homology due to horizontal gene transfer (HGT). The existence of dormant forms among bacteria and the high incidence of HGT have been proposed to be driving forces of bacterial evolution, and they might have a role in the low level of conservation of essential genes among related bacteria by non-orthologous gene displacement, but this correlation has not been recognized. The aim of this mini-review is to give a brief overview of the approaches that have been taken to define and study essential genes, and the implications of non-orthologous gene displacement in bacterial evolution, focusing mainly in the case of Escherichia coli. To this end, we reviewed the available literature, and we searched for the presence of the essential genes defined by mutagenesis in the genomes of the 63 best-sequenced E. coli genomes that are available in NCBI database. We could not document specific cases of non-orthologous gene displacement among the E. coli strains analyzed, but we found that the quality of the genome-sequences in the database is not enough to make accurate predictions about the conservation of essential-genes among members of this bacterial species.

The microRNA landscape of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived skin tumor. It is the second-most-common cancer affecting the Caucasian population and is responsible for >20% of all skin-cancer-related deaths. The estimated incidence of non-melanoma skin cancer in the USA is >1000000 cases per year, of which roughly 20-30% are squamous cell carcinoma. To better understand and treat this challenging cancer, current research focuses on development of novel strategies to improve the understanding of tumor biogenesis on an individual basis. microRNAs are becoming important biomarkers in the diagnosis, prognosis and treatment of cSCC. This review describes the current knowledge on miRNA expression in cSCC and its role as a biomarker for personalized medicine.

The Transcriptional Regulators of the CRP Family Regulate Different Essential Bacterial Functions and Can Be Inherited Vertically and Horizontally.

One of the best-studied transcriptional regulatory proteins in bacteria is the Escherichia coli catabolite repressor protein (CRP) that when complexed with 3'-5'-cyclic AMP (cAMP) changes its conformation and interacts with specific DNA-sequences. CRP DNA-binding can result in positive or negative regulation of gene expression depending on the position of its interaction with respect to RNA polymerase binding site. The aim of this work is to review the biological role and phylogenetic relations that some members of the CRP family of transcriptional regulators (also known as cAMP receptor protein family) have in different bacterial species. This work is not intended to give an exhaustive revision of bacterial CRP-orthologs, but to provide examples of the role that these proteins play in the expression of genes that are fundamental for the life style of some bacterial species. We highlight the conservation of their structural characteristics and of their binding to conserved-DNA sequences, in contrast to their very diverse repertoire of gene activation. CRP activates a wide variety of fundamental genes for the biological characteristic of each bacterial species, which in several instances form part of their core-genome (defined as the gene sequences present in all members of a bacterial species). We present evidence that support the fact that some of the transcriptional regulators that belong to the CRP family in different bacterial species, and some of the genes that are regulated by them, can be inherited by horizontal gene transfer. These data are discussed in the framework of bacterial evolution models.

Pseudomonas aeruginosa quorum-sensing response in the absence of functional LasR and LasI proteins: the case of strain 148, a virulent dolphin isolate.

Pseudomonas aeruginosa is an opportunistic pathogen that presents a complex regulatory network called 'quorum-sensing', which is responsible for the transcription of genes coding for several traits implicated in its pathogenicity. Strain 148 is a dolphin isolate that has been shown to produce quorum-sensing-regulated virulence traits and to be virulent in a mouse model, despite the fact that it contains a 20-kbp deletion that eliminates from the chromosome the lasR gene and the lasI promoter. LasR is a key quorum-sensing transcriptional regulator that, when coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) produced by LasI, activates transcription of genes coding for some virulence-associated traits such as elastase, lasI, rhlI and rhlR. RhlR is also a key quorum-sensing transcriptional regulator that, when interacting with the autoinducer butanoyl homoserine lactone (C4-HSL) that is produced by the synthase RhlI, activates the genes involved in the synthesis of some virulence-associated traits, as rhamnolipids and pyocyanin. We describe that in P. aeruginosa 148, the LasR/3O-C12-HSL-independent rhlR transcriptional activation is due to the release of the negative effect of Vfr (a CRP-ortholog) caused by the insertion of an IS element in vfr, and that rhlI transcription is driven from the rhlR promoter, forming the rhlR-I operon.

Use of Mature miRNA Strand Selection in miRNAs Families in Cervical Cancer Development.

Aberrant miRNA expression is well recognized as a cancer hallmark, nevertheless miRNA function and expression does not always correlate in patients tissues and cell lines studies. In addition to this issue, miRNA strand usage conduces to increased cell signaling pathways modulation diversifying cellular processes regulation. In cervical cancer, 20 miRNA families are involved in carcinogenesis induction and development to this moment. These families have 5p and 3p strands with different nucleotide (nt) chain sizes. In general, mature 5p strands are larger: two miRNAs of 24 nt, 24 miRNAs of 23 nt, 35 miRNAs of 22 nt and three miRNAs of 21 nt. On the other hand, the 3p strands lengths observed are: seven miRNAs of 23 nt, 50 miRNAs of 22 nt, six miRNAs of 21 nt and four miRNAs of 20 nt. Based on the analysis of the 20 miRNA families associated with cervical cancer, 67 3p strands and 65 5p strands are selected suggesting selectivity and specificity mechanisms regulating cell processes like proliferation, apoptosis, migration, invasion, metabolism and Warburg effect. The insight reviewed here could be used in the miRNA based therapy, diagnosis and prognosis approaches.