Abigail González-Valdez

Transcriptional regulation of Pseudomonas aeruginosa rhlR: role of the CRP orthologue Vfr (virulence factor regulator) and quorum-sensing regulators LasR and RhlR.

The production of many virulence factors by Pseudomonas aeruginosa is regulated by the quorum-sensing (QS) response. In this regulatory network LasR and RhlR, bound to their corresponding autoinducers, play a central role. The QS response has a hierarchical structure: LasR/3O-C12-HSL activates the transcription of rhlR, and RhlR/C4-HSL activates the transcription of several genes, including the rhlAB operon, which encodes the enzymes responsible for rhamnolipid synthesis. The rhlAB operon is located immediately upstream of the rhlR gene. rhlR has four transcription start sites, two of which are located in the rhlB coding region. Vfr directly activates transcription of lasR, and has been reported to be also involved in rhlR expression. The aim of this work was to characterize the details of the mechanism of rhlR transcriptional regulation. We show that Vfr directly regulates rhlR transcription through its binding to several Vfr-binding sites (VBSs) present in the rhlR promoter region, one of which has a negative effect on transcription. Two of the VBSs overlap with las boxes where LasR/3O-C12-HSL binds to activate rhlR transcription. We also show that rhlR transcription is subject to positive-feedback autoregulation through RhlR/C4-HSL activation of the rhlA promoter. This positive autoregulation plays a major role in rhlR expression.

Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity

BackgroundPseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits.ResultsIn this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes.ConclusionsContrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed.

The Stability of G6PD Is Affected by Mutations with Different Clinical Phenotypes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The kcat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.

High variability in quorum quenching and growth inhibition by furanone C-30 in Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis patients causing severe damage. This bacterium is intrinsically resistant to antibiotics and shows resistance against new antimicrobials and its virulence is controlled by the quorum-sensing response. Thus, attenuating its virulence by quorum quenching instead of inhibiting its growth has been proposed to minimize resistance; however, resistance against the canonical quorum quencher furanone C-30 can be achieved by mutations leading to increased efflux. In the present work, the effect of C-30 in the attenuation of the QS-controlled virulence factors elastase and pyocyanin was investigated in 50 isolates from cystic fibrosis patients. The results demonstrate that there is a high variability in the expression of both elastase and pyocyanin and that there are many naturally resistant C-30 strains. We report that the main mechanism of C-30 resistance in these strains was not due to enhanced efflux but a lack of permeability. Moreover, C-30 strongly inhibited the growth of several of the isolates studied, thus imposing high selective pressure for the generation of resistance.

Glucose-6-Phosphate Dehydrogenase: Update and Analysis of New Mutations around the World

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the g6pd gene; recognizing 186 mutations. In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein.

Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production.

Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.

Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.

The effect of specific rhlA-las-box mutations on DNA binding and gene activation by Pseudomonas aeruginosa quorum-sensing transcriptional regulators RhlR and LasR.

Pseudomonas aeruginosa is a free-living bacterium and an important opportunistic pathogen. The genes coding for virulence-associated traits are regulated at the level of transcription by the quorum-sensing response. In this response, the regulator LasR coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) activates transcription of genes for several virulence factors. LasR/3O-C12-HSL also activates transcription of rhlR, the gene coding for the transcriptional regulator RhlR, and of rhlI that encodes the synthase that produces the autoinducer butanoyl-homoserine lactone (C4-HSL) that interacts with RhlR. Genes activated by RhlR/C4-HSL include those involved in rhamnolipids production (like the rhlAB operon) and lecA, coding for PA-I lectin. The molecular basis of LasR/3O-C12-HSL- and RhlR/C4-HSLDNA-binding specificity (at the so-called las-boxes) has not been clearly determined, and the aim of this work was to contribute to its understanding. Therefore, we analyzed the interaction of LasR and RhlR to variants of the rhlA-las-box that were constructed based on the comparison of this las-box to the las-box of lecA. We conclude that LasR and RhlR DNA-binding specificity is a complex multifactorial phenomenon in which both positive and negative effects are involved and that binding of these proteins does not necessarily result in gene activation.

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

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

Abstract 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.