Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Role of Arf-like proteins (Arl1 and Arl2) of Mucor circinelloides in virulence and antifungal susceptibility.

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.

Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans.

The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent Km values for Na+ and Li+ were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent Km for K+ Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea IMPORTANCE: The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria A mechanism is proposed in which an MrpAD subcomplex catalyzes Na+/H+ antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways.

Non-ribosomal Peptide Synthases from Pseudomonas aeruginosa Play a Role in Cyclodipeptide Biosynthesis, Quorum-Sensing Regulation, and Root Development in a Plant Host.

Diverse molecules mediate cross-kingdom communication between bacteria and their eukaryotic partners and determine pathogenic or symbiotic relationships. N-acyl-L-homoserine lactone-dependent quorum-sensing signaling represses the biosynthesis of bacterial cyclodipeptides (CDPs) that act as auxin signal mimics in the host plant Arabidopsis thaliana. In this work, we performed bioinformatics, biochemical, and plant growth analyses to identify non-ribosomal peptide synthase (NRPS) proteins of Pseudomonas aeruginosa, which are involved in CDP synthesis. A reverse genetics strategy allowed the identification of the genes encoding putative multi-modular-NRPS (MM-NRPS). Mutations in these genes affected the synthesis of the CDPs cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-Tyr), while showing wild-type-like levels of virulence factors, such as violacein, elastase, and pyocyanin. When analyzing the bioactivity of purified, naturally produced CDPs, it was found that cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val) were capable of antagonizing quorum-sensing-LasR (QS-LasR)-dependent signaling in a contrasting manner in the cell-free supernatants of the selected NRPS mutants, which showed QS induction. Using a bacteria-plant interaction system, we further show that the pvdJ, ambB, and pchE P. aeruginosa mutants failed to repress primary root growth, but improved root branching in A. thaliana seedlings. These results indicated that the CDP production in P. aeruginosa depended on the functional MM-NRPS, which influences quorum-sensing of bacteria and plays a role in root architecture remodeling.

Transkingdom signaling based on bacterial cyclodipeptides with auxin activity in plants.

Microorganisms and their hosts communicate with each other through an array of signals. The plant hormone auxin (indole-3-acetic acid; IAA) is central in many aspects of plant development. Cyclodipeptides and their derivative diketopiperazines (DKPs) constitute a large class of small molecules synthesized by microorganisms with diverse and noteworthy activities. Here, we present genetic, chemical, and plant-growth data showing that in Pseudomonas aeruginosa, the LasI quorum-sensing (QS) system controls the production of three DKPs--namely, cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Phe), and cyclo(L-Pro-L-Tyr)--that are involved in plant growth promotion by this bacterium. Analysis of all three bacterial DKPs in Arabidopsis thaliana seedlings provided detailed information indicative of an auxin-like activity, based on their efficacy at modulating root architecture, activation of auxin-regulated gene expression, and response of auxin-signaling mutants tir1, tir1 afb2 afb3, arf7, arf19, and arf7arf19. The observation that QS-regulated bacterial production of DKPs modulates auxin signaling and plant growth promotion establishes an important function for DKPs mediating prokaryote/eukaryote transkingdom signaling.

Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from Pseudomonas aeruginosa are involved in its catalytic and thermal properties.

Isocitrate lyase, encoded by the aceA gene, plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids. Phylogenetic analysis indicated that the ICL superfamily is divided in two families: the ICL family, which includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. ICL from P. aeruginosa (ICL-Pa) was identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes (grouped in subfamily 1). Analysis also showed that psychrophilic bacteria are mainly grouped in ICL subfamily 3, whose ICL proteins contain the highly conserved catalytic pattern QIENQVSDEKQCGHQD. We performed site-directed mutagenesis, enzymatic activity, and structure modeling of conserved residues in mutated ICLs by using ICL-Pa as a model. Our results indicated that the N214 residue is essential for catalytic function, while mutating the Q211, E219, and Q221 residues impairs its catalytic and thermostability properties. Our findings suggest that conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation.

Co-expression of α and ß subunits of the 3-methylcrotonyl-coenzyme A carboxylase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and ß), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αß)(4) complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30º C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K(m) for its substrate and V(max) of 168 µM and 430 nmoles mg(-1)min(-1), respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.

Bacterial transport of sulfate, molybdate, and related oxyanions.

Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.

Phylogenetic analysis of the chromate ion transporter (CHR) superfamily.

ChrA is a membrane protein that confers resistance to the toxic ion chromate through the energy-dependent chromate efflux from the cytoplasm. In the protein databases, ChrA is a member of the chromate ion transporter (CHR) superfamily, composed of at least several dozens of members, distributed in the three domains of life. The aim of this work was to perform a phylogenetic analysis of the CHR superfamily. An exhaustive search for ChrA homologous proteins was carried out at the National Center for Biotechnology Information database. One hundred and thirty-five sequences were identified as members of the CHR superfamily [77 long-chain sequences, or bidomains (LCHR), and 58 short-chain sequences, or monodomains (SCHR)], organized mainly as tandem pairs of genes whose resultant proteins probably possess oppositely oriented membrane topology. LCHR sequences were split into amino and carboxyl domains, and the resultant domains were aligned with the SCHR proteins. A phylogenetic tree was reconstructed using four different methods, obtaining similar results. The domains were grouped into three clusters: the SCHR proteins cluster, the amino domain cluster of LCHR proteins and the carboxyl domain cluster of LCHR proteins. These results, as well as differences in the genomic context of CHR proteins, enabled the proteins to be sorted into two families (SCHR and LCHR), and 10 subfamilies. Evidence was found suggesting an ancient origin of LCHR proteins from the fusion of two SCHR protein-encoding genes; however, some secondary events of fusion and fission may have occurred later. The separate distribution of the LCHR and SCHR proteins, differences in the genomic context in both groups and the fact that chromate transport has been demonstrated only in LCHR proteins suggest that the CHR proteins comprise two or more paralogous groups in the CHR superfamily.

Identification of the aceA gene encoding isocitrate lyase required for the growth of Pseudomonas aeruginosa on acetate, acyclic terpenes and leucine.

Pseudomonas aeruginosa PAO1 mutants affected in acyclic monoterpenes, n-octanol, and acetate assimilation were isolated using transposon mutagenesis. The isocitrate lyase gene (aceA) corresponding to ORF PA2634 of the PAO1 strain genome was identified in one of these mutants. The aceA gene encodes a protein that is 72% identical to the isocitrate lyase (ICL) characterized from Colwellia maris, but is less than 30% identical to their homologues from pseudomonads. The genetic arrangement of aceA suggests that it is a monocistronic gene, and no adjacent related genes were found. The ICL protein was detected as a 60-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis from cultures grown on acetate, but not in glucose-grown PAO1 cultures. Genetic complementation further confirmed that the aceA gene encodes the ICL enzyme. The ICL enzyme activity in crude extracts from cultures of the PAO1 strain was induced by acetate, citronellol and leucine, and repressed by growth on glucose or citrate. These results suggest that ICL is involved in the assimilation of acetate, acyclic monoterpenes of the citronellol family, alkanols, and leucine, in which the final intermediary acetyl-coenzyme A may be channelled to the glyoxylate shunt.

Leak and Scattering Assessment by Optically Stimulated Luminescence Dosimetry in a Helical Linear Accelerator / Valoración de Fuga y Dispersión por medio de Dosimetría de Luminiscencia Estimulada Ópticamente (OSL) en un Acelerador Lineal Helicoidal

Radiation absorbed doses to organs outside the radiation therapy treatment beam can be significant and therefore of clinical interest. Two sets of out-of-beam measurements were performed measuring the leak dose and the scattered dose, at 5 points within the accelerator components (accelerator tube and collimator) and at 21 points on the equipment and surroundings based on a positioning scheme. For this purpose, 52 Optically Stimulated Luminescence (OSL) dosimeters were used in a latest generation helical linear accelerator. Of the 200 cGy fired at a cheese-like phantom, 0.332% of the out-of-beam dose contribution was found to come from the leak and 0.784% was transformed into scattering. For these dose values, estimates of the risk of second tumors in long-term survivors indicate a reduced probability of acquiring a second secondary radiation malignancy, based on information from the 1990 BEIR Committee report.

La dosis absorbida de radiación a órganos fuera del haz de tratamiento de radioterapia puede ser significativa y, por lo tanto, de interés clínico. Se realizaron dos sets de mediciones fuera del haz para determinar la dosis de fuga y la dosis dispersa, en 5 puntos dentro de los componentes del acelerador (tubo de aceleración y colimador) y 21 puntos en el equipo y alrededores basado en un esquema de posicionamiento. Para este fin se utilizaron 52 dosímetros de luminiscencia estimulada ópticamente (OSL, Optically Stimulated Luminescence), en un acelerador lineal helicoidal de última generación. De los 200 cGy disparados a un maniquí tipo queso, se encontró que el 0.332% de la contribución de dosis fuera del haz provenía de la fuga y 0.784% se transforma en dispersión. Para estos valores de dosis, las estimaciones del riesgo de segundos tumores en los supervivientes a largo plazo indican una reducida probabilidad de contraer una segunda malignidad por radiación secundaria, según la información del informe del Comité BEIR de 1990.

Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides.

In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gß (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gß and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gß and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gß), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis.

Structural evidence for the involvement of the residues Ser187 and Tyr422 in substrate recognition in the 3-methylcrotonyl-coenzyme A carboxylase from Pseudomonas aeruginosa.

The enzyme 3-methylcrotonyl-CoA carboxylase from Pseudomonas aeruginosa (Pa-MCCase) is essential for the assimilation of leucine and acyclic monoterpenes. The structure of the Pa-MCCase was analysed by computational modelling to establish the molecular basis of substrate recognition. The active site is composed of two zones, which may play important roles in substrate recognition and catalysis. To further understand the interactions of the active site with the substrate, site-directed mutagenesis of the conserved residues S187 and R51 located in zone I, and F417, Y422 and G423 from zone II of the Pa-MCCase was carried out. The residue substitutions S187A and Y422D completely abolished the Pa-MCCase activity, whereas substitutions R51A, F417Y and G423A indicated that these residues are not essential. Interestingly, the residues R47, R51 and S187 form a well-defined pocket that may play important roles in substrate coupling to the Co-A motif. At zone one, mutation S187A was essential, but mutant R51A retained activity, suggesting that the R51 function could be relegated to neighbouring positive residues. Residue Y422 instead of contributing to substrate discrimination, it may participate in deprotonation of methyl group on MC-CoA, because it is located at adequate distances from the 3-methylcrotonyl-chain and carboxybiotin groups in the Pa-MCCase carboxylation site.

Mechanisms of bacterial resistance to chromium compounds.

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.