Gracia Morales

Population structure of Pseudomonas aeruginosa

The metabolically versatile Gram-negative bacterium Pseudomonas aeruginosa inhabits terrestrial, aquatic, animal-, human-, and plant-host-associated environments and is an important causative agent of nosocomial infections, particularly in intensive-care units. The population genetics of P. aeruginosa was investigated by an approach that is generally applicable to the rapid, robust, and informative genotyping of bacteria. DNA, amplified from the bacterial colony by circles of multiplex primer extension, is hybridized onto a microarray to yield an electronically portable binary multimarker genotype that represents the core genome by single nucleotide polymorphisms and the accessory genome by markers of genomic islets and islands. The 240 typed P. aeruginosa strains of diverse habitats and geographic origin segregated into two large nonoverlapping clusters and 45 isolated clonal complexes with few or no partners. The majority of strains belonged to few dominant clones widespread in disease and environmental habitats. The most frequent genotype was represented by the sequenced strain PA14. Core and accessory genome were found to be nonrandomly assembled in P. aeruginosa. Individual clones preferred a specific repertoire of accessory segments. Even the most promiscuous genomic island, pKLC102, had integrated preferentially into a subset of clones. Moreover, some physically distant loci of the core genome, including oriC, showed nonrandom associations of genotypes, whereas other segments in between were freely recombining. Thus, the P. aeruginosa genome is made up of clone-typical segments in core and accessory genome and of blocks in the core with unrestricted gene flow in the population.

The Pseudomonas putida Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.

Induction of axonal growth by heterophilic interactions between the cell surface recognition proteins Fll and Nr-CAM/Bravo

F11 and Nr-CAM/Bravo are two axon-associated glycoproteins belonging to different subgroups of the immunoglobulin superfamily. In this report we have investigated the interaction of both proteins using neurite outgrowth and binding assays. Antibody blocking experiments demonstrate that neurite extension of tectal cells on immobilized F11 is mediated by Nr-CAM/Bravo. Binding studies further reveal a direct heterophilic interaction between F11 and Nr-CAM/Bravo. This activity can be mapped to the amino-terminal second or third immunoglobulin-like domain within F11 with domain-specific monoclonal antibodies and deletion mutant proteins expressed on COS cells. Furthermore, perturbation experiments with domain-specific monoclonal antibodies demonstrate that this region is required for adhesion and neurite extension.

Structure of Pseudomonas aeruginosa Populations Analyzed by Single Nucleotide Polymorphism and Pulsed-Field Gel Electrophoresis Genotyping

Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.

The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator

Pseudomonas putida KT2440 contains a branched aerobic respiratory chain with multiple terminal oxidases. Their relative proportion varies according to environmental conditions. The role of the oxygen-responsive ANR global regulator on expression of these terminal oxidases was analysed. During exponential growth in a highly aerated complete medium, ANR activated expression of the Cbb3-1 terminal oxidase (equivalent to Pseudomonas aeruginosa Cbb3-2), but had little role on expression of other terminal oxidases. In early stationary phase, or under oxygen limitation, inactivation of the anr gene led to increased expression of the bo(3)-type cytochrome (Cyo) and cyanide-insensitive (CIO) terminal oxidases, and to a much lower expression of Cbb3-1. DNase I footprints identified ANR binding sites at the promoters for these oxidases. Their location suggests that ANR is a transcriptional activator of Cbb3-1 genes and a repressor of CIO genes, consistent with expression data. ANR binding sites at the promoter for Cyo genes suggests a complex regulation in combination with other factors. Therefore, ANR coordinates expression of Cyo, CIO and Cbb3-1, but does not influence cytochrome aa3 and Cbb3-2 terminal oxidases under the conditions analysed. Functional assays showed that Cyo has a leading role during aerobic exponential growth, while Cbb3-1 becomes very important in stationary phase.

The expression of the Leishmania infantum KMP-11 protein is developmentally regulated and stage specific.

Transcription of the gene coding for the KMP-11 protein of Leishmania infantum results in the production of a mature RNA transcript of 1.3 kb in length. The expression of KMP-11 mRNA is strongly down-regulated not only during the parasite growth from the logarithmic to the stationary phase but also during the differentiation transit from promastigotes to amastigotes. The estimated concentration of KMP-11 is one order of magnitude higher in promastigotes than in amastigotes. The analysis of the Triton X-114 phase partition of the protein shows that, in agreement with its predicted secondary structure, KMP-11 has an amphipathic nature since it is found in the aqueous as well as in the detergent phase. By fluorescence microscopy a defined pattern of distribution of the protein was observed only in promastigotes where KMP-11 is mainly located in the flagellum and the flagellar pocket.

The Leishmania infantum histone H3 possesses an extremely divergent N-terminal domain

The isolation of a Leishmania cDNA clone coding for an antigen identified as the histone H3 is described. The nucleotide sequence of the cDNA predicts that the Leishmania histone H3 contains 129 residues and that it has a molecular mass of 14,620 Da. Comparison of the amino acid sequence with the consensus sequence of the eukaryotic histone H3 shows that the Leishmania protein has a highly conserved globular region and an extremely divergent amino-terminal portion.

Synergistic Neurite-outgrowth Promoting Activity of Two Related Axonal Proteins, Bravo/Nr-CAM and G4/Ng-CAM in Chicken Retinal Explants

In the developing chicken retina, optic fibres migrating to the tectum express on their surfaces several cell adhesion molecules, including Bravo/Nr‐CAM and G4/Ng‐CAM. We have previously described differential distribution along the retinotectal projection and differential modulation by environmental cues for Bravo and G4 and here we further compare the characteristics of these immunoglobulin superfamily molecules. From day 6 of embryonic development (E6) to day 20 (E20), Bravo and G4 were found to coexist in the retinal optic fibre layer. However, while G4 staining was confined to that layer, as development proceeded Bravo staining spread to the plexiform layers and some radial structures of the retina. G4 displayed a dose‐dependent neurite‐outgrowth promoting activity for E6 retinal explants, while Bravo did not support neurite growth. Surprisingly, when the retinal explants were grown on mixtures of the two molecules, a much more vigorous growth of neurites was seen, revealing a synergistic effect. We propose that Bravo and G4, as well as other axonal surface molecules, affect axonal growth in different ways when they are present in combination than when they are alone.

Both B and γ δ TCR+ lymphocytes regulate α β TCR+ lymphocytes involved in superantigen specific responses

Endogenous superantigens (SAg) presented by MHC class II IA molecules induce slow‐evolving negative selection of α β T cells. The role of both B and γ δ T cells on the regulation of these SAg‐specific α β T cell responses was addressed in IAb+IEb‐ C57BL/6 mice bearing genetically induced B cell and γ δ T cell deficiencies. B lymphocytes were required in the negative selection of Vβ5+/Vβ12+ CD4+ T cells. In contrast, γ δ T cells positively stimulated the utilization of the same SAg‐responsive α β T cell subsets. These differences started in mature CD4+ thymocytes and extended to naive T cell pools for B cell negative selection, and up to memory T cells for γ δT cell influences. The levels of SAg‐responsive T cells did not vary between C57BL/6 and double deficient (B cell and γ δ T cell‐deficient) congenic mice, implying that both B and γ δ T cells acted through independent mechanisms.