Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Epiphytic fitness of a biological control agent of fire blight in apple and pear orchards under Mediterranean weather conditions

The behaviour of Pseudomonas fluorescens EPS62e was investigated in apple and pear orchards under Mediterranean climatic conditions. The trials studied the influence of weather conditions, plant host species, presence of indigenous microbial community and spread from treated to nontreated trees on colonization and survival. Population dynamics were assessed by real-time PCR and CFU-counting methods. With inoculated flowers, weather conditions were optimal for colonization, and EPS62e established high and stable population levels around 10(8) CFU per organ, according to both methods of analysis. The plant host species did not influence the colonization rate, and the biocontrol agent dominated the microbial communities of blossoms, representing up to 100% of the total cultivable population. With inoculated leaves, the EPS62e population decreased to nondetectable levels 30 days after treatment according to both methods used. EPS62e spread moderately in the orchard, being detected in nontreated flowers of trees 15-35 m from the inoculation site. The combined use of real-time PCR and CFU-counting methods of analysis permitted the identification of three physiological states for EPS62e in the field, which consisted of active colonization, survival and entry into a viable but nonculturable state, and cell death.     

Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii

Most higher plants and microalgae are not able to synthesize ketocarotenoids. In this study the unicellular chlorophyte Chlamydomonas reinhardtii has been genetically engineered with the beta-carotene ketolase cDNA from Haematococcus pluvialis, bkt1 (GeneBank accession no. X86782), involved in the synthesis of astaxanthin, to obtain a transgenic microalga able to synthesize ketocarotenoids. The expression of bkt1 was driven by the Chlamydomonas constitutive promoter of the rubisco small subunit (RbcS2) and the resulting protein was directed to the chloroplast by the Chlamydomonas transit peptide sequences of Rubisco small subunit (RbcS2) or Ferredoxin (Fd). In all transformants containing the bkt1 gene fused to the RbcS2 or the Fd transit peptides a new pigment with the typical ketocarotenoid spectrum was detected. Surprisingly this ketocarotenoid was not astaxanthin nor canthaxanthin. The ketocarotenoid was identified on the basis of its mass spectrum as 3,3'-dihydroxy-beta,varepsilon-carotene-4-one (4-keto-lutein) or its isomer ketozeaxanthin.     

The crystal structure of human E-cadherin domains 1 and 2, and comparison with other cadherins in the context of adhesion mechanism

Cell adhesion mediated by type I cadherins involves homophilic "trans" interactions that are thought to be brought about by a strand exchange mechanism involving the N-terminal extracellular domain. Here, we present the high-resolution crystal structure of the N-terminal two domains of human E-cadherin. Comparison of this structure with other type I cadherin structures reveals features that are likely to be critical to facilitate dimerization by strand exchange as well as dimer flexibility. We integrate this structural knowledge to provide a model for type I cadherin adhesive interactions. Intra-molecular docking of the conserved N-terminal "adhesion arm" into the acceptor pocket in monomeric E-cadherin appears largely identical to inter-molecular docking of the adhesion arm in adhesive trans dimers. A strained conformation of the adhesion arm in the monomer, however, may create an equilibrium between "open" and "closed" forms that primes the cadherin for formation of adhesive interactions, which are then stabilized by additional dimer-specific contacts. By contrast, in type II cadherins, strain in the adhesion arm appears absent and a much larger surface area is involved in trans adhesion, which may compensate the activation energy required to peel off the intra-molecularly docked arm. It seems that evolution has selected slightly different adhesion mechanisms for type I and type II cadherins.     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.