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.     

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67

Cryptococcus neoformans is an opportunistic human pathogen that causes life‐threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal–host relationship is discussed.     

How belowground interactions contribute to the coexistence of mycorrhizal and non-mycorrhizal species in severely phosphorus-impoverished hyperdiverse ecosystems

Background

Mycorrhizal strategies are very effective in enhancing plant acquisition of poorly-mobile nutrients, particularly phosphorus (P) from infertile soil. However, on very old and severely P-impoverished soils, a carboxylate-releasing and P-mobilising cluster-root strategy is more effective at acquiring this growth-limiting resource. Carboxylates are released during a period of only a few days from ephemeral cluster roots. Despite the cluster-root strategy being superior for P acquisition in such environments, these species coexist with a wide range of mycorrhizal species, raising questions about the mechanisms contributing to their coexistence.

Scope

We surmise that the coexistence of mycorrhizal and non-mycorrhizal strategies is primarily accounted for by a combination of belowground mechanisms, namely (i) facilitation of P acquisition by mycorrhizal plants from neighbouring cluster-rooted plants, and (ii) interactions between roots, pathogens and mycorrhizal fungi, which enhance the plants’ defence against pathogens. Facilitation of nutrient acquisition by cluster-rooted plants involves carboxylate exudation, making more P available for both themselves and their mycorrhizal neighbours. Belowground nutrient exchanges between carboxylate-exuding plants and mycorrhizal N₂-fixing plants appear likely, but require further experimental testing to determine their nutritional and ecological relevance. Anatomical studies of roots of cluster-rooted Proteaceae species show that they do not form a complete suberised exodermis.

Conclusions

The absence of an exodermis may well be important to rapidly release carboxylates, but likely lowers root structural defences against pathogens, particularly oomycetes. Conversely, roots of mycorrhizal plants may not be as effective at acquiring P when P availability is very low, but they are better defended against pathogens, and this superior defence likely involves mycorrhizal fungi. Taken together, we are beginning to understand how an exceptionally large number of plant species and P-acquisition strategies coexist on the most severely P-impoverished soils.

     

Specificity of a DNA-based (DNAzyme) peroxidative biocatalyst

A complex formation between hemin and a congruous oligonucleotide not only greatly enhances the former’s peroxidative activity but also results in a biocatalyst (DNAzyme) with a novel specificity. Herein substrate, regio-, enantiomeric, and diastereomeric selectivities of heme, the DNAzyme, and the enzyme horseradish peroxidase are comparatively examined.     

Identifying protein construct variants with increased crystallization propensity--a case study

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.     

Plasma-initiated graft polymerization as an immobilization platform for metal free Russian propolis ethanol extracts designed specifically for biomaterials

The antibacterial and anti-biofilm activities of propolis have been intensively reported. However, the application of this folk remedy as a means to prevent biomedical implant contamination has yet to be completely evaluated. In response to the significant resistant and infectious attributes of biofilms, biomaterials engineered to possess specific chemical and physical properties were immobilized with metal free Russian propolis ethanol extracts (MFRPEE), a known antibacterial agent. The results obtained from this study begin to examine the application of MFRPEE as a novel alternative method for the prevention of medical and biomedical implant infections. When constructed under specific experimental conditions, immobilized biomaterials showed excellent stability when subjected to simulated body fluid and fetal bovine serum. The ability of immobilized biomaterials to specifically target pathogens (both Gram-positive and Gram-negative biofilm forming bacteria), while promoting tissue cell growth, renders these biomaterials as potential candidates for clinical applications.     

Focus: Zoonotic Disease: Zoonotic Ocular Onchocercosis by Onchocerca lupi

The parasitic filarioid Onchocerca lupi causes ocular disease characterized by conjunctivitis and nodular lesions. This nematode was first described in 1967 in a wolf from Georgia, and since then cases of infection from dogs and cats with ocular onchocercosis and sporadically from humans also with subcutaneous and cervical lesions caused by O. lupi have been reported from the Middle East, Europe, and North America. Due to its zoonotic potential, this parasitic infection has gained attention in the past 20 years. Phylogenetic studies have highlighted the recent divergence of O. lupi from other Onchocerca spp. and the importance of domestication in the evolutionary history of this worm. Moreover, the finding of an O. lupi genotype associated with subclinical and mild infection in the Iberian Peninsula, raises important questions about the pathogenicity of this presently enigmatic parasite.     

Persistence of Mhc heterozygosity in homozygous clonal killifish,Rivulus marmoratus: implications for the origin of hermaphroditism

The mangrove killifish Rivulus marmoratus, a neotropical fish in the order Cyprinodontiformes, is the only known obligatorily selfing, synchronous hermaphroditic vertebrate. To shed light on its population structure and the origin of hermaphroditism, major histocompatibility complex (Mhc) class I genes of the killifish from seven different localities in Florida, Belize, and the Bahamas were cloned and sequenced. Thirteen loci and their alleles were identified and classified into eight groups. The loci apparently arose approximately 20 million years ago (MYA) by gene duplications from a single common progenitor in the ancestors of R. marmoratus and its closest relatives. Distinct loci were found to be restricted to different populations and different individuals in the same population. Up to 44% of the fish were heterozygotes at Mhc loci, as compared to near homozygosity at non-Mhc loci. Large genetic distances between some of the Mhc alleles revealed the presence of ancestral allelic lineages. Computer simulation designed to explain these findings indicated that selfing is incomplete in R. marmoratus populations, that Mhc allelic lineages must have diverged before the onset of selfing, and that the hermaphroditism arose in a population containing multiple ancestral Mhc lineages. A model is proposed in which hermaphroditism arose stage-wise by mutations, each of which spread through the entire population and was fixed independently in the emerging clones.