Study of pyoverdine type and production by Pseudomonas aeruginosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and accumulation of pyoverdine-negative mutations

The lungs of cystic fibrosis patients are frequently colonized by Pseudomonas aeruginosa, which produces high-affinity fluorescent peptidic siderophores, pyoverdines. Three pyoverdines which differ in their peptide chain and are easily differentiated by isoelectric focusing exist, only one being produced by a given strain. P. aeruginosa isolates from cystic fibrosis patients of a German hospital were analyzed by sequential, pulse-field gel electrophoresis (PFGE) and for pyoverdine production and type. Only producers of type I and type II pyoverdine were found. There was a perfect correlation between the type of pyoverdine produced and the clonality determined by PFGE. PFGE clone C, the most prevalent among cystic fibrosis patients, and found in an aquatic environment, produced type II pyoverdine. Pyoverdine-negative mutants seemed to increase as a function of the lung colonization time, but retained the capacity to take up pyoverdines. Most isolates that took up type II pyoverdine were also able to utilize type I pyoverdine as judged by growth stimulation experiments. No correlation was observed between the loss of pyoverdine production and mucoidy.     

Concordance of Epileptic Networks Associated with Epileptic Spikes Measured by High-Density EEG and Fast fMRI

Objective

The present study aims to investigate whether a newly developed fast fMRI called MREG (magnetic resonance encephalography) measures metabolic changes related to interictal epileptic discharges (IED). For this purpose BOLD changes are correlated with the IED distribution and variability.

Methods

Patients with focal epilepsy underwent EEG-MREG using a 64 channel cap. IED voltage maps were generated using 32 and 64 channels and compared regarding their correspondence to the BOLD response. The extents of IEDs (defined as number of channels with >50% of maximum IED negativity) were correlated with the extents of positive and negative BOLD responses. Differences in inter-spike variability were investigated between interictal epileptic discharges (IED) sets with and without concordant positive or negative BOLD responses.

Results

17 patients showed 32 separate IED types. In 50% of IED types the BOLD changes could be confirmed by another independent imaging method. The IED extent significantly correlated with the positive BOLD extent (p = 0.04). In 6 patients the 64-channel EEG voltage maps better reflected the positive or negative BOLD response than the 32-channel EEG; in all others no difference was seen. Inter-spike variability was significantly lower in IED sets with than without concordant positive or negative BOLD responses (with p = 0.04).

Significance

Higher density EEG and fast fMRI seem to improve the value of EEG-fMRI in epilepsy. The correlation of positive BOLD and IED extent could suggest that widespread BOLD responses reflect the IED network. Inter-spike variability influences the likelihood to find IED concordant positive or negative BOLD responses, which is why single IED analysis may be promising.     

Multilocus Sequence Analysis of the Marine Bacterial Genus Tenacibaculum Suggests Parallel Evolution of Fish Pathogenicity and Endemic Colonization of Aquaculture Systems

The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Complex recombination patterns arising during geminivirus coinfections preserve and demarcate biologically important intra-genome interaction networks

Genetic recombination is an important process during the evolution of many virus species and occurs particularly frequently amongst begomoviruses in the single stranded DNA virus family, Geminiviridae. As in many other recombining viruses it is apparent that non-random recombination breakpoint distributions observable within begomovirus genomes sampled from nature are the product of variations both in basal recombination rates across genomes and in the over-all viability of different recombinant genomes. Whereas factors influencing basal recombination rates might include local degrees of sequence similarity between recombining genomes, nucleic acid secondary structures and genomic sensitivity to nuclease attack or breakage, the viability of recombinant genomes could be influenced by the degree to which their co-evolved protein-protein and protein-nucleotide and nucleotide-nucleotide interactions are disreputable by recombination. Here we investigate patterns of recombination that occur over 120 day long experimental infections of tomato plants with the begomoviruses Tomato yellow leaf curl virus and Tomato leaf curl Comoros virus. We show that patterns of sequence exchange between these viruses can be extraordinarily complex and present clear evidence that factors such as local degrees of sequence similarity but not genomic secondary structure strongly influence where recombination breakpoints occur. It is also apparent from our experiment that over-all patterns of recombination are strongly influenced by selection against individual recombinants displaying disrupted intra-genomic interactions such as those required for proper protein and nucleic acid folding. Crucially, we find that selection favoring the preservation of co-evolved longer-range protein-protein and protein DNA interactions is so strong that its imprint can even be used to identify the exact sequence tracts involved in these interactions.     

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.     

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.