Transformation of Bacillus subtilis by DNA bound on clay in non-sterile soil

Abstract Chromosomal DNA from Bacillus subtilis and different forms of plasmid pHV14 (covalently closed circular (CCC), linear monomer (M), and linear multimer (LM)) were adsorbed and bound on the clay mineral montmorillonite. After extensive washing of the clay-DNA complexes with DNA buffer (pH 7.5), approx. 25% of the chromosomal DNA, and approx. 30, 90, and 5%, respectively, of the CCC, M and LM form remained bound. Chromosomal and plasmid DNA bound on clay were capable of transforming competent cells, with different specific activities. The clay-DNA complexes persisted in non-sterile soil and retained transforming ability up to 15 days after their addition to the soil. DNA bound on montmortillonite was protected from the activity of Eco RI, supporting the evidence that DNA adsorbed on soil components was resistant to degradation by nucleases.     

GC–MS volatolomic approach to study the antimicrobial activity of the antarctic bacterium Pseudoalteromonas sp. TB41

Many bacteria produce a wide range of volatile info-chemicals compounds (mVOCs) that constitute an important regulatory factor in the interrelationships among different organisms in microbial ecosystems. It has been shown that Antarctic bacteria isolated from three different sponge species, by producing mVOCs, are able to inhibit specifically the growth of Burkholderia cepacia complex (Bcc) strains (i.e. opportunistic pathogens of cystic fibrosis patients) as demonstrated by cross-streaking inhibition assays. This study reports a metabolomics approach to investigate the volatile profile of both the Antarctic sponge-associated Pseudoalteromonas sp. TB41 (P-sp-TB41) and Burkholderia cenocepacia strain LMG16654 (Bc-LMG16654) under aerobic conditions. Solid phase micro extraction (SPME) in head space of biological samples allowed an in vivo sampling of mVOCs with minimal specimen disturbance. The SPME fiber was termically desorbed in the injection port of gas chromatography–mass spectrometer (GC–MS) system setted in EI scan mode. The raw data were processed using both an automated mass spectra deconvolution and identification system and a metabolomic approach, which allowed a selection of 30 compounds presumably responsible for the inhibition of Bc-LMG16654 growth. The results obtained from samples prepared under cross-streaking conditions also suggest that the presence of Bc-LMG16654 cells neither interferes with the production of mVOCs nor induces the synthesis of different mVOCs. The employing of mass spectrometry played a key role in tuning the experimental system and in the evaluation of results. The use of this approach to study the interaction, in aerobic condition, among other Antarctic bacteria and Bcc and the possibility to extend this approach to other pathogen-antagonist relationship, is currently in progress.     

VEGF signalling controls GnRH neuron survival via NRP1 independently of KDR and blood vessels

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.     

Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model

This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms’ intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms.     

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.     

Genetic Structure of Marchalina hellenica (Hemiptera: Margarodidae) Populations from Turkey: Preliminary mtDNA Sequencing Data

The scale insect Marchalina hellenica (Gennadius) (Hemiptera: Margarodidae) contributes to the production of pine honey in Turkey and Greece via the honeydew excreted when it feeds on pine trees. Although it is an insect of prime economic importance, there is no information on its genetic structure. Preliminary data were obtained based on sequencing analysis of 12s rDNA and COI mtDNA gene segments from samples from four areas of Turkey. Sequences of the 12s rDNA gene segment from Greek samples available in GenBank were also included. No variability was detected concerning the COI mtDNA gene segment analysis, although 13 haplotypes were revealed based on the 12s rDNA gene segment. The most distant population was from Mudanya-Bursa Province (Turkey). Further research is necessary on the genetic structure and variability of M. hellenica populations from the two neighboring countries.     

The role of gene fusions in the evolution of metabolic pathways: the histidine biosynthesis case

Background

Histidine biosynthesis is one of the best characterized anabolic pathways. There is a large body of genetic and biochemical information available, including operon structure, gene expression, and increasingly larger sequence databases. For over forty years this pathway has been the subject of extensive studies, mainly in Escherichia coli and Salmonella enterica, in both of which details of histidine biosynthesis appear to be identical. In these two enterobacteria the pathway is unbranched, includes a number of unusual reactions, and consists of nine intermediates; his genes are arranged in a compact operon (hisGDC [NB]HAF [IE]), with three of them (hisNB, hisD and hisIE) coding for bifunctional enzymes. We performed a detailed analysis of his gene fusions in available genomes to understand the role of gene fusions in shaping this pathway.

Results

The analysis of HisA structures revealed that several gene elongation events are at the root of this protein family: internal duplication have been identified by structural superposition of the modules composing the TIM-barrel protein.Several his gene fusions happened in distinct taxonomic lineages; hisNB originated within γ-proteobacteria and after its appearance it was transferred to Campylobacter species (ε-proteobacteria) and to some Bacteria belonging to the CFB group. The transfer involved the entire his operon. The hisIE gene fusion was found in several taxonomic lineages and our results suggest that it probably happened several times in distinct lineages.Gene fusions involving hisIE and hisD genes (HIS4) and hisH and hisF genes (HIS7) took place in the Eukarya domain; the latter has been transferred to some δ-proteobacteria.

Conclusion

Gene duplication is the most widely known mechanism responsible for the origin and evolution of metabolic pathways; however, several other mechanisms might concur in the process of pathway assembly and gene fusion appeared to be one of the most important and common.

     

Phylogeny of the genus Azospirillum based on 16S rDNA sequence

Abstract The 16S rDNA of 17 strains of Azospirillum , 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum , and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens ; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.