The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies

Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment

Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit

Background: The currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments. Results: We demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features. Conclusions: Differential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning

Erwinia oleae sp. nov., isolated from olive knots caused by Pseudomonas savastanoi pv. savastanoi

Three endophytic bacterial isolates were obtained in Italy from olive knots caused by Pseudomonas savastanoi pv. savastanoi. Phenotypic tests in combination with 16S rRNA gene sequence analysis indicated a phylogenetic position of these isolates in the genus Erwinia or Pantoea, and revealed two other strains with highly similar 16S rRNA gene sequences (> 99 %), CECT 5262 and CECT 5264, obtained in Spain from olive knots. Rep-PCR DNA fingerprinting of the five strains from olive knots with BOX, ERIC and REP primers revealed three groups of profiles that were highly similar to each other. Multilocus sequence analysis (MLSA) based on concatenated partial atpD, gyrB, infB and rpoB gene sequences, indicated that the strains constitute a single novel species in the genus Erwinia. The strains showed general phenotypic characteristic of Erwinia, and whole genome DNA-DNA hybridization data confirmed they represent a single novel Erwinia species. The strains showed a DNA G+C base composition ranging from 54.7 to 54.9 mol%. They could be discriminated from the phylogenetically related Erwinia species by their ability to utilise potassium gluconate, L-rhamnose and D-arabitol, but not glycerol, inositol and D-sorbitol. The name Erwinia oleae (type strain DAPP-PG 531T = LMG 25322T = DSM 23398T) is proposed for this new taxon

On the Geometric Interplay Between Goodness-of-Fit and Estimation: Illustrative Examples

We show how information geometry throws new light on the interplay between goodness-of-fit and estimation, a fundamental issue in statistical inference. A geometric analysis of simple, yet representative, models involving the same population parameter compellingly establishes the main theme of the paper: namely, that goodness-of-fit is necessary but not sufficient for model selection. Visual examples vividly communicate this. Specifically, for a given estimation problem, we define a class of least-informative models, linking these to both nonparametric and maximum entropy methods. Any other model is then seen to involve an informative rotation, often embodying extra-data considerations. We also look at the way that translation of models generates a form of bias-variance trade-off. Overall, our approach is a global extension of pioneering local work by Copas and Eguchi which, we note, was also geometrically inspired

Therapeutic Strategies for Modulating the Extracellular Matrix to Improve Pancreatic Islet Function and Survival After Transplantation

PURPOSES OF REVIEW: Extracellular matrix (ECM) components modulate the interaction between pancreatic islet cells. During the islet isolation prior to transplantation as treatment for type 1 diabetes, the ECM is disrupted impacting functional graft survival. Recently, strategies for restoring ECM have shown to improve transplantation outcomes. This review discusses the current therapeutic strategies to modulate ECM components to improve islet engraftment. RECENT FINDINGS: Approaches applied are seeding islets in ECM of decellularized organs, supplementation of specific ECM components in polymeric scaffolds or immunoisolating capsules, and stimulating islet ECM production with specific growth factors or ECM-producing cells. These strategies have shown success in improving functional islet survival. However, the same experiments show that caution should be taken as some ECM components may negatively impact islet function and engraftment. ECM restoration resulted in improved transplantation outcomes, but careful selection of beneficial ECM components and strategies is warranted