Comparative Analysis of the Orphan CRISPR2 Locus in 242 Enterococcus faecalis Strains

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.     

Evaluation of the fossil fish‐specific diversity in a chadian continental assemblage: Exploration of morphological continuous variation in Synodontis (Ostariophysi,Siluriformes)

In the fossil record, the quantification of continuous morphological variation has become a central issue when dealing with species identification and speciation. In this context, fossil taxa with living representatives hold great promise, because of the potential to characterise patterns of intraspecific morphological variation in extant species prior to any interpretation in the fossil record. The vast majority of catfish families fulfil this prerequisite, as most of them are represented by extant genera. However, although they constitute a major fish group in terms of distribution, and ecological and taxonomic diversity, the quantitative study of their past morphological variation has been neglected, as fossil specimens are generally identified based on the scarcest remains, that is, complete neurocrania that bear discrete characters. Consequently, a part of freshwater catfish history is unprospected and unknown. In this study, we explored the morphological continuous variation of the humeral plate shape in Synodontis catfishes using Elliptic Fourier Analysis (EFA), and compared extant members and fossil counterparts. We analysed 153 extant specimens of 11 Synodontis species present in the Chad basin, in addition to 23 fossil specimens from the Chadian fossiliferous area of Toros Menalla which is dated around 7 Ma. This highly speciose genus, which is one of the most diversified in Africa, exhibits a rich fossil record with several hundred remains mostly identified as Synodontis sp. The analysis of the outline of the humeral plate reveals that some living morphological types were already represented in the Chad Basin 7 My ago, and allows for the discovery of extinct species. Beside illuminating the complex Neogene evolutionary history of Synodontis, these results underline the interest in the ability of isolated remains to reconstruct a past dynamic history and to validate the relevance of EFA as a tool to explore specific diversity through time. J. Morphol. 277:1486–1496, 2016. © 2016 Wiley Periodicals, Inc.     

Study of non-consumptive mortality of Crustacean zooplankton in a Siberian reservoir using staining for live/dead sorting and sediment traps

We studied non-consumptive (non-predatory) mortality of Daphnia and Cyclops vicinus during four sampling seasons. Mortality estimations were based on live/dead sorting using special staining and measurements of sedimentation rates for dead individuals, depended on wind speed. Original equations were used for calculations. The estimated specific non-consumptive mortality never had biologically senseless negative values, which were often obtained on the basis of the other ways of mortality estimations, and was in a good agreement with other components of population dynamics. As found, the non-consumptive mortality was the important, often the determinant component of the zooplankton population dynamics.     

Influence of Nutrient Concentrations on MPN Quantification and Enrichment of Nitrate-Reducing Fe(II)-Oxidizing and Fe(III)-Reducing Bacteria from Littoral Freshwater Lake Sediments

The application of culture-dependent studies to quantify Fe-metabolizing microorganisms from the environment is a necessity, as there are so far no universal functional marker genes for application in culture-independent studies. Media composition can vary between studies, therefore, we determined the effects of three different growth media on the quantification (MPNs) and identity (via cloning and sequencing of dominant DGGE bands) of nitrate-reducing Fe(II)-oxidizers and lactate- or acetate-oxidizing Fe(III)-reducers from a lacustrine sediment: low sulphate freshwater medium (FWM), sterile filtered bicarbonate-buffered lake water (BLW) and a mixture of both (MIX). We consistently found fewer cells in the BLW than in the FWM and the MIX. The DGGE banding patterns of the microbial communities enriched in different media types clustered together according to the e^? donor and acceptor couples and not according to the medium used. Thus, although the medium composition significantly influenced the quantification and thereby conclusions on the abundance and potential significance of the targeted group within the ecosystem, biodiversity assessments through enrichment cultures were less influenced by the medium, but instead were affected by the type and concentration of the e^? donor/acceptor.     

The influence of neuropeptide Y and norepinephrine on ovulation in the rat ovary.

Neuropeptide Y (NPY) was measured in tissue extracts from ovaries of rats treated with pregnant mare serum gonadotropin (PMSG). The extracted NPY-immunoreactive material was identical to synthetic human NPY with regard to size and hydrophobicity as evaluated by gel filtration and high performance liquid chromatography. The concentration of NPY was related to the estrous cycle and a maximum was observed in relation to the endogenous luteinizing hormone (LH) peak. NPY immunoreactivity was demonstrated by immunohistochemistry to be localized within nerve fibers supplying blood vessels and follicles. The increase in the NPY content could not be related to accumulation around specific ovarian structures. Employing an in vitro set-up, NPY (10(-7) M) was unable to induce ovulation and did not increase the ovulation rate in LH-stimulated ovaries. The combination of NPY (10(-7) M) and NE (10(-7) M) did not significantly increase the number of ovulations compared to that induced by NE (10(-7) M) alone. In conclusion, NPY content in the ovary is related to the estrous cycle, but NPY does not seem to have any direct effect on the ovulatory process.     

Microbial production of specifically ring-13C-labelled 4-hydroxybenzoic acid

When transformed with a recombinant vector carrying the ubiC gene (encoding chorismate pyruvate-lyase, EC 4.1.3.27) the triple mutant (Phe^–, Trp^–, Tyr^–) Klebsiella pneumoniae 62-1 excretes 4-hydroxybenzoic acid instead of chorismic acid. The recombinant strain can be used to produce in high yield specifically ring-labelled 4-hydroxybenzoic acid from isotopically labelled glucose.