Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Analysis of genome-specific sequences inPhleum species: Identification and use for study of genomic relationships

Sau3AI shot gun cloning and colony hybridization with total genomic probes were used to isolate genome-specific sequences inPhleum species. The total DNA isolated from diploid speciesP. alpinum andP. bertolonii was partially digested withSau3AI and cloned using pUC19 as a vector to anE. coli strain DH5mcr. A partial genomic DNA library consisting of 3030 colonies for the genome ofP. alpinum and one consisting of 3240 colonies for the genome ofP. bertolonii were constructed. Twelve hundred and thirty colonies from the DNA library ofP. alpinum and 1320 from that ofP. bertolonii were respectively blotted to membrane filters and hybridized to the total genomic probes from these two species. Eight clones specific toP. alpinum and 13 specific toP. bertolonii were isolated through colony hybridization and further dot-blot hybridization. Most of these clones may carry highly or moderately repetitive sequences. Three sequences specific toP. alpinum and 3 specific toP. bertolonii were used as probes to hybridize theEcoRI-digested DNA samples from four species,P. alpinum,P. bertolonii,P. pratense andP. montanum, on Southern blot. The results from these hybridization experiments showed that all 3P. bertolonii-specific probes and 2 of the 3P. alpinum-specific probes hybridized to the DNA ofP. pratense, thus confirming the conclusion of the close relationships between the cultivated timothy and its two wild relatives that was drawn in our previous study using the C-banding technique.     

短杆菌属海洋环境适应机制的泛基因组学

[目的]为了探究短杆菌属对海洋环境的适应机制.[方法]本研究通过对6株分离自不同洋区、属于不同分类单元的短杆菌菌株进行测序、拼接和注释,结合23株从美国国家生物技术信息中心(NCBI)下载的短杆菌属模式菌株及非模式菌株的基因组数据,进行泛基因组学分析和物种进化分析.[结果]泛基因组学分析表明短杆菌属具有开放型泛基因组,...     

DNA extraction from processed wood: A case study for the identification of an endangered timber species (Gonystylus bancanus)

We applied human forensic techniques to the extraction of whole genomic DNA from processed wood samples to explore the possibility of identifying an endangered tropical timber species by using DNA sequencing technology. High-yield and high-quality DNA samples were obtained from 2 commercial wood and 3 herbarium samples. Large PCR fragments ranging from 500–800 bp were successfully amplified from 2 chloroplast and 1 mitochondrial regions in all 5 samples, indicating limited degradation of the cytoplasmic genomes. DNA extraction from stem wood taken from herbarium specimens appeared superior to that from stem wood with bark intact or from leaf samples. DNA sequences from thetrn regions allowed for easy identification of the focal species based on GenBank Blast search. Little sequence variation was observed in the 3 regions, with the mitochondrialcox3 region completely conserved. Extraction of high-quality and large intact DNA fragments makes dry wood materials amenable to various DNA marker-based applications, including fingerprinting and historical approaches. By sampling stemwood, the wealth of historical information housed in international herbaria can be explored with minimal damage to taxonomically important features.     

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

The potential use of identification of skeletal remains for the early detection of Ammotragus: an exotic ungulate species in Southern Spain

The main aim of our work was to evaluate osteometry as a complementary tool for the early detection of Ammotragus lervia, an exotic ungulate, which currently shows an expanding trend in southeastern Spain. For this purpose, 142 metacarpi and 123 metatarsi from seven Iberian ungulate species were determined by means of a classification function. In a general way, this function works, but regarding related species (those very similar from a morphometrical view), correct determination was reached in only 44.4–90% of the cases. However, in these cases, we can use auxiliary criteria like sexually dimorphic traits, and reach 100% correct identification of bones.     

Microhabitat use by rodent species in a central Brazilian cerrado

We analysed microhabitat use by the rodents Calomys tener, Necromys lasiurus and Thalpomys lasiotis and the factors that may influence their abundances in “murundu” grasslands (open fields with termite mounds) at Aguas Emendadas Ecological Station, Planaltina, Federal District, Brazil. Two grids with 100 sampling points were established and traps were placed at each intersection of the grid, where five microhabitat variables were also measured. Rodents were trapped from June through October 2008. Microhabitat explained 21% of the variation in community structure, with grass density and the number of termite mounds explaining most of the variation. Necromys lasiurus was most often captured in areas with dense grasses, whereas T. lasiotis and C. tener were most often in areas less dense with grasses.     

Genetic and molecular events in transformation ofHaemophilus influenzae with plasmid RSF 0885 carrying cloned segments of chromosomal DNA

InHaemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ inHaemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. Therec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different clones. The reduced transformation frequencies seen inrec 1 ^- strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells can increase the transformation efficiency onrec 1 ⁺ strain.     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004     

Tool use inCebus apella: A case study

The case of a captive Cebus apella,capable of utilizing detached objects as true hammering tools in cracking nuts open, is investigated. In the experimental task administered, the monkey was offered nuts of two different sizes, hazelnuts and walnuts, and a choice among three “hammers” of different percussional efficacy. Its performance was compared to that of one of its cagemates that did not use tools and to its own performance in the absence of tools. Results show that there was a consistent choice of the most efficient tool, that tool use greatly reduces the amount of time needed to crack a nut open (in comparison with either the action of the teeth or pounding the nut against a hard substrate), and that the efficacy of tool use is not conditioned by the relative hardness or size of the nut. In the light of these results, the role of tool use in the exploitation of food resouces is discussed.     

Genus-wide Bacillus species identification through proper artificial neural network experiments on fatty acid profiles

Gas chromatographic fatty acid methyl ester analysis of bacteria is an easy, cheap and fast-automated identification tool routinely used in microbiological research. This paper reports on the application of artificial neural networks for genus-wide FAME-based identification of Bacillus species. Using 1,071 FAME profiles covering a genus-wide spectrum of 477 strains and 82 species, different balanced and imbalanced data sets have been created according to different validation methods and model parameters. Following training and validation, each classifier was evaluated on its ability to identify the profiles of a test set. Comparison of the classifiers showed a good identification rate favoring the imbalanced data sets. The presence of the Bacillus cereus and Bacillus subtilis groups made clear that it is of great importance to take into account the limitations of FAME analysis resolution for the construction of identification models. Indeed, as members of such a group cannot easily be distinguished from one another based upon FAME data alone, identification models built upon this data can neither be successful at keeping them apart. Comparison of the different experimental setups ultimately led to a few general recommendations. With respect to the routinely used commercial Sherlock Microbial Identification System (MIS, Microbial ID, Inc. (MIDI), Newark, Delaware, USA), the artificial neural network test results showed a significant improvement in Bacillus species identification. These results indicate that machine learning techniques such as artificial neural networks are most promising tools for FAME-based classification and identification of bacterial species.     

Rationalising germplasm collections: a case study for wheat

In total 70 genebank accessions comprising 50 hexaploid, 12 tetraploid and 8 diploid wheats of the Gatersleben collection were selected based on the screening of the passport data for identical cultivar names or accession numbers of the donor genebanks. Twelve potential duplicate groups consisting of three to nine accessions with identical names/numbers were selected and analysed with DNA markers (microsatellites). A bootstrap approach based on re-sampling of both microsatellite markers and alleles within marker loci was used to test for homogeneity. Although several homogeneous groups were identified it became clear that cultivar name identity alone did not allow the determination of duplicates. A combination of SSR-analysis followed by the bootstrap method and database survey considering the botanical classification and other data (origin, growth habit and donor) available is recommended in order to determine duplicates. A procedure for the identification of duplicates and their further handling in ex situ genebanks is discussed.     

The use of antlers to monitor temporal variation in environmental lead levels: a case study from an industrialized area in Germany

In order to assess temporal changes in ambient lead levels, Pb concentrations were determined in roe deer (Capreolus capreolus) antlers (n=116) that had been collected in the industrialized area of Siegen (western Germany) in the period 1948–2000. Lead concentrations ranged between 0.3 and 166.3 mg/kg dry weight. An overall decline in antler Pb concentration occurred from the mid-1970s onward, and lead concentrations in the samples from the periods 1980–1989 and 1990–2000 were significantly lower than those in samples from previous periods. Our findings indicate a marked decline in lead exposure of the roe deer inhabiting the study area in the recent past. This decline is attributed to a decrease in atmospheric lead deposition, caused mainly by the phase-out of leaded gasoline, and, in addition, by a reduction of lead emissions from stationary sources. The results of the present study and of previous investigations demonstrate that analysis of antler samples obtained in an area over a certain period can be used to reconstruct temporal changes in environmental lead levels.     

The importance of temporal dynamics of edge effect in reedbed design: a 12-year study on five bird species

Although edge effect is a key topic of conservation biology, we have no data on the temporal dynamics of it. I investigated the distribution of five passerine bird species across reedbed (Phragmites australis) edges during large-scale construction work in the Kis-Balaton marshland, Hungary. The construction provided an experimental approach to study the effects of large timescale changes within a shorter period, because neither the locality nor the vegetation type changed. The water level was increased in the study area, which homogenised the internal structure of reedbed by declining the scattered small willow bushes (Salix) and the grass/sedge layer. The sedge (Acrocephalus schoenobaenus) and reed warblers (Acrocephalus scirpaceus) preferred edges. The sedge warbler, however, declined after inundation, while the reed warbler did not respond. Savis warbler (Locustella luscinioides) sharply declined during the study with changing edge effect. The number of great reed warblers (Acrocephalus arundinaceus) increased during the study, mainly in the reedbed interior, where the stands became patchy with open water. Reed bunting (Emberiza schoeniclus) avoided interiors, and declined over the study. Therefore, there were significant changes in the distribution of reedbed birds across the edge, although the location of edges and the basic habitat, reedbed, did not change. The results highlight the need to incorporate edge effect as a dynamic process in wetland planning.     

Moth pollination of woody species in the Cerrados of Central Brazil: a case of so much owed to so few?

We present a survey of moth pollination in woody species of the Cerrado of Central Brazil. Although with the exception of Roupala montana (which has simple polysepalous flowers arranged in dense cymes) all moth pollinated species in this community have tubular flowers, or a pseudo-tube formed from a single folded petal in Qualea grandiflora, settling moth flowers (tube less than 15 mm) vs. hawkmoth flowers (tube more than 50 mm) are markedly different in size. Moths visit some 20 woody Cerrado species, but they are probably effective pollinators of only 13 species or ca.14% of the woody taxa studied, and even in these latter species they are often very sparse visitors. Nevertheless, it is notable that moths are pollinators for 21% of the 38 most commonly distributed woody Cerrado species. Moreover, the reproductive efficacy of the studied moth pollinated species was similar to that of species pollinated by other kind of vectors.     

Notch signalling in Drosophila: three ways to use a pathway

Cell-cell interactions mediated by Notch are critical at multiple stages of development. Our current understanding of the Notch signalling pathway suggests a comparatively simple transduction mechanism. However, this core pathway can be deployed in three different types of developmental process: lateral inhibition, lineage decisions and boundary formation. These illustrate how the activity of the pathway can be modulated both at the cell surface, through availability and effectiveness of ligand interactions, and inside the cell, through effects on the transduction pathway and the responsiveness of target genes.     

Ectomycorrhizal syntheses with Picea abies and three fungal species: a case study on the use of an in vitro technique to identify naturally occurring ectomycorrhizae

Ectomycorrhizal syntheses between Picea abies and the fungal associates Scleroderma citrinum, Boletus luridus, and Tricholoma vaccinum were carried out using Melin's Erlenmeyer flask technique. The symbioses of S. citrinum were characterized by a mantle composed of an outer prosenchymatous and an inner synenchymatous layer. The mantles of B. luridus and T. vaccinum were solely prosenchymatous. Rhizomorphs were produced in all treatments, but only in association with S. citrinum were they differentiated with additional, enlarged hyphae. All synthesized ectomycorrhizae were white or whitish to light orange and greyishorange. On large-scale root sampling in two differing Picea abies forests in Switzerland, nine out of a total of 22 morphological types of ectomycorrhizae were white or yellow in colour and were, therefore, comparable with the synthesized ectomycorrhizae. These nine natural types generally had distinct mantle features (irregular synenchyma, gelatinous matrix, cystides, thick-walled hyphae), but mostly lacked clamp connections. Synthesized ectomycorrhizae, on the other hand, lacked distinct mantle characteristics and always had clamp connections. Natural and synthesized white or yellow ectomycorrhizae did not coincide morphologically and thus identification of the fungal partners of natural symbioses by means of in vitro-synthesis with potential ectomycorrhizal fungi was not possible in the present study.     

The use of a triploid hybrid for introgression in Lolium species

Summary Triploid hybrids of Lolium multiflorum (4x) x L. perenne (2x) behaved cytologically as autotriploids but the segregation of isozyme variants did not always agree with the expected trisomic ratios. The overall effect of these deviations from expectation was a greater proportion than expected of diploid progeny from the cross L. multiflorum (2x) x triploid hybrid which did not include any of the L. perenne alleles at the three marker isozyme loci. The possible mechanisms for these aberrant segregation ratios are discussed together with the advantages of the crossing scheme to accelerate the recovery of the genotype of the recurrent parent in a backcrossing programme to transfer characters from one species to another.     

Pyrolysis patterns of 5 close Corynebacterium species analyzed by artificial neural networks

In the present study, an artificial neural network was trained with the Stuttgart Neural Networks Simulator, in order to identify Corynebacterium species by analyzing their pyrolysis patterns. An earlier study described the combination of pyrolysis, gas chromatography and atomic emission detection we used on whole cell bacteria. Carbon, sulfur and nitrogen were detected in the pyrolysis compounds. Pyrolysis patterns were obtained from 52 Corynebacterium strains belonging to 5 close species. These data were previously analyzed by Euclidean distances calculation followed by Unweighted Pair Group Method of Averages, a clustering method. With this early method, strains from 3 of the 5 species (C. xerosis, C. freneyi and C. amycolatum) were correctly characterized even if the 29 strains of C. amycolatum were grouped into 2 subgroups. Strains from the 2 remaining species (C. minutissimum and C. striatum) cannot be separated. To build an artificial neural network, able to discriminate the 5 previous species, the pyrolysis data of 42 selected strains were used as learning set and the 10 remaining strains as testing set. The chosen learning algorithm was Back-Propagation with Momentum. Parameters used to train a correct network are described here, and the results analyzed. The obtained artificial neural network has the following cone-shaped structure: 144 nodes in input, 25 and 9 nodes in 2 successive hidden layers, and then 5 outputs. It could classify all the strains in their species group. This network completes a chemotaxonomic method for Corynebacterium identification.     

Multigenic and morphometric differentiation of ground squirrels (Spermophilus, Scuiridae, Rodentia) in Turkey, with a description of a new species

This study reports on the molecular phylogenetics of ground squirrels, genus Spermophilus, in Turkey using cytochrome b (1140bp), part of the D-loop and flanking tRNAs (572bp), X chromosome (867-1051bp) and Y chromosome (983-989bp) DNA sequences. Individuals also were characterized by karyotype and with geometric morphometric analyses of mandibles and skulls. Two hundred fourteen individuals from 91 localities were studied. All the data support the recognition of a new species in SW Anatolia: the Taurus ground squirrel Spermophilus taurensis sp. nov. The new species has a small distribution in the Taurus Mountains in an area that is a hotspot for biodiversity. Molecular clock analysis suggests that the new species diverged from the European ground squirrel, Spermophilus citellus, about 2.5 million years ago and that the ancestor of these two species diverged from the widespread Anatolian ground squirrel, Spermophilus xanthoprymnus, about 5 million years ago. Morphometric differentiation in skull and mandible shape among the three species is incomplete, but statistically significant. S. xanthoprymnus is subdivided into five cytochrome b phylogroups and we use these data to infer the location of glacial refugia where the species lived during the last glacial maximum. This study illustrates the potential of combined molecular and morphometric studies to uncover new Anatolian species and to reconstruct their phylogeographic history. The new species is important for squirrel taxonomy and for understanding Eurasian mammal evolution.