A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterisation of type 1 cell surface-associated antigens

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment.

Mangrove ecosystems are environments subject to substantial degradation by anthropogenic activities. Its location, in coastal area, interfacing the continents and the oceans makes it substantially important in the prospection for biotechnological applications. In this study, we assessed the diversity of culturable bacteria present over the seasons at two depths (0-10 and 30-40 cm) in a mangrove sediment and in a transect area from the land to the sea. In total, 238 bacteria were isolated, characterized by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and further identified, by Fatty Acid Methyl Esther (FAME-MIDI), into the orders of Vibrionales, Actinomycetales and Bacillales. Also the ability of the isolates in producing economically important enzymes (amylases, proteases, esterases and lipases) was evaluated and the order Vibrionales was the main enzymatic source.

Microsatellite analysis of pacu broodstocks used in the stocking program of Paranapanema River, Brazil

O monitoramento da diversidade genética é fundamental em um programa de repovoamento. Avaliouse a diversidade genética de pacu Piaractus mesopotamicus (Holmberg, 1887) em duas estações de piscicultura em Andirá -Paraná, Brasil, utilizadas no programa de repovoamento do Rio Paranapanema. Foram amplificados seis loci microssatélite para avaliar 60 amostras de nadadeira. O estoque de reprodutores B apresentou maior número de alelos e heterozigose (alelos: 22 e H O: 0,628) que o estoque de reprodutores A (alelos: 21 e H O: 0,600). Alelos com baixos níveis de frequência foram observados nos dois estoques. Os coeficientes positivos de endogamia no locus Pme2 (estoque A: F IS = 0,30 e estoque B: F IS = 0,20), Pme5 (estoque B: F IS = 0,15), Pme14 (estoque A: F IS = 0,07) e Pme28 (estoque A: F IS = 0,24 e estoque B: F IS = 0,20), indicaram deficiência de heterozigotos. Foi detectada a presença de um alelo nulo no lócus Pme2. As estimativas negativas nos loci Pme4 (estoque A: F IS = -0,43 e estoque B: F IS= -0,37), Pme5 (estoque A: F IS = - 0,11), Pme14 (estoque B: F IS = - 0,15) e Pme32 (estoque A: F IS = - 0,93 e estoque B: F IS = - 0,60) foram indicativas de excesso de heterozigotos. Foi evidenciado desequilíbrio de ligação e riqueza alélica baixa só no estoque A. A diversidade genética de Nei foi alta nos dois estoques. A distância (0,085) e identidade (0,918) genética mostraram similaridade entre os estoques, o qual reflete uma possível origem comum. 6,05% da variância genética total foi devida a diferenças entre os estoques. Foi observado um recente efeito gargalo nos dois estoques. Os resultados indicaram uma alta diversidade genética nos estoques de reprodutores e baixa diferenciação genética entre eles, o que foi causado pelo manejo reprodutivo das pisciculturas, redução do tamanho populacional e intercâmbio genético entre as pisciculturas.

Study on DNA Damage Induced by Neon Beam Irradiation in Saccharomyces Cerevisiae

Yeast strain Saccharornyces cerevisiae was irradiated with different doses of 85 MeV/u Ne-20(10+) to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks (DSBs) and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Cy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T-->G and T-->C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.

Characterization of the genetic variability among Caesalpinia pulcherrima (L.) Sw. (Fabaceae) plants using RAPD molecular markers

O flamboyanzinho (Caesalpinia pulcherrima, Fabaceae) é muito utilizado como cerca-viva e na arborização de ruas, parques e jardins. de florescimento exuberante, suas flores podem apresentar coloração rosa, laranja ou amarela, conforme a variedade. Como características florais são essenciais para a definição do valor comercial de espécies ornamentais, o objetivo principal do trabalho foi verificar a existência de polimorfismo entre plantas de C. pulcherrima, que produzem flores de diferentes colorações, por marcadores moleculares RAPD. Foram escolhidas aleatoriamente 30 plantas adultas, cultivadas no município de Jaboticabal, Estado de São Paulo, para a retirada de amostras foliares para a extração de DNA. Dentre essas matrizes, 20 possuem flores alaranjadas, oito, flores amarelas, e apenas duas produzem flores de coloração rosa. Dos 140 primers de RAPD avaliados, 94 foram capazes de ampliar fragmentos definidos, gerando 246 bandas, das quais se observou 100% de bandas monomórficas, indicando que nenhum dos primers utilizados detectou polimorfismo entre os tratamentos. Concluiu-se que a técnica para detecção de polimorfismo por marcadores RAPD não foi eficiente, e que existe a necessidade de se testarem marcadores moleculares mais específicos. Além disso, a característica morfológica cor de flor é, possivelmente, controlada por vários genes ou pela combinação deles.

A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.

Temporal genetic structure patterns in tropical maize populations under reciprocal recurrent selection.

In order to investigate the effect of long term recurrent selection on the pattern of gene diversity, thirty randomly-selected individuals from the progenitors (p) and four selection cycles (C0, C3, C6 and C11) were sampled for DNA analysis from the tropical maize (Zea mays L.) breeding populations, Atherton 1 (AT1) and Atherton 2 (AT2). Fifteen polymorphic Simple Sequence Repeat markers amplified a total of 284 and 257 alleles in AT1 and AT2 populations, respectively. Reductions in the number of alleles were observed at advanced selection cycles. About 11 and 12% of the alleles in AT1 and AT2 populations respectively, were near to fixation. However, a higher number of alleles (37% in AT1 and 33% in AT2) were close to extinction. Fisher's exact test and analysis of molecular variance (AMOVA) showed significant population differentiations. Gene diversity estimates and AMOVA revealed increased genetic differentiations at the expense of loss of heterozygosity. Population differentiations were mainly due to fixation of complementary alleles at a locus in the two breeding populations. The estimates of effective population at an advanced selection cycle were close to the population size predicted by the breeding method.

Genetic structure and low-genetic diversity suggesting the necessity for conservation of the Chinese Longsnout catfish, Leiocassis longirostris (Pisces : Bagriidae)

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Development of polymorphic EST markers in Penaeus monodon: applications in penaeid genetics

The objective of this study was to develop type I markers for genome mapping and other genetic studies of Penaeus monodon. Primers were designed based on expressed sequence tags (ESTs) from a P monodon cephalothorax cDNA library to amplify 100-300 bp products. 34 of the primer pairs successfully amplified PCR products from genomic DNA. Single-strand conformation polymorphism analysis showed that similar to 30% of the ESTs tested exhibit polymorphism in a test panel of P monodon individuals. Mendelian inheritance of the EST-derived markers has been established in two international reference mapping families of P monodon, and mapping of these markers is in progress. Some ESTs were successfully amplified from other Penaeus species (P. chinensis, P japonicus and P vannamei), indicating that the markers are applicable in cross-species comparison. Two populations of P. japonicus could be differentiated using one of the ESTS. In conclusion, the polymorphic EST markers developed in this study are applicable in genome mapping and population genetic studies of penaeid shrimp. (C) 2002 Elsevier Science B.V. All rights reserved.

Amplified DNA detection sensitivity using streptavidin-biotinylated protein complex: Characterization by electrochemical impedance spectroscopy

Thiol-terminated oligonucleotide was immobilized to gold surface by self-assembly method. A novel amplification strategy was introduced for improving the sensitivity of DNA. hybridization using biotin labeled protein-streptavidin network complex. This complex can be formed in a cross-linking network of molecules so that the amplification of the response signal will be realized due to the big molecular size of the complex. It could be proved from the impedance technique that this amplification strategy caused dramatic improvement of the detection sensitivity. These results give significant advances in the generality and sensitivity as it is applied to biosensing.

Molecular identification of 16 Porphyra lines using sequence-related amplified polymorphism markers

Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs). The SRAP analytic system was set up and applied to Porphyra germplasm identification in this study for the first time. Sixteen Porphyra lines were screened by SRAP technique with 30 primer combinations. In the analysis, 14 primer combinations produced stable and reproducible amplification patterns in three repetitive experiments. Among the total 533 amplified fragments, 522 (98%) were polymorphic, with an average of 38 fragments for each primer combination, ranging in size from 50 to 500 bp. The 533 fragments were visually scored one by one and then used to develop a dendrogram with Unweighted Pair-Group Method Arithmetic Average (UPGMA), and the 16 Porphyra lines were divided into two major groups at the 0.68 similarity level. From the total 533 fragments, I I amplified by two primer combinations, ME1/EM1 and ME4/EM6, were used to develop the DNA fingerprints of the 16 Porphyra lines. The DNA fingerprints were then converted into binary codes, with I and 0 representing presence and absence of the corresponding amplified fragment, respectively. In the DNA fingerprints, each of the 16 Porphyra lines has its unique binary code and can be easily distinguished from the others. This is the first report on the development of SRAP technique and its utilization in germplasm identification of seaweeds. The results demonstrated that SRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the classification and identification of Porphyra lines. (c) 2007 Elsevier B.V. All rights reserved.

Genetic mapping of laminaria japonica and l. longissima using amplified fragment length polymorphism markers in a "two-way pseudo-testcross" strategy

With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F, cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.