Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Abstract Background From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.

DNA block copolymers – from micelles to machines

DNA block copolymer, a new class of hybrid material composed of a synthetic polymer and an oligodeoxynucleotide segment, owns unique properties which can not be achieved by only one of the two polymers. Among amphiphilic DNA block copolymers, DNA-b-polypropylene oxide (PPO) was chosen as a model system, because PPO is biocompatible and has a Tg < 0 °C. Both properties might be essential for future applications in living systems. During my PhD study, I focused on the properties and the structures of DNA-b-PPO molecules. First, DNA-b-PPO micelles were studied by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). In order to control the size of micelles without re-synthesis, micelles were incubated with template-independent DNA polymerase TdT and deoxynucleotide triphosphates in reaction buffer solution. By carrying out ex-situ experiments, the growth of micelles was visualized by imaging in liquid with AFM. Complementary measurements with FCS and polyacrylamide gel electrophoresis (PAGE) confirmed the increase in size. Furthermore, the growing process was studied with AFM in-situ at 37 °C. Hereby the growth of individual micelles could be observed. In contrast to ex-situ reactions, the growth of micelles adsorbed on mica surface for in-situ experiments terminated about one hour after the reaction was initiated. Two reasons were identified for the termination: (i) block of catalytic sites by interaction with the substrate and (ii) reduced exchange of molecules between micelles and the liquid environment. In addition, a geometrical model for AFM imaging was developed which allowed deriving the average number of mononucleotides added to DNA-b-PPO molecules in dependence on the enzymatic reaction time (chapter 3). Second, a prototype of a macroscopic DNA machine made of DNA-b-PPO was investigated. As DNA-b-PPO molecules were amphiphilic, they could form a monolayer at the air-water interface. Using a Langmuir film balance, the energy released owing to DNA hybridization was converted into macroscopic movements of the barriers in the Langmuir trough. A specially adapted Langmuir trough was build to exchange the subphase without changing the water level significantly. Upon exchanging the subphase with complementary DNA containing buffer solution, an increase of lateral pressure was observed which could be attributed to hybridization of single stranded DNA-b-PPO. The pressure versus area/molecule isotherms were recorded before and after hybridization. I also carried out a series of control experiments, in order to identify the best conditions of realizing a DNA machine with DNA-b-PPO. To relate the lateral pressure with molecular structures, Langmuir Blodgett (LB) films were transferred to highly ordered pyrolytic graphite (HOPG) and mica substrates at different pressures. These films were then investigated with AFM (chapter 4). At last, this thesis includes studies of DNA and DNA block copolymer assemblies with AFM, which were performed in cooperation with different group of the Sonderforschungsbereich 625 “From Single Molecules to Nanoscopically Structured Materials”. AFM was proven to be an important method to confirm the formation of multiblock copolymers and DNA networks (chapter 5).

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Rapid analysis of GSTM1, GSTT1 and GSTP1 polymorphisms using real-time polymerase chain reaction

Polymorphisms of glutathione transferases (GST) are important genetic determinants of susceptibility to environmental carcinogens (Rebbeck, 1997). The GSTs are a multigene family of dimeric enzymes involved in detoxification, and, in a few cases, the bioactivation of a variety of xenobiotics (Hayes et al., 1995). The cytosolic GST enzyme family consists of four major classes of enzymes, referred to as alpha, mu, pi and theta. Several members of this family (for example, GSTM1, GSTT1 and GSTP1) are polymorphic in human populations (Wormhoudt et al., 1999). Molecular epidemiology studies have examined the role of GST polymorphisms as susceptibility factors for environmentally and/or occupationally induced cancers (Wormhoudt et al., 1999). In particular, case-control studies showed a relationship between the GSTM1 null genotype and the development of cancer in association with smoking habits, which has been shown for cancers of the respiratory and gastrointestinal tracts as well as other cancer types (Miller et al., 1997). Only a few molecular epidemiological studies addressed the role of GSTT1 and GSTP1 polymorphisms in cancer susceptibility. Since GSTP1 is a key player in biotransformation/bioactivation of benzo(a)pyrene, GSTP1 may be even more important than GSTM1 in the prevention of tobacco-induced cancers (Harries et al., 1997; Harris et al., 1998). To date, this relationship has not been sufficiently addressed in humans. Comprehensive molecular epidemiological studies may add to the current knowledge of the role of GST polymorphisms in cancer susceptibility and extent of the knowledge gained from approaches that used phenotyping, such as GSTM1 activity as it relates to trans-stilbene oxide, or polymerase chain reaction (PCR) based genotyping of polymorphic isoenzymes (Bell et al., 1993; Pemble et al., 1994; Harries et al., 1997).

Perfil imunológico e microbiológico de pacientes com periodontites crônica e agressiva

O objetivo deste estudo foi determinar os níveis de interleucina-1β (IL-1 β), IL-2, IL-4, IL-8, interferon-γ (IFN-γ) e a atividade de elastase no fluido gengival (FG) de pacientes com periodontite crônica generalizada (PCG) e periodontite agressiva generalizada (PAgG), e correlacionar com indivíduos de um grupo controle com gengivite apenas. Um objetivo secundário foi analisar o perfil microbiológico subgengival destes indivíduos. Dados clínicos transversais foram obtidos de 20 pacientes com PCG, 17 pacientes com PAgG e 10 indivíduos com gengivite. Amostras de FG foram coletadas com tiras de papel e os níveis de: IL-1β, IL-2, IL-4, IL-8 e IFN-γ foram medidos, utilizando um imunoensaio do tipo multiplex (Luminex). Atividade da elastase foi avaliada por um ensaio enzimático. Amostras de placa subgengival foram analisadas através do checkerboard DNA-DNA hybridization. As diferenças de significância entre os grupos para dados imunológicos e microbiológicos foram realizadas utilizando o teste Kruskal-Wallis, ajustando para múltiplas comparações. As médias dos parâmetros clínicos e os volumes de FG foram maiores nos pacientes com PCG e PAgG comparados ao grupo gengivite. Níveis mais elevados de IL-1β e atividade de elastase foram encontrados em sítios profundos quando comparado a sítios rasos em ambos os grupos com periodontite (p <0,05). Os dados microbiológicos apresentaram níveis significativamente mais elevados das espécies do complexo vermelho em pacientes com PCG e PAgG, quando comparados aos indivíduos com gengivite (p <0,05). Não houve diferença estatisticamente significante nos níveis de biomarcadores no FG e nos níveis de espécies bacterianas subgengivais entre pacientes com PCG e pacientes com PAgG. Sendo assim, concluímos que os dados do presente estudo não mostraram diferença estatisticamente significante nos parâmetros imunológicos e microbiológicos medidos entre indivíduos com PCG e PAgG.

Efeito do tratamento periodontal não cirúrgico em pacientes com periodontite crônica e agressiva: achados microbiológicos e imunológicos

O objetivo do presente estudo foi comparar a expressão de IL-1β, IL-4, IL-8, interferon-γ, atividade de elastase e a composição do perfil microbiano subgengival antes e depois do tratamento periodontal não cirúrgico em pacientes com doença peridontal crônica generalizada (PC) e agressiva generalizada (PA). Vinte pacientes com PC e quatorze com PA foram avaliados. Dados clínicos, fluido gengival e biofilme subgengival foram analisados na visita inicial (VI) e 3 meses (3M) após o tratamento periodontal não cirúrgico. Amostras de fluido gengival (FG) foram coletadas com tiras de papel e os níveis de: IL-1β, IL-4, IL-8 e INF-γ foram medidos, utilizando um tipo de imunoensaio multiplexado (Luminex). Atividade da elastase foi avaliada por um ensaio enzimático. Amostras de placa subgengival foram analisadas através do checkerboard DNA-DNA hybridization. Na avaliação de 3 meses após terapia periodontal foi encontrado melhora significativa para todos os parâmetros clínicos em ambos os grupos. Foram encontradas reduções significativas na atividade de elastase nos sítios rasos e profundos dos pacientes do grupo PA e nos sítios profundos do grupo PC, também foi achado um aumento significativo de INF-γ nos sítios rasos do grupo PA. Os dados microbiológicos, mostraram reduções significativas para os níveis dos membros do complexo vermelho (P. gingivalis, T. forsythia, T.denticola), e para as espécies E.nodatum e P.micra no grupo PC. No grupo PA, ocorreram reduções significativas no níveis de P. gingivalis, T. forsythia, Fusobacterium nucleatum ss polymorphum e Fusobacterium periodonticum. Quando as respostas clínica e imunológica 3M após terapia foram comparadas entre os grupos, apenas diferenças sutis foram observadas. Nenhuma diferença microbiológica foi encontrada entre os grupos após a terapia. Em conclusão, os achados suportam nossa hipótese de que as periodontites cronica e agressiva respondem de forma semelhante ao tratamento periodontal nao cirúrgico.

Studies on 4S and 5S RNA of HeLa cells

SECTION I

Section I is concerned with a partial sequence analysis conducted on 5S RNA from HeLa cells. Analysis of the oligonucleotide pattern after pancreatic ribonuclease digestion of a highly-purified preparation of 5S RNA gave results which were in general agreement with those published for KB cells, both with respect to the identity and the frequency of the partial sequences. However, the presence of a trinucleotide not found in the KB 5S pattern, together with the reproducibly much lower than expected molar yield of the larger oligonucleotides strongly suggested the occurrence of alternate sequences at various sites in the 5S molecules of human cells. The presence of ppGp and pppGp at the 5'-terminus of HeLa 5S RNA was clearly demonstrated. The implications of this finding with regard to the origin of 5S RNA are discussed.

SECTION II

In Section II the proportion of the HeLa cell genome complementary to tRNA was investigated by using RNA- DNA hybridization. The value for saturation of the HeLa DNA by tRNA was found to be 1.1 x 10^-5, which corresponds to about 4900 sites for tRNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized tRNA revealed significant differences from the nucleotide composition of the input tRNA, with the purine to pyrimidine ratio indicating, however, that these differences were not produced by excessive RNase attack of the hybrid. The size of the hybridized tRNA was only moderately smaller than that of the input RNA; the average S value in formaldehyde was 2.7 (corresponding to a length of about 65 nucleotides), suggesting that a relatively small portion near the ends of the hybridized 4S chains had been removed by RNase.

SECTION III

The proportion of the HeLa cell genome complementary to 5S RNA was investigated by using RNA-DNA hybridization. The value for saturation of the HeLa DNA by 5S RNA was found to be 2.3 x 10^-5, which corresponds to about 7,000 sites for 5S RNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized 5S RNA revealed no significant difference from the nucleotide composition of the input RNA. At the RNA to DNA input ratio of 1:1000, the average S value in formaldehyde of the hybridized 5S RNA corresponded to a polynucleotide chain about two-thirds the size of the input RNA.

Biologically sensitive field-effect devices using polysilicon TFTs

The authors present a review of recent developments in the detection of biomolecular interactions with field-effect devices. Ion-sensitive field-effect transistors (ISFETs) and enzyme field-effect transistors (EnFETs), based on polycrystalline silicon (poly-Si) TFTs, are discussed. Label-free electrical detection of DNA hybridization has been achieved by a new method, by using MOS capacitors or poly-Si TFTs. In principle, the method can be extended to other chemical or biochemical systems, such as proteins and cells.

Perfil microbiano de infecções endodônticas primárias e sua relação com o tamanho de lesões periradiculares associadas

O presente trabalho tem por objetivo investigar a microbiota de canais radiculares que apresentem lesão perirradicular e relacionar o perfil microbiano detectado com a área/volume destas lesões visualizadas por radiografias periapicais e tomografias computadorizadas tipo cone-beam. Foram selecionados 19 dentes com infecção endodôntica primária. As amostras microbiológicas foram coletadas dos canais com o auxílio de limas tipo Hedströen e cones de papel absorvente estéril. A técnica do Checkerboard DNA-DNA hybridization foi utilizada para detecção de até 79 espécies bacterianas em cada amostra, utilizando sondas de DNA específicas. Os dados microbiológicos foram expressos em percentagem média (prevalência), proporção e nível médio de cada espécie em cada amostra. Os testes t independente e de correlação de Pearson foram usados para correlacionar a contagem das bactérias testadas com os dados clínicos (p≤ 0,05). Foi encontrada uma média de 17 espécies por amostra. E. brachy (70%), S. pneumonia (67,5%), P. oris (67,5%), E. faecium (65%), N. gonorrhoeae (62,5%), K. pneumoniae (62,5%), P. melaninogenica (62,5%), P. nigrescens (62,5%) e P. micra (62,5%) foram as espécies mais prevalentes, e as espécies encontradas em níveis médios mais altos foram P. oris (7,5 x 105), E. brachy (7,3 x 105), E. faecium (7,2 x 105), K. pneumoniae (7,0 x 105), N. gonorrhoeae (6,8 x 105), S. epidermidis (6,5 x 105) e H. pylori (6,5 x 105). Houve correlação positiva entre as lesões periapicais de maior área e contagens significativamente mais altas da carga bacteriana total e de bactérias Gram-negativas (p<0,05). Baseado nos resultados obtidos é possível concluir que a microbiota presente em dentes com periodontite apical primária possui perfil misto e complexo, e que uma maior tamanho de lesão perirradicular pode estar associada a contagem elevada espécie totais e bactérias Gram-negativas.

Remodelação alveolar e perfil bacteriano em implantes instalados em diferentes níveis relacionados à crista óssea

A substituição clínica de dentes naturais perdidos por implantes osteointegrados tem representado uma das primeiras opções terapêuticas para a reabilitação de pacientes total ou parcialmente edêntulos. Apesar dos excelentes índices de sucesso demonstrados pelas restaurações implanto-suportadas, alguns fatores permanecem não esclarecidos, principalmente no que diz respeito à remodelação óssea ao redor dos implantes osteointegrados. Desta forma, o objetivo deste estudo foi avaliar a relação do nível de instalação dos implantes dentários com os parâmetros clínicos, com a remodelação óssea peri-implantar e com a colonização bacteriana, em implantes de plataforma regular, submetidos à carga imediata. Implantes de plataforma regular foram instalados em dois diferentes níveis em relação à crista óssea ao nível ósseo e supra ósseo (1 mm). No total, trinta e cinco implantes em 9 pacientes (idade média de 62,4 11,2 anos) foram avaliados radiograficamente no momento da instalação dos implantes (T1) e 6 meses após (T2), momento no qual também foram feitas análises clínicas e coleta de amostras para o teste microbiológico. Nos exames radiográficos foram analisadas a perda óssea, a partir de mensurações lineares da distância entre um ponto fixo do componente protético e o ponto mais coronário do contato osso-implante, e a densidade óptica alveolar obtida a partir de regiões ósseas de interesse (ROIs). As análises clínicas consistiram na avaliação da profundidade de sondagem e na mensuração do volume do fluido gengival peri-implantar. O perfil bacteriano dos sítios avaliados foi caracterizado por meio do método de análise de checkerboard DNA-DNA hybridization. Os testes estatísticos realizados mostraram não haver relação entre o nível de instalação dos implantes em relação à crista óssea e a remodelação óssea alveolar, tanto com relação à perda óssea (p = 0,725), como com relação à densidade óptica alveolar (p = 0,975). Também não foi possível estabelecer uma correlação entre a remodelação óssea e parâmetros clínicos como profundidade de sondagem e volume do fluido gengival peri-implantar. Com relação ao perfil bacteriano, não foram encontradas diferenças estatisticamente significativas entre os grupos avaliados para nenhuma das 40 bactérias analisadas.

Análise da diversidade microbiana em infecções endodônticas persistentes

O presente trabalho teve por objetivo investigar a microbiota de canais radiculares relacionadas ao insucesso do tratamento endodôntico, buscando a identificação e a quantificação destes micro-organismos. Foram selecionados 36 dentes com infecção endodôntica persistente. O material obturador foi removido do canal radicular e amostras microbiológicas foram coletadas dos canais com o auxílio de limas tipo Hedströen e cones de papel absorvente estéril. A técnica do Checkerboard DNA-DNA hybridization foi utilizada para detecção de até 79 espécies bacterianas em cada amostra, utilizando sondas de DNA específicas. Os dados microbiológicos foram expressos em percentagem média (prevalência), proporção e nível médio de cada espécie em cada amostra. Os testes t independente e de correlação de Pearson foram usados para correlacionar a contagem das bactérias testadas com os dados clínicos (p≤ 0,05). Foi encontrada uma média de 11 espécies por amostra. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) e S. warneri (28%) foram as espécies mais prevalentes, e as espécies encontradas em níveis médios mais altos foram E. faecium, D. pneumosintes, S. epidermidis, H. pylori e C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%), S. ixodetis (3%) apresentaram prevalências mais baixas. E. faecium e S. epidermidis apresentaram os maiores valores de prevalência, níveis médios e proporção. Não houve correlação entre a microbiota detectada nas amostras com os sinais e sintomas clínicos apresentados pelos pacientes, porém nas lesões periapicais de maior área foi detectada contagem significativamente maior de bacilos e espécies Gram-negativas (p<0,05). Baseado nos resultados obtidos é possível concluir que a microbiota presente em dentes com periodontite apical persistente possui perfil misto e complexo, e que uma maior área de lesão perirradicular pode estar associada a contagem elevada de bacilos e de espécies Gram-negativas.

Pseudomonas duriflava sp nov., isolated from a desert soil

A Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain HR2(T) was isolated from a soil sample from the Talklimaken Desert in Xinjiang Province, China. Strain HR2(T) grew optimally at pH 7.0-8.0 and 30-37 degrees C in the presence of 0-1% (w/v) NaCl. An analysis of 16S rRNA gene sequences revealed that strain HR2(T) fell within the radiation of the genus Pseudomonas, the highest level of similarity being found with respect to Pseudomonas luteola IAM 13000(T) (97.5%); the levels of sequence similarity with respect to other recognized Pseudomonas species were < 96.4%. DNA-DNA hybridization showed that the genetic relatedness between strain HR2(T) and P. luteola IAM 13000(T) was 53.2%. The G + C content of the genomic DNA of strain HR2(T) was 55.2 mol%. The major fatty acids were 18: 1, summed feature 3 and 16:0. The hydroxylated fatty acids 10:0 3-OH, 12:0 3-OH and 12:0 2-OH were also present. The data obtained in this polyphasic study indicated that this isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas duriflava sp. nov. is proposed, The type strain is HR2(T) (=KCTC 221129(T) =CGMCC 1.6858(T)).

Meiothermus rosaceus sp nov isolated from Tengchong hot spring in Yunnan, China

A rosy-pigmented Gram-negative, thermophilic bacterium with an optimum growth temperature of about 55degreesC was isolated from Tengchong hot springs in Yunnan province, China. Its growth scarcely occurred below 40degreesC or above 70degreesC. Phylogenetic and secondary structural analyses of 16S rRNA and DNA-DNA hybridization showed that the organism represented a new species of the genus Meiothermus. This new species could be distinguished easily from other species of the genus Meiothermus by the following phenotypic characteristics: rosy pigment, expanded body, sucrose and maltose were not utilized, gelatin and starch were not hydrolyzed. On the basis of the above data, the name Meiothermus rosaceus sp. nov. was proposed for the species represented by the strain RH9901(T)(CCTCC-AB200291). (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-head method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 mug/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis.

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 165 rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204(T)), Planktothrix rubescens (type strain CCAP 1459/22(T)) Planktothrix pseudagardhii sp. nov. (type strain T1-8-4(T)), Planktothrix mougeotii (type strain TR1-5(T)), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207(T)), Tychonema bourrellyi (type strain CCAP 1459/11B(T)) and Limnothrix redekei (type strain NIVA CYA 277/1(T)).