Past studies characterizing the feline intestinal microflora have used traditional bacterial culture techniques. However, in recent years it has been recognized that the majority of intestinal bacteria are non cultivable. Therefore, the aim of this study was to describe the microflora along the intestinal tract in healthy cats using comparative 16S ribosomal DNA (16S rDNA) analysis. Intestinal content from the stomach, duodenum, jejunum, ileum, and colon was collected from 4 healthy cats and one specific pathogen free cat (SPF) and the bacterial composition was identified by direct sequencing of bacterial 16S rDNA amplicons. A predominant anaerobic microflora was observed in all evaluated segments of the intestine. Fourteen different bacterial orders were identified with the majority of all sequences classified in the class Clostridiales. Six different Clostridium clusters were identified with the majority of sequences affiliated with Clostridium cluster I. Comparative 16S rDNA analysis was also used to evaluate differences in the fecal microflora between healthy cats (n=6), cats with histopathologically confirmed inflammatory bowel disease (IBD; n=6), and cats with intestinal neoplasia (n=3). Compared to the IBD group, cats in the control group showed a significantly higher number of sequences classified as Firmicutes, Bacteroidetes, and Actinobacteria (p<0.0001). The control group had a significantly higher proportion of clones affiliated with Clostridium cluster XI, and a significantly lower proportion affiliated with cluster I (both p<0.0001). In the neoplasia group, the majority of sequences were classified in the phylum Firmicutes (97.9%) and clones were predominately affiliated with Clostridium clusters I and XI. These data indicate that the feline intestinal microflora is highly diverse and is comprised predominantly of anaerobic bacteria. Further studies are warranted to evaluate the clinical significance of the observed differences in intestinal microflora between healthy cats and cats with gastrointestinal disease.

Abstract This research used molecular biological techniques such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to analyze the dynamic microbial community and biodiversity in the groundwater contaminated with petroleum-hydrocarbons. The 16S rDNA sequences from all water samples were compared with the sequences of relative bacteria in the Ribosomal Database Project Bank to construct a phylogenetic tree. The results allowed us to understand the composition of the microbial communities in the petroleum-hydrocarbon contaminated groundwater. In this study, groundwater samples taken from the Chinese Petroleum Corporation Kaohsiung Refinery (CPCKR), Chinese Petroleum Corporation at Ciaotou fuel Tank Farm (CPCCTF) and China Petrochemical Development Corporation at Kaohsiung Factory (CPDCKF) were analyzed. The contaminated sites at CPDCKR and CPCCTF are remeated by natural attenuation. While the CPDCKF site is remeated by an enhanced air sparging bioremediation. In CPDCKR, we found that the low polluted area contained the richest microbial community, followed by the non-polluted area, and the high polluted area. At the CPCCTF site, the microbial community in the non-polluted area was richer than the high-polluted area. Increased microbial populations and variation in microbial community have beenobserved in non-polluted, less polluted, and highly polluted areas. The microbial community showed a dynamic succession of complexity during the bioremediation process at the CPDCKF site. From the 16S rDNA sequence analysis, it is possible that all samples contained petroleum-hydrocarbon degrading bacteria. These petroleum-hydrocarbon degrading bacteria include Methylobacterium, Xanthobacter, Xanthomonas and Pseudomonas at CPCKR site, Flavobacterium at CPCCTF site, Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, and Candida at CPDCKF site. This study also demonstrates that it is more economic and reliable of using molecular techuiques to analyze the groundwater. Thus, groundwater samples can be used to replace soil samples for future work.

Fifty two cellulolytic thermophilic microorganisms were analyzed for their physiological characterization and phylogenetic systematics. Based on 16S rDNA sequence analysis, 3 strains from DCB and 4 novel isolates from southern Taiwan are close related to the genera of Bacillus and Geobacillus respectively. Among 4 new Geobacillus strains, strain T4, a Gram negative, motile, aerobically growing sporulating rod, can secrete thermostable endoglucanase. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70¢XC. Based on 16S rDNA sequence analysis, DNA G+C content, phenotypic and physiological characteristics, as well as DNA-DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). Furthermore, a phylogenetic tree of 20 related microorganisms was also constructed based on their thermostable cellulase amino acid sequences. Our sequence analysis shows that cellulases belonging to the large family of glycoside hydrolases (GHs) can be divided into four subfamilies: TC-1 (GH family 12 group), TC-2 (bacterial group £L in which fungal species Thermoascus aurantiacus fits), TC-3 (bacterial group £L£L), and TC-4 (GH family 1 group). Together with the 16S rDNA sequence analysis of strain T4 and 10 related microorganisms, strain T4 has a close phylogenetic relationship with subfamily TC-4 but far from subfamily TC-1.

In this study, we had established a 16S rDNA-DGGE analys is system to detect the microbial community in petroleum polluted soil and assess the feasibility of using this system to monitor the bioremediation process. Three genomic DNA extracted methods, the KIT, the Bead-beating system, and the Freeze-thaw method were used to evaluate the DNA extraction efficiency and purity. These DNA samples were further tested by DGGE to analysis the microbial community in soil samples. The results showed that KIT method performed advantageous not only in the DNA extraction efficiency and purity, but also expressed the richest bacterial community in its PCR products. From the DGGE analysis, our data indicated that composition of bacterial community were different in the soil samples that were taken from the same site but at different time. This indicated that the species and number of microorganisms in a polluted soil were under a dynamic transition. The combination of DGGE and 16S rDNA gene sequence analysis system were also proven useful in identifying the predominant microbes in a soil sample and monitoring its bacterial community.

Abstract The genetic diversity and identification of slow- and fast- growing soybean root nodule bacterial isolates from different agro-climatic regions in Mpumalanga, Limpopo and Gauteng Provinces of South Africa were evaluated. The 16S-rDNA-RFLP analysis of 100 rhizobial isolates and eight reference type strains placed the isolates into six major clusters, and revealed their site-dependent genomic diversity. Sequence analysis of single and concatenated housekeeping genes (atpD, glnII and gyrB), as well as the symbiotic gene nifH captured a considerably higher level of genetic diversity and indicated the dominance of Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum in Mpumalanga, Limpopo and Gauteng Provinces. Gene sequence similarities of isolates with type strains of Bradyrhizobium ranged from 97.3 to 100% for the 16S rDNA, and 83.4 to 100% for the housekeeping genes. The glnII gene phylogeny showed discordance with the other genes, suggesting lateral gene transfer or recombination events. Concatenated gene sequence analysis showed that most of the isolates did not align with known type strains and might represent new species from South Africa. This underscores the high genetic variability associated with soybean Bradyrhizobium in South African soils, and the presence of an important reservoir of novel soybean-nodulating bradyrhizobia in the country. In this study, the grouping of isolates was influenced by site origin, with Group I isolates originating from Limpopo Province and Group II and III from Mpumlanga Province in the 16S rDNA-RFLP analysis.

Abstract Acetes is a kind of macroholozooplankton. We can use the "genetic analysis to know its evolution, zooplankton population structure and taxa. The step is :ampified the 16S rDNA of the mitochondrial DNA and the COI fragment DNA in Acetes, and then sequence. We get the genetic distance between species of the 16S rDNA is 2.65~13.16%, and it's 1.43~25.42% of the COI fragment gene in Acetes. These results show that the diversity of COI fragment DNA in Acetes is more than the 16S rDNA of the mitochondria DNA. If we translated the COI gene DNA to amino acid sequence, the genetic distance between species of the amino acid sequence is about 1.83%~8.97%. By the way, we know that the genetic distance between species of the amino acid sequences are clearly less then the DNA sequences. Using the genetic data, we constructure a phylogenetic tree. The tree divided the Acetes into two group : Japonicus group and Erythraeus group, we can find the Acetes japonicus is the most permitive species and the A. erythraeus is the last specation species in the Acetes under the DNA sequences. But under the tree which was made from the amino acid sequence, we found the Acetes erythraeus is the most permitive species in the Acetes. The genetic distance between A. intermedius and A. erythreaus were 5.44% and 18.01% of the 16S rDNA and COI gene which indicated that the A. intermedius is true species. The population of A. japonicus between Japon Sea and Taiwan showed 0.24% and 2.36% of the 16S rDNA and COI gene that reveled two population should have gene-flow. The populations diversity of the A. intermedius in Taiwan showed the same way which had no different and the populations in Taiwan should be a single population.

Abstract Recent researches have pointed out that most of marine bacteria are uncultivable. However, majority of prior researches about bacteria flora in cultivated ponds used cultivating method to researches. That means those researches ignored uncultivable bacteria in ponds and caused inaccuracy in counting bacteria. Therefore we used analyzing bacteria 16S rDNA sequences to study composition of bacteria in cultivated ponds in place of traditional bacteria taxonomy. The phylogenetic diversity of bacteria examined by analyzing the 16S rDNA sequences permits the characterization of environmental bacteria community without culturing and has been used widely. This research is to adopt both MMA medium cultivation and direct recovery of bacteria 16S rDNA sequences to investigate bacteria flora in a closed Penaeus monodon pond. We sampled from A2 (10¡Ñ8¡Ñ1.5m) test pond at the Department of Marine Resource in Sun Yat-Sen University on August 17, 1999. Then, we adopted AO (Acridine Orange) epifluorescent microscopic technique to count total direct count (TDC) and direct count of viable bacteria cell (DVC). Respective results were 2.846¡Ñ107/ml, 1.029¡Ñ107/ml, and plate count (PC) determined by MPN count method were 1.130¡Ñ105cfu/ml. In the part of cultivable bacteria, they could be separated into 9 groups by their morphologic after culturing in MMA plate at 25¢Jfor 5 days. We isolated 15 strains to analyze their 16S rDNA sequences, and separated respectively into 4 groups after comparing with the genebank. Those four groups are CFB group, low G+C Gram-positive bacteria, Alpha proteobacteria and Gamma proteobacteria. The genus Vibrio (47%) in the Gamma proteobacteria group is the dominant. In the part of uncultivable bacteria, we filtered bacteria from the water in the same pond, amplified the 16S rDNA by the polymerase chain reaction (PCR) and then cloned. After that, we randomly isolated 40 clones for sequence analysis. The bacteria belong to following groups, cyanobacteria, CFB group, Verrucomicrobia, Gram-positive eubacteria, Alpha proteobacterium, Beta proteobacterium, Gamma proteobacterium and Delta proteobacterium.

16S rDNA sequences from 23 heterocytous cyanobacteria were obtained and phylogenetic tree from these sequences and sequences available in the GenBank was constructed. Relationships between traditional taxonomy and phylogenetic clusters were discussed.

Made available in DSpace on 2014-06-12T23:13:15Z (GMT). No. of bitstreams: 2 arquivo3053_1.pdf: 2529352 bytes, checksum: ea122352da45d79a2b72d5634d4999af (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco / O zoantídeo Palythoa caribaeorum é um cnidário colonial cujo os pólipos são conectados por um espesso tecido que possui partículas minerais incorporadas e habita extensas áreas dos recifes costeiros no Brasil. Este zoantídeo é popularmente conhecido por "baba-de-boi", por secretar um muco muito viscoso. O muco produzido por corais, animais que são filogeneticamente próximos ao zoantídeos, consiste de um conjunto complexo de Eukarya Archaea e Bacteria. O objetivo deste estudo foi realizar uma caracterização taxonômica da microbiota presente no muco de colônias saudáveis e branqueadas de P. caribaeorum. O muco foi coletado nos recifes costeiros de Porto de Galinhas e de Suape, litoral sul de Pernambuco, em 2010. O isolamento das bactérias foi realizada utilizando o meio Ágar Marinho. O PCR dos segmentos 16S rDNA foi realizado utilizando os primers universais 27F e 1492R. O Sequênciamento dos fragmentos foi realizado no sequênciador ABI 3100 e a qualidade das sequências foram verificadas usando o programa Sequencing analysis 3.4 e, então, comparadas com as sequencias depositadas no GenBank usando o algoritmo Blastn. Um total de 50 amostras de bactérias isoladas de colônias saudáveis e branqueadas foram analisadas. O grupo dominante foi -Proteobacteria com 36 isolados (72%), seguido por - Proteobacteria e Actinobacteria com seis isolados (12%) cada, e Firmicutes com dois isolados (4%). O gênero Vibrio foi o mais comum (50%), corroborando trabalhos anteriores em que este gênero foi o mais comum associado aos cnidários. Sequências de alguns isolados foram relacionados ao nível de espécie (97%) aos já depositados no GenBank, entre elas, espécies com potencial biotecnológico interessante, como bactérias do gênero Alcanivorax, que usa hidrocarbonetos derivados do petróleo como fonte de carbono e energia; e bactérias dos gêneros Vibrio, Photobacterium, Pseudomonas, Micrococcus e Pseudoalteromonas, grupos conhecidos por produzir compostos antimicrobianos e potentes toxinas marinhas. Algumas das sequências dos isolados foram relacionados com patógenos de seres humanos como V. alginolyticus e V. proteolyticus, e outras relacionadas a espécies do gênero Pseudoalteromonas, que podem ter participação no fenômeno do branqueamento. Outros isolados podem representar novas espécies uma vez que suas seqüências mostrou uma baixa similaridade com os taxa já conhecidos e muitos deles foram registrados pela primeira vez no muco de P. caribaeorum. É importante intensificar os estudos de diversidade microbiana neste zoantídeo para entender melhor o papel dessas bactérias nas propriedades farmacológicas do muco

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-29T12:07:11Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Livia Araujo.pdf: 1983609 bytes, checksum: cbba526737c10f6743b3fd015372721a (MD5) / Made available in DSpace on 2016-06-29T12:07:11Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Livia Araujo.pdf: 1983609 bytes, checksum: cbba526737c10f6743b3fd015372721a (MD5) Previous issue date: 2014-02-20 / CAPEs / Zymomonas mobilis vem despertando um grande interesse no meio científico, industrial e biotecnológico devido ao seu alto potencial fermentativo. Do ponto de vista taxonômico, Z. mobilis é a única espécie do gênero Zymomonas, e é subdivida em três subespécies: Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae e Z. mobilis subsp. francensis. A diferenciação destas subespécies é baseada em testes fisiológicos. Estes testes consomem tempo e são frequentemente duvidosos. Por isso, técnicas moleculares são propostas como uma alternativa rápida e confiável para diferenciação destas bactérias. O presente estudo teve como objetivo realizar a caracterização molecular das 32 linhagens de Zymomonas mobilis depositadas na Coleção de Microrganismos UFPEDA, através da análise das sequências do gene 16S rDNA e ARDRA. As linhagens foram cultivadas em meio SSDL por 24 horas à 30º, seguida de centrifugação e extração de DNA cromossômico. As reações de PCR foram realizadas com iniciadores e condições específicas para a amplificação do gene 16S rDNA. Os produtos do gene 16S rDNA amplificados foram purificados, sequenciados e clivados com as enzimas de restrição Hae III, NdeII e StuI. Os dados obtidos pelo sequenciamento do gene 16S rDNA foram analisados, comparados e alinhados, pelo programas BLASTn e MultiAlin, com sequências de linhagens de Z. mobilis previamente depositadas no banco de dados GenBank. Um dendograma foi construido através do programa ClustalW pelo método de neighbor-joining Os perfis de restrição teórico das enzimas de restrição Hae III, NdeII e StuI foram gerados a partir do WebCutter 2.0. Dendogramas foram construídos a partir da matriz de similaridade genética de Jaccard, calculada pela análise dos perfis de restrição teóricos de cada enzima. A análise das sequências obtidas no presente estudo revelou o elevado grau de conservação no gene 16SrDNA, confirmando a relação de proximidade das linhagens de Zymomonas mobilis depositadas na Coleção de Micro-organismos UFPEDA e a aproximidade com a Z. mobilis subsp. mobilis LMG445, sugerindo que as linhagens desta coleção pertencem a esta subespécie.Além disso, conclui-se que a análise do perfil restrição teórico do gene 16S rDNA possibilita a diferenciação de Z. mobilis,a nível de subespécie, mas não é eficaz para analisar a variabilidade genética entre as linhagens de Z. mobilis UFPEDA. Baseados nestes resultados, outros marcadores filogenéticos devem ser empregados para analisar a variabilidade genética destas linhagens, possibilitando um melhor conhecimento da diversidade desta bactéria. / Zymomonas mobilis has attracted great interest in the scientific, industrial and biotechnological medium due to its high fermentation potential. The taxonomic viewpoint, Z. mobilis is the only species of the genus Zymomonas , and is subdivided into three subspecies : Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae and Z. mobilis subsp. francensis. The differentiation of these subspecies is based on physiological tests. These tests are time consuming and often unreliable. Therefore, molecular techniques are proposed as a fast and reliable alternative to differentiation of these bacteria. This study aims to perform molecular characterization of 32 strains of Zymomonas mobilis deposited in the Collection of Microorganisms UFPEDA by sequence analysis of 16S rDNA gene and the theoretical restriction profile of this gene. The strains were grown in SSDL for 24 hours at 30 ° , followed by centrifugation and extraction of chromosomal DNA . PCR reactions were performed with primers and specific conditions for amplification of the 16S rDNA gene. The amplified products of 16S rDNA were purified, sequenced, and cleaved with restriction enzymes Hae III, NdeII and StuI . The data obtained by 16S rDNA gene were analyzed, compared and aligned by BLASTn and MultiAlin programs with sequences of strains of Z. mobilis previously deposited in the GenBank databas . A dendogram was constructed using the program ClustalW method by neighbor-joining. Profiles theoretical restriction of restriction enzyme Hae III, NdeII and StuI were generated from WebCutter 2.0. Dendrograms were constructed from the genetic Jaccard similarity matrix, calculated by analyzing the theoretical restriction profiles of each enzyme. The analysis of the sequences obtained in this study revealed the high degree of conservation in 16SrDNA gene, confirming the close relationship of strains of Zymomonas mobilis deposited in the Collection of Micro-organisms UFPEDA and closeness with Z. mobilis subsp. mobilis LMG445, suggesting that the strains in this collection belong to this subespécie. In addition, it is concluded that the theoretical restriction profile analysis of the 16S rDNA gene allows differentiation of Z. mobilis , the level of subspecies , but it is not effective to analyze the genetic variability between strains of Z. mobilis UFPEDA . Based on these results , other phylogenetic markers should be employed to analyze the genetic variability of these strains , allowing a better understanding of the diversity of this bacteria.