This dissertation examines the microbial and functional diversity in Kartchner Caverns, a limestone cave in Arizona, USA. Kartchner is highly oligotrophic due to the lack of photosynthesis and the limited inputs of organic material from the surface. This characteristic poses a challenge for microbial life in the cave. The first objective of this work was to evaluate the bacterial richness, diversity and taxonomic composition of speleothems surfaces within Kartchner Caverns in order to gain insight into the distribution patterns associated with these communities. Secondly, the metabolic strategies used by cave communities to survive harsh cave conditions were investigated based on phylogenetic associations and metagenomics. Both objectives were directed toward answering the questions "who are there?" and "what are they doing?". The 454-pyrotag analysis of the V6 region of the 16S rRNA gene revealed an unexpectedly high bacterial diversity with each speleothem supporting a unique bacterial community profile. A focused study on one room of the cave revealed three community types: Type 1 was dominated by the phylum Proteobacteria; Type 2 by Actinobacteria; and Type 3 by Acidobacteria. Phylogenetic associations of the sequences generated by the 454 sequencing and by a Sanger clone library suggested cave microbial communities are supported by chemoautotrophic activities such as nitrite and iron oxidation. Results from the phylogenetic associations guided the metagenomic analysis which supports the presence of chemoautotrophic activities in the cave. Genes for two complete CO2 fixation mechanisms, the Calvin-Benson-Bashan and the rTCA cycles were identified in the cave metagenome, as well as genes for ammonia and nitrite oxidation. These genes are associated with both Bacteria and Archaea suggesting members of both domains are acting as primary producers in the cave ecosystem. Comparative analysis of cave samples to other environments suggests an overabundance of DNA repair mechanisms which could be potentially used by cave communities to overcome the toxicity due to high concentrations of calcium on the speleothem surfaces. This work provides the first comprehensive analysis of the microbial diversity and potential strategies used by microbial communities to survive under the extreme conditions found in a semi-arid limestone cave environment.

A Molecular Approach to Assessing Meiofauna Diversity in Marine Sediments Heather C. Hamilton Abstract The purpose of this study was to determine if a molecular approach could be applied to calculating the diversity of meiofauna in marine sediments from two sites in Tampa Bay, FL, similar to the approach of McCaig et al, 1999 in calculating the diversity of microbes in pastureland soils. The approach includes extracting total DNA directly from the sediment and amplifying the 18S rRNA gene by PCR. Clone libraries from the 18S gene would be created for each site and 300 sequences from each clone library would be obtained. These sequences would then be phylogenetically analyzed and assigned to an OTU, from which diversity indices can be calculated. The phylogenetic analysis of the sequences from the two sites revealed that of the 102 OTUs assigned from the sequences, only 7 OTUs included sequences from both sites, while 93 OTUs contained sequences from one site or from the other. Thus the sites were phylogenetically different from each other. Shannon diversity indices calculated for each site showed a difference between the two sites and paralleled diversity indices for macrofauna data for each site collected by the Hillsborough County Environmental Protection Commission. Sequences from 30 OTUs were completely sequenced and identified by phylogenetic comparison with a metazoan reference alignment. A discrepancy between the sequence data and data collected from preserved samples taken at each site was evident upon analysis: roughly 60% of each preserved sample consisted of nematodes and 10% consisted of copepods, while roughly 30% of the identified OTUs consisted of copepods and 10% consisted of nematodes. This discrepancy could be explained if the OTUs that were not identified consisted of nematode sequences or if a primer bias were present in the PCR amplification such that the regions flanking the primer site in the nematode sequences inhibited primer annealing.

16S rRNA gene analysis has shown that bacterial diversity in the GAB bores studied was limited to the genera Hydrogenobacter in the phylum Aquificae, Thermus in the phylum Deinococcus-Thermus, Desulfotomaculum in the phylum Firmicutes, the alpha-, beta- and gamma-classes of the phylum Proteobacteria and the phylum Nitrospirae. There was no clone closely related to members of the delta-proteobacteria and epsilon-proteobacteria classes detected. The number of bacterial strains directly isolated from the Fairlea and the Cooinda bores were far less than the numbers of distinctive phylotypes detected by the 16S rRNA gene characterisation. In addition none of the bacterial strains directly isolated from the water samples were represented in the 16S rRNA gene clone libraries. Similar discrepancies between the bacterial populations obtained from the 16S rRNA gene analysis and those obtained from direct isolation have been reported in the literature (Dunbar et al., 1999; Kampfer et al., 1996; Suzuki et al., 1997; Ward et al., 1998; Ward et al., 1997). However, in general, the phyla with which the isolates were affiliated were the same as those phyla to which the clones belonged. The environmental changes introduced (by bringing the artesian water up to the surface and exposing it to four types of metal coupons made of carbon steels identified by codes ASTM-A53B, ASTM-A53, AS-1074 and AS-1396 and commonly used in bore casings) led to changes in the bacterial community structures. In general, the species which proliferated in the communities before and after the changes were different. The diversity of the bacterial species in the community decreased following the environmental changes. Clones dominating the clone libraries constructed from newly established bacterial communities also differed from the clones dominating the libraries constructed from the bacterial communities which had existed naturally in the bores. These trends toward change in the bacterial communities were observed at both the Fairlea and the Cooinda bore sites. All four metal types incubated in the Fairlea bore water lost between 3.4 and 4.7% of their original weight. In contrast none of the metals incubated in Cooinda bore water lost weight. Clone library A1 showed that the natural population of the Fairlea bore was dominated by clone A1-3, which represented a novel species related to the isolate boom-7m-04. But after metal incubation (and recording of the metal weight loss), the bacterial community was dominated by clone PKA34B, which has a 95% similarity in its 16S rRNA gene sequence with Desulfotomaculum putei. Desulfotomaculum species are known to cause metal corrosion due to their byproduct H2S. But the low level of phylogenetic relatedness found does not provide enough information to speculate on whether the species represented by clone PKA34B is a member of the genus Desulfotomaculum or not. However, the fact that clone PKA34B dominated the PKA clone library by 50% makes the species it represents a suspected candidate likely to be involved with the metal weight loss at the Fairlea bore. In contrast, clone library 4381 showed that the natural population of the Cooinda bore was dominated by clone 4381-15 representing a species distantly related to a hydrogen oxidiser Hydrogenophaga flava (95% similarity). The dominating clone of the new community formed after metal incubation was clone COO25, which has 99% similarity with Thermus species that have not been reported to be involved with metal corrosion to my knowledge. In this project detection, identification and comparative quantification by 16S rRNA gene-targeted PCR probing with probes 23B and 34B were successfully developed for a Leptothrix-like species and for a Desulfotomaculum-like species represented by clones PKA23B and PKA34B respectively. This method of probing permits a fast, sensitive and reproducible detection, identification and at least a comparative quantification of the bacteria in the environment without the need for culturing. Therefore it is extremely suitable for use in bacterial population monitoring. PCR probing with the 34B probe has a potential commercial use as a means of screening for bores with a potential high risk of corrosion due to this Desulfotomaculum-like species. Direct isolation of bacteria from the GAB water has resulted in the isolation of seven strains from the Fairlea bore and eight from the Cooinda bore. Among these isolates, three novel strains were studied in detail. Reports on the characterisation of strain FaiI4T (T=Type strain) from the Fairlea bore (Kanso & Patel, 2003) and strain CooI3BT from the Cooinda bore have been published (Kanso et al., 2002). The data generated during this project add to our current information and extend our knowledge about the bacterial communities of the GAB's sub-surface environment. This information will provide a basis for further ecological studies of the GAB. Studies on involvement of certain groups of bacteria with the corrosion of metals used in bore casings could provide a foundation for further studies to develop maintenance and managing strategies for the GAB bores.

Probiotics are viable microorganisms that show beneficial effects on the health of the host by improving their intestinal microflora. The microorganisms applied as probiotics mainly include Lactobacillus and Bifidobacterium species. Probiotics can inhibit the bacterial pathogens, reduce serum cholesterol levels, improve lactose tolerance and stimulate the immune response. They also have other properties such as / tolerance to acid and bile salts, adherence to gastrointestinal cells for colonization, resistance to antibiotics and &beta / -galactosidase acitivity. The properties of probiotic products are determined by the characteristics of the microorganisms they contain. For that reason, isolation and characterization of new strains having probiotic properties is an important issue. New strains are generally isolated from their natural habitats which are fermented dairy products such as kefir. In order to exert beneficial health affects in the digestive system, commercial probiotic products should contain adequate numbers of viable cells. Probiotic microorganisms should protect their viability during their shelf storage. Therefore, the viability of probiotics is especially important for food manufacturers that search for new probiotic strains with good survival and stability properties upon storage. In this study, probiotic microorganisms were isolated from traditional kefir grains known as a &lsquo / complex probiotic&rsquo / . The isolates were firstly identified using biochemical tests, then the putative species belonging to &lsquo / Lactobacillus acidophilus group&rsquo / were identified with 16S rRNA gene sequencing. Analysis of sequencing resulted in differentiation of &ldquo / L. acidophilus group&rdquo / organisms, namely L. amylovorus and L. acidophilus. Moreover, typing of commercial and traditional L. acidophilus strains and L. amylovorus strains were performed with RAPD-PCR by using primer M13. While several L. acidophilus strains showed different RAPD fingerprints most of the L. acidophilus and L. amylovorus strains could not be differentiated due to high similarity of their RAPD fingerprints. Following identification, survival of these isolates in probiotic yogurt preparations were investigated and compared to the survival of commercial probiotics. Consequently, although the survival of kefir grain isolates were less than commercial probiotics, they sustained the minimum recommended level for probiotics (106 cfu/ml) during cold storage. Such level of survival makes them considerably good candidates to be used as commercial probiotic cultures.

Pienarūgščių bakterijų paieška ir jų identifikavimas migruojančių didžiųjų ančių (Anas platyrhynchos) žarnyne naudojant dalinių 16S rRNR geno sekų analizę ir kultivavimu paremtus metodus Klaidas VARNA Vilniaus Universiteto Ekologijos Institutas, Hidrobiontų Ekologijos ir Fiziologijos Laboratorija bei Populiacinės Genetikos Laboratorija, Akademijos-2, Vilnius-21, 08412, Lietuva. Šiame tyrime pavasarinių ir rudeninių didžiųjų ančių (Anas platyrhynchos) migrantų iš Nemuno deltos virškinamojo trakto pieno rūgšties bakterijų įvairovė buvo ištirta naudojant molekulinius metodus (polimerazės grandininės reakcijos amplifikacija ir dalinių 16S rRNR geno sekų sekvenavimas) ir kultivavimu paremtus metodus. Migruojančių didžiųjų ančių (Anas platyrhynchos) pieno rūgšties bakterijų paieška buvo atlikta pirmą kartą. Rudeniniai didžiųjų ančių migrantai plonojo žarnyno sienelėse (1.2×107 iki 2.1×107 k.f.v./g) ir jų turinyje (nuo 3.4×107 iki 1.1×108 k.f.v./g) turi didesnį pieno rūgšties bakterijų skaičių nei pavasariniai migrantai (atitinkamai nuo 3.2×106 iki 4.8×106 k.f.v./g ir nuo 1.0×107 iki 2.2×107 k.f.v./g). Tiek rudeninių tiek ir pavasarinių didžiųjų ančių migrantų plonojo žarnyno sienelėse ir jų turinyje dominavo kokinės pieno rūgšties bakterijų formos (atitinkamai 65% ir 83.5% bei 81.4% ir 91.6%), o lazdelių buvo mažiau (atitinkamai 35% ir 16.5% bei 18.6% ir 8.4%). Manoma, kad minėtus skirtumus įtakoja keli veiksniai: ilgai trunkanti migracija, perėjimo periodas, skirtingas maistas ir... [toliau žr. visą tekstą] / Identification of lactic acid bacteria in the migrant mallard ducks Anas platyrhynchos intestinal tract by partial 16S rRNA gene sequence analysis and using culture-based techniques Klaidas VARNA Institute of Ecology of Vilnius University, Laboratory of Hydrobionts Ecology and Physiology, Laboratory of Population Genetics, Akademijos-2, Vilnius-21, 08412, Lithuania. In this study the lactic acid bacteria diversity of the intestinal tract content of the vernal and autumnal migrant mallard ducks (Anas platyrhynchos) from Nemuno delta has been investigated by molecular methods: polymerase chain reaction amplification and sequencing of partial 16S rRNA genes and using culture-based techniques. The investigation of the lactic acid bacteria of the migrant mallard ducks has been performed the first time. Autumnal migrant mallard ducks in the small intestine walls (from 1.2×107 until 2.1×107 c.f.u./g) and in their content (from 3.4×107 until 1.1×108 c.f.u./g have the greatest number of the lactic acid bacteria then vernal migrants (respectively from 3.2×106 until 4.8×106 c.f.u./g and from 1.0×107 until 2.2×107 c.f.u./g). In the small intestine walls and in their content of the autumnal and vernal migrant mallard ducks, dominated cocci-shaped lactic acid bacteria (respectively 65% and 83.5%, 81.4% and 91.6%), whereas rod-shaped was under (respectively 35% and 16.5%, 18.6% and 8.4%). Supposedly, that these defferences determine some factors: a long migration, period of incubate... [to full text]

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

Made available in DSpace on 2015-04-20T12:31:09Z (GMT). No. of bitstreams: 1 Juliana Vianna Pereira.pdf: 4570056 bytes, checksum: c660fca0452deacfa10d23ee9bc79a2b (MD5) Previous issue date: 2009-08-26 / The complex microbiota of the oral cavity has been intensively studied and saliva is characterized by microorganisms which colonize different regions of mouth, such as tongue, supragengival and subgingival biofilm. Considering this, the purpose of this study was to evaluate the bacterial diversity of the saliva of patients with different levels of oral hygiene according to the Silness, Löe index. In this research, two genomic libraries of saliva source from 15 patients each were constructed. The pooled samples differ in the average index of Silness, Löe being considered as high or low index within the rate 1.0 to 3.0 and 0 to 0.5, respectively. The DNA saliva was extracted by phenol / chloroform method and 16S rRNA gene for the microorganisms of each sample were amplified and cloned. The sequences obtained were compared to those from sequences of the GenBank NCBI and RDP. The library resultant from saliva of patients with high level of dental biofilm showed 23 OTUs grouped as five known genus: Streptococcus, Granulicaella, Gemella, Peptostreptococcus and Veillonella besides 33.3% of uncultured bacteria. The Library made from saliva of patients with low level of dental biofilm, was significantly different from its counterpart (p = 0.000) and was composed by 42 OTUs, distributed among 11 known genus: Streptococcus, Granulicaella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces, and 24.87% of uncultured bacteria. The genus Streptococcus was the more prevalent in the two libraries, constituting 79.08% of the first and 73.64% the second. In conclusion, patients with low dental biofilm index have saliva with higher bacterial diversity than patients with high dental biofilm index, and despite most uncultivated species aggregate with Steptococcus, they still are new and unknown microorganisms / A complexa microbiota da cavidade bucal tem sido intensivamente estudada e a saliva destaca-se por apresentar microrganismos de diferentes regiões, como a língua, biofilme subgengival e supragengival. Diante disto, o objetivo do presente estudo foi avaliar a diversidade bacteriana da saliva de pacientes com diferentes índices de higiene bucal e para isto, foram construídas duas bibliotecas genômicas da saliva, que foram constituídas por amostras de 15 pacientes cada uma, com a média de índice de biofilme de Silness; Löe diferenciado, sendo a primeira com índice de 1,0 a 3,0 (denominada alto índice) e a segunda, entre 0 a 0,5 (denominada baixo índice). O DNA da saliva foi extraído pelo método fenol/clorofórmio e o gene 16S rRNA para cada biblioteca foi amplificado e clonado. As sequências obtidas foram comparadas com aquelas armazenadas no GenBank do NCBI e RDP. A biblioteca composta pela saliva de pacientes com Alto índice de biofilme dental apresentou cinco Gêneros conhecidos: Streptococcus, Granulicaella, Gemella, Veillonella e Peptostreptococcus e 33,3% de bactérias não-cultivadas, agrupados em 23 OTUs. A Biblioteca, composta pela saliva de pacientes com Baixo índice de biofilme dental, foi diferente siguinificativamente da primeira (p=0,000) e foi composta de 42 OTUs, distribuídas em 11 Gêneros conhecidos: Streptococcus, Granulicaella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces, alem de 24,87% de bactérias não-cultivadas. O Gênero Streptococcus foi o mais prevalente nas duas bibliotecas, constituindo 79,08% da primeira e 73,63% da segunda. Conclui-se que existe maior diversidade bacteriana na saliva de pacientes com Baixo índice de biofilme dental em relação à pacientes com Alto índice de biofilme dental e que apesar da maioria das espécies não-cultivadas agruparem-se com os Streptococcus, ainda contituem-se de microrganismos novos e desconhecidos

The giant barrel sponge, Xestospongia muta is an iconic and essential species of the coral reefs in South Florida. The sponge has primary roles providing ecosystem services and creating unique habitats for diverse microbial communities. On April 27, 2012 an outbreak of Sponge Orange Band Disease (SOB) was detected off the coast of South Florida. The disease begins with sponge bleaching, followed by mesohyl or “mesohyl” necrosis and often total mesohyl disintegration. Sampling from two diseased populations at Boynton Beach and Fort Lauderdale, FL took place on May 11th and May 29th, 2012. Each of the nine diseased sponges from Boynton Beach and the five diseased sponges from Fort Lauderdale had three separate mesophyl samples collected to examine the effects of disease progression on the microbial community. These included healthy mesohyl from a diseased sponge (HoD), the boundary layer which captured the advancing line of diseased mesohyl (BL) and diseased mesohyl from a diseased sponge (D). Mesohyl from three sponges with no visible signs of SOB disease were also collected from each sampling location to use for healthy controls (HC). Sequencing of the V4 region of the 16S rRNA gene was performed on all of these samples via the “454” pyrosequencing on a Titanium GS FLX platform. The microbial communities associated with the diseased samples revealed a microbiome shift that followed the progression of Sponge Orange Band Disease (SOB) and was dominated by Bacteroidetes, Protebacteria and Chloroflexi. No singular or group of microbes were solely found within the infected mesohyl of Xestospongia muta from both sampling site populations; therefore there is no unequivocal candidate as a definite microbial causative SOB agent. But there were bacteria associated with disease progression that included Armatimonadetes, Caldithrix, Chlorobi, Fibrobacteres, Fusobacteria, GN02, KSB3, OP1, OP2, OP8, Planctomycetes, SR1, TM6, Tenericutes, Verrucomicrobia, WPS-2 and ZB3. Verrucomicrobia and Plantomycetes increased significantly within the D and the BL populations, which was consistent within all the diseased sponges. This study provides a deep sequencing profile of microbial communities within Xestospongia muta affected with SOB Disease and provides a new insight into the sponge healthy microbiome.