Genotypic characterization of extracellular enzyme producing thermophilic bacteria in Balçova Geothermal Region

Thesis (Master)--İzmir Institute of Technology, Biotechnology and Bioengineering, İzmir, 2003Includes bibliographical references (leaves:75-86)Text in English;Abstract: Turkish and Englishxii, 86 leavesThermophiles are the organisms which are adapted to live at high temperatures. The enzymes from thermophiles find a number of commercial applications because of their thermostability and thermoactivity. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes.In keeping with this view, Balçova Geothermal Region could serve as a good source for new thermophilic microorganisms with novel industrially important properties.The aim of this research was therefore the isolation of industrially important extracellular enzyme producing thermophilic bacteria from Balçova Geothermal Region and their identification by genetical means. 16S-ITS rDNA RFLP, plasmid profiling and pulsed field gel electrophoresis studies were performed for this purpose.112 thermophilic strains were isolated from various environmental samples collected within Balçova Geothermal Region. These strains were screened for the existence of 6 extracellular enzyme activities. These were, lipases, amylases, proteases, xylanases, cellulases and pectinases. In total, 110 lipase (tween 20 as substrate), 106 amylase, 55 protease, 28 xylanase, 10 cellulase and 3 pectinase activities were detected.Some other phenotypic tests were also performed for these isolated strains. Since all the isolated strains were Gram (+), endospore forming rods, they were identified as Bacillus sp.16S-ITS rDNA RFLP and plasmid RFLP profiles were produced by using two restriction endonucleases Taq I and Hae III . The isolated strains were clustered into eleven groups by Taq I restriction profiles of 16S-ITS rDNA while nine groups were obtained by Hae III digestion profiles. When these groups were compared, it was concluded that 17 genotypically different strains existed in total 112 isolates. Two of the isolated strains yielded similar RFLP profiles to those of Bacillus stearothermophilus (CECT 43) reference strain.Plasmid profiling was also performed. It was found that 23 of the isolated strains contained plasmid DNA. Hae III restriction profiles indicated the existence of three different types of plasmids.PFGE optimization studies by Sma I restriction endonuclease for thermophilic Bacilli were also performed. A new method for preparation of agarose plugs was developed

Horizontal gene transfer in Thermus thermophilus: mechanisms and barriers

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 12-08-2019La autora no permite ningún tipo de modificación en el excelComparative genome analysis have evidenced that fluent genetic exchange is mainly mediated by Horizontal Gene Transfer (HGT), thus recognized as a leading force of prokaryotic evolution and microbial genetic diversity. Transduction, transformation and conjugation are the three standard mechanisms driving HGT. The ancient thermophilic bacteria Thermus thermophilus laterally transfers DNA in a really efficient way due to a highly sophisticated competence machinery as well as by a conjugation-like process. However, conjugation in T. thermophilus emerges rather unconventional as it is efficient between isogenic cells, thus, bidirectional, and no type IV secretion systems seems to be involved. Besides, genes associated to the megaplasmid are transferred with higher frequencies (~10 fold) than those localized in the chromosome. No evident order in the transfer among megaplasmid markers contrasts with the identification of multiple hotspots of transfer in the chromosome. Several of these loci showing higher transfer frequencies are encoded near putative Tth111 type II restriction sites which might work as OriTs. The competence machinery actively participates in conjugation, being required in the receptor cell but not in the donor. Hence, a two-step model (push-pull) is proposed, where the donor energetically pumps DNA to a receptor cell, which actively pulls in the DNA transferred with its competence apparatus. Two active hexameric ATPases, named CptA and HerA, paralogs to the helicases HerA and Ftsk from other bacteria, have been identified as chief components of the pushing step. Therefore, T. thermophilus is able to proficiently exchange DNA by transformation (including DNA-protected vesicles which could work as long distance vehicles), and largely by conjugation. This fruitful dynamic gene flow is compatible with a battery of protective strategies to prevent potentially harmful invasion of genetic parasites. Among them, the Argonaute protein elicits a DNA-DNA interference on DNA taken up by transformation. We proved larger insights of Argonaute-mediated interference, acting, in vivo, against virtually any kind of DNA template. However, when such DNA is transferred by a conjugation-like process, Argonaute is not activated, suggesting a selective immunity towards the way in which DNA is acquired. This fact, together with the higher efficiency shown by conjugation compared to natural competence when the same DNA was transferred, enforcedthe proposal of conjugation as the major motor of shared traits among populations of Thermus spp in thermal environments. Finally, we analyse the role of the PrimPol polymerase as a ssDNA guide supplier for the Argonaute protein

Genomic data mining for the computational prediction of small non-coding RNA genes

The objective of this research is to develop a novel computational prediction algorithm for non-coding RNA (ncRNA) genes using features computable for any genomic sequence without the need for comparative analysis. Existing comparative-based methods require the knowledge of closely related organisms in order to search for sequence and structural similarities. This approach imposes constraints on the type of ncRNAs, the organism, and the regions where the ncRNAs can be found. We have developed a novel approach for ncRNA gene prediction without the limitations of current comparative-based methods. Our work has established a ncRNA database required for subsequent feature and genomic analysis. Furthermore, we have identified significant features from folding-, structural-, and ensemble-based statistics for use in ncRNA prediction. We have also examined higher-order gene structures, namely operons, to discover potential insights into how ncRNAs are transcribed. Being able to automatically identify ncRNAs on a genome-wide scale is immensely powerful for incorporating it into a pipeline for large-scale genome annotation. This work will contribute to a more comprehensive annotation of ncRNA genes in microbial genomes to meet the demands of functional and regulatory genomic studies.Ph.D.Committee Chair: Dr. G. Tong Zhou; Committee Member: Dr. Arthur Koblasz; Committee Member: Dr. Eberhard Voit; Committee Member: Dr. Xiaoli Ma; Committee Member: Dr. Ying X

Isolation of haloalkaliphilic microorganisms from leather industry

Thesis (Master)--İzmir Institute of Technology, Biology, İzmir, 2004Includes bibliographical references (leaves: 43-49)Text in English; Abstract: Turkish and Englishxiii, 68 leavesHaloalkalophiles are extremophilic microorganisms that are adapted to saline and alkaline conditions. Different species of them have been isolated so far from soda lakes and soil samples. Haloalkalophilic microorganisms have significant adaptive mechanisms to avoid denaturing effect of salts and to balance their interior pH. Extracellular enzymes that are produced by these halophilic and alkalophilic microorganism are applicable for industrial purposes. Therefore isolation of these organisms from their habitats and study on genotypic characterization constitute initial steps for furter biotechnological studies. In this study, processing steps of leather factories and their wastewater were chosen for sampling. In order to isolate target microorganisms Horikoshi-I medium including 12% NaCl was used. After isolation microorganisms were purified. Phenotypic tests were applied (Gram staining, catalase and oxidase behaviors, spore observation by phase contrast microscope, sensitivity to antibiotics and extracellular enzyme screenings). For genotypic characterization, genomic DNA was isolated and 16S-ITS rDNA region was amplified. Differentiation was achieved according to Restriction Fragment Length Polymorphism (RFLP) method by using Hae III and Taq I endonucleases. Isolates, which represented the different RFLP patterns, were chosen for building up the haplotype groups. As a result of the study seven different RFLP haplotypes were identified.Moreover, 16S ribosomal DNA partial sequencing was also performed on some of the strains in. These haloalkalophilic microorganisms and their enzymes could be used in different biotechnological studies in the future for various industrial applications

Expanding the repertoire of bacterial (non-)coding RNAs

The detection of non-protein-coding RNA (ncRNA) genes in bacteria and their diverse regulatory mode of action moved the experimental and bio-computational analysis of ncRNAs into the focus of attention. Regulatory ncRNA transcripts are not translated to proteins but function directly on the RNA level. These typically small RNAs have been found to be involved in diverse processes such as (post-)transcriptional regulation and modification, translation, protein translocation, protein degradation and sequestration. Bacterial ncRNAs either arise from independent primary transcripts or their mature sequence is generated via processing from a precursor. Besides these autonomous transcripts, RNA regulators (e.g. riboswitches and RNA thermometers) also form chimera with protein-coding sequences. These structured regulatory elements are encoded within the messenger RNA and directly regulate the expression of their “host” gene. The quality and completeness of genome annotation is essential for all subsequent analyses. In contrast to protein-coding genes ncRNAs lack clear statistical signals on the sequence level. Thus, sophisticated tools have been developed to automatically identify ncRNA genes. Unfortunately, these tools are not part of generic genome annotation pipelines and therefore computational searches for known ncRNA genes are the starting point of each study. Moreover, prokaryotic genome annotation lacks essential features of protein-coding genes. Many known ncRNAs regulate translation via base-pairing to the 5’ UTR (untranslated region) of mRNA transcripts. Eukaryotic 5’ UTRs have been routinely annotated by sequencing of ESTs (expressed sequence tags) for more than a decade. Only recently, experimental setups have been developed to systematically identify these elements on a genome-wide scale in prokaryotes. The first part of this thesis, describes three experimental surveys of exploratory field studies to analyze transcript organization in pathogenic bacteria. To identify ncRNAs in Pseudomonas aeruginosa we used a combination of an experimental RNomics approach and ncRNA prediction. Besides already known ncRNAs we identified and validated the expression of six novel RNA genes. Global detection of transcripts by next generation RNA sequencing techniques unraveled an unexpectedly complex transcript organization in many bacteria. These ultra high-throughput methods give us the appealing opportunity to analyze the complete RNA output of any species at once. The development of the differential RNA sequencing (dRNA-seq) approach enabled us to analyze the primary transcriptome of Helicobacter pylori and Xanthomonas campestris. For the first time we generated a comprehensive and precise transcription start site (TSS) map for both species and provide a general framework for the analysis of dRNA-seq data. Focusing on computer-aided analysis we developed new tools to annotate TSS, detect small protein-coding genes and to infer homology of newly detected transcripts. We discovered hundreds of TSS in intergenic regions, upstream of protein-coding genes, within operons and antisense to annotated genes. Analysis of 5’ UTRs (spanning from the TSS to the start codon of the adjacent protein-coding gene) revealed an unexpected size diversity ranging from zero to several hundred nucleotides. We identified and validated the expression of about 60 and about 20 ncRNA candidates in Helicobacter and Xanthomonas, respectively. Among these ncRNA candidates we found several small protein-coding genes that have previously evaded annotation in both species. We showed that the combination of dRNA-seq and computational analysis is a powerful method to examine prokaryotic transcriptomes. Experimental setups are time consuming and often combined with huge costs. Another limitation of experimental approaches is that genes which are expressed in specific developmental stages or stress conditions are likely to be missed. Bioinformatic tools build an alternative to overcome such restraints. General approaches usually depend on comparative genomic data and evolutionary signatures are used to analyze the (non-)coding potential of multiple sequence alignments. In the second part of my thesis we present our major update of the widely used ncRNA gene finder RNAz and introduce RNAcode, an efficient tool to asses local protein-coding potential of genomic regions. RNAz has been successfully used to identify structured RNA elements in all domains of life. However, our own experience and the user feedback not only demonstrated the applicability of the RNAz approach, but also helped us to identify limitations of the current implementation. Using a much larger training set and a new classification model we significantly improved the prediction accuracy of RNAz. During transcriptome analysis we repeatedly identified small protein-coding genes that have not been annotated so far. Only a few of those genes are known to date and standard proteincoding gene finding tools suffer from the lack of training data. To avoid an excess of false positive predictions, gene finding software is usually run with an arbitrary cutoff of 40-50 amino acids and therefore misses the small sized protein-coding genes. We have implemented RNAcode which is optimized for emerging applications not covered by standard protein-coding gene annotation software. In addition to complementing classical protein gene annotation, a major field of application of RNAcode is the functional classification of transcribed regions. RNA sequencing analyses are likely to falsely report transcript fragments (e.g. mRNA degradation products) as non-coding. Hence, an evaluation of the protein-coding potential of these fragments is an essential task. RNAcode reports local regions of high coding potential instead of complete protein-coding genes. A training on known protein-coding sequences is not necessary and RNAcode can therefore be applied to any species. We showed this with our analysis of the Escherichia coli genome where the current annotation could be accurately reproduced. We furthermore identified novel small protein-coding genes with RNAcode in this extensively studied genome. Using transcriptome and proteome data we found compelling evidence that several of the identified candidates are bona fide proteins. In summary, this thesis clearly demonstrates that bioinformatic methods are mandatory to analyze the huge amount of transcriptome data and to identify novel (non-)coding RNA genes. With the major update of RNAz and the implementation of RNAcode we contributed to complete the repertoire of gene finding software which will help to unearth hidden treasures of the RNA World

Identification and characterization of thermostable uracil glycosylases from the archaeon Methanobacterium thermoautotrophicum and the bacterium Thermus thermophilus

Die hydrolytischen Deaminationsreaktionen von Cytosin und 5-Methylcytosin bewirken hochmutagene U/G und T/G "Fehlpaarungen". Diese Prozesse sind besonders bei hohen Temperaturen betont und stellen eine ernste Bedrohung der genomischen Integrität der Thermophiles dar. Als der Hauptreparaturmechanismus wurde die "Basenexcisionsreparatur" (BER) vorgeschlagen und die Wirkung von Uracil und T/G, U/G "Fehlpaarungen" spezifischen DNA-Glycosylasen postuliert. Mit dem Ziel das Wissen über U/G und T/G entfernende Enzyme zu vertiefen, lag der Fokus dieser Arbeit in der Isolierung und enzymatischen Charakterisierung unterschiedlicher und neuartiger Uracylglycosylasen zweier thermophiler Mikroorganismen: Archeon Methanobacterium thermoautotrophicum DH und Bakterium Thermus thermophilus HB27. Die in vivo Rolle der charakterisierten Proteine wurde anhand der Kombination der enzymatischen Daten mit der vergleichenden Genomanalyse diskutiert

Statistical Algorithms and Bioinformatics Tools Development for Computational Analysis of High-throughput Transcriptomic Data

Next-Generation Sequencing technologies allow for a substantial increase in the amount of data available for various biological studies. In order to effectively and efficiently analyze this data, computational approaches combining mathematics, statistics, computer science, and biology are implemented. Even with the substantial efforts devoted to development of these approaches, numerous issues and pitfalls remain. One of these issues is mapping uncertainty, in which read alignment results are biased due to the inherent difficulties associated with accurately aligning RNA-Sequencing reads. GeneQC is an alignment quality control tool that provides insight into the severity of mapping uncertainty in each annotated gene from alignment results. GeneQC used feature extraction to identify three levels of information for each gene and implements elastic net regularization and mixture model fitting to provide insight in the severity of mapping uncertainty and the quality of read alignment. In combination with GeneQC, the Ambiguous Reads Mapping (ARM) algorithm works to re-align ambiguous reads through the integration of motif prediction from metabolic pathways to establish coregulatory gene modules for re-alignment using a negative binomial distribution-based probabilistic approach. These two tools work in tandem to address the issue of mapping uncertainty and provide more accurate read alignments, and thus more accurate expression estimates. Also presented in this dissertation are two approaches to interpreting the expression estimates. The first is IRIS-EDA, an integrated shiny web server that combines numerous analyses to investigate gene expression data generated from RNASequencing data. The second is ViDGER, an R/Bioconductor package that quickly generates high-quality visualizations of differential gene expression results to assist users in comprehensive interpretations of their differential gene expression results, which is a non-trivial task. These four presented tools cover a variety of aspects of modern RNASeq analyses and aim to address bottlenecks related to algorithmic and computational issues, as well as more efficient and effective implementation methods

Analysis of DNA-binding Proteins in Yeast Saccharomyces Cerevisiae

Gene expression is an elaborate and finely tuned process involving the regulated interactions of multiple proteins with promoter and enhancer elements. A variety of approaches are currently used to study these interactions in vivo, in vitro as well as in silico. With the genome sequences of many organisms now readily available, a plethora of DNA functional elements have been predicted, but the process of identifying the proteins that bind to them in vivo remains a bottleneck. I developed two high-throughput assays to address this issue. The first is a modification of the yeast one-hybrid assay. The second is probing protein microarrays with DNA sequence elements. Using these methods, I identified two proteins, Sef1 and Yjl103c, that bind to the same DNA sequence element. Sef1 and Yjl103c are little-characterized members of the zinc cluster family of transcription factors of S. cerevisiae. Characterization of their mechanism of action as well as identification of some of their target genes leads to the conclusion that they play a pivotal role in the transcriptional regulation of utilization of nonfermentable carbon sources by budding yeast

Biochemical and molecular characterization of extracellular enzyme producing Staphylococci isolated from different origins

Thesis (Master)--Izmir Institute of Technology, Molecular Biology and Genetics, Izmir, 2006Includes bibliographical references (leaves: 53-60)Text in English; Abstract: Turkish and Englishxi, 78 leavesStaphylococci are pathogenic bacteria known to cause diseases among diferrent organisms including human. The two species Staphylococcus aureus and Staphylococcus epidermidis are well defined in human diseases although their exact mechanism of pathogenesis is still not fully understood. These pathogenic bacteria could be isolated from soil, water, air, as well as from the living organisms and they are both pathogenic and saprophytic.Extracellular enzymes of the organisms are used for the industrial purposes. The isolation and characterization of these enzymes are crucial steps in biotechnology. The extracellular enzymes derived from the bacteria serve for many purposes in the industry. In this project 128 Staphylococcus sp. were used. Of these 128 bacteria, 12 were isolated from patients, 40 were isolated from the foodhandler.s hygiene detections, 27 were isolated from pygeons and 49 of them were reference strains. They were searched for the presence of some of the industrially important extracellular enzymes: protease, lipase, cellulase, xylanase, amylase, laccase, urease, DNase and pectinase with biochemical tests. They were also searched for the presence of the lipase, protease and thermonuclease amplifications by PCR. The bacteria apart from the refence strains were also tried to be identified by 16S-ITS-rRNA RFLP analysis. The results would indicate the extracellular enzyme production among these pathogenic bacteria and would also be used as a guide in further studies to correlate between Staphylococcal pathogenity and enzyme production