published_or_final_version / Microbiology / Master / Master of Philosophy

A dissertation submitted to the Faculty of Health science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg 2013 / The causative pathogen of tuberculosis, Mycobacterium tuberculosis (Mtb), is equipped with several DNA repair mechanisms for continued survival within the host. One such mechanism is Base Excision Repair (BER) that repairs DNA damage caused by reactive oxygen and nitrogen species (ROS/RNS) generated by the host immune cells during infection. BER is dependent on DNA glycosylases namely: formamidopyrimidine (Fpg/MutM/Fapy), endonucleaseVIII (Nei) and endonucleaseIII (Nth) with Nei being structurally similar to Fpg but functionally similar to Nth. Bioinformatics analysis of the genome sequences of Mtb and its non-pathogenic relative Mycobacterium smegmatis (Msm) identified a unique duplication of Fpg and Nei glycosylases and a single nth gene in the same chromosomal context in both organisms. Previously, it has been shown that the lack of Fpg/Nei glycosylases in Msm display no differences in growth and survival under normal and oxidative stress conditions with no increase in spontaneous mutation rates as compared to the parental strain, suggesting that nth maybe significant for mycobacterial genome maintenance. Hence, in this study the nth gene was site specifically inactivated by homologous recombination in the parental Msm strain and in selected combinatorial mutant strains deficient in the Fpg/Nei glycosylases. Loss of the nth allele in the panel of mutants was genotypically confirmed by PCR and southern blot analyses. Inactivation of the nth gene did not affect the in vitro growth of the mutant strains under normal culture conditions. Interestingly, UV induced DNA damage of the single nth mutant resulted in a dramatic increase in mutation frequency that was not observed in any of the mutants. The progressive loss of fpg, nei and nth genes showed exaggerated reduced survival under oxidative stress. The subsequent deletion of nth in mutants deficient in fpg/nei resulted in a dramatic increase in spontaneous mutation rates and frequencies, implying that nth is integral for the repair of both spontaneous and induced DNA damage. Undoubtedly, these results indicate that Msm nth encoding the Nth glycosylase is involved in DNA repair and has anti-mutator properties. Furthermore, nth together with fpg and nei is part of a robust DNA repair system that maintains the integrity of the mycobacterial genome.

The sequence of the rightmost 4,626 bp of the Bacillus phage φ29 genome is presented and analyzed. Nine large open reading frames (ORF's) have been found. Three of these ORF's are correlated with the late genes 14, 15 and 16. The remaining six ORF's are in the right early region. One of these early ORF's has been identified as gene 17 (g17), the only early gene to have been genetically mapped in this region. The remaining ORF's (16.5, 16.6, 16.7, 16.8 and 16.9) were previously unknown. The biological efficacies of some of these putative early ORF's were demonstrated using an in vitro E. coli transcription-translation system. The primary amino acid sequences, molecular weights, translational initiation sequences and genetic organization of these nine genes are presented and discussed. Gene product 15 (gp15) was found to have strong homology with Salmonella phage P22 gp19, a lysozyme. gp15 also has a lesser but possibly significant homology with T4 gene product e (gpe), also a lysozyme. Using a clone containing φ29 g15 it was shown that gp15 can complement T4 gene e (ge) mutant infections, leading to the conclusion that φ29 g15 encodes a lysozyme. Three transcriptional initiation sites (P(E)3, P(EC)3 and B2) were previously mapped in this region. The sequences of the putative P(EC)3 and B2 promoter sites are presented and shown to have homology with the Bacillus σ⁵⁵ concensus sequence. Sequences having homology to a minor Bacillus sigma factor recognition site, σ³², are also presented and discussed. The region between the last late gene (g16) and the last early gene (ORF-16.5) consists of only 30 bp. Analysis of potential secondary structures of transcripts across this region suggests that the same sequences may be involved in the termination of both late and early transcription.

The project was concerned with the construction of defined attenuated derivatives of P. multocida serotype B:2 strains, causative agents of haemorrhagic septicaemia, and attempts were made to construct defined mutations in genes such as aroA, cya, and galE loci that have been used to induce attenuation in other bacterial strains. Mutants defective in the aroA gene were constructed by allelic exchange of the locus in the chromosome of the wild-type strains with a cloned aroA gene interrupted with a cassette encoding kanamycin resistance (KmR). The aroA defective strains were confirmed by PCR, Southern blotting, lack of growth on minimal medium and by enzyme assay. KmR inactivated aroA mutants JRMT1 and JRMT2 strains derived from P. multocida 85020 and Quetta strains, respectively, were highly attenuated in a mouse model, with an LD50 108 C.F.U./mouse after injection intraperitoneally (i.p.). In contrast, the wild-type strains had LD50 <50 C.F.U./mouse by this route. Vaccination once by the i.p. route or twice by the i.n. route with these aroA mutants gave complete protection to the mice against subsequent challenge i.p. with 10,000 LD50 of the homologous wild-type strain or 1000 LD50 of the heterologous wild-type strain. Vaccination with these by the s.c. route was not protective. When high doses of the attenuated strains were inoculated by the i.p. or i.n. routes, there was some spread to the internal organs but the organisms were cleared by 24 and 72 hrs respectively. In contrast, the wild-type parent strains spread rapidly and multiplied in high numbers and killed the mice by 24 and 96 hrs respectively.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Botany / Master / Master of Philosophy

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Esterases play a variety of roles in nearly every aspect of life ranging from cellular metabolism, signal transduction to defence mechanisms in plants. One aspect of esterases that recently is receiving more attention is the role esterases play in the .degradation of plant material. With fossil fuels (coal and oil) estimated to run out in the next 20 to 30 years, renewable sources such as plant biomass are becoming increasingly important. Plant biomass contains hemicellulosic and cellulosic materials that need to be degraded to their different constituents before they can be optimally used for the production of commodities. Although the enzymes needed to hydrolyse the xylan backbone (xylanases and P-xylosidases) are important, enzymes that remove side chains from the polymer are equally important. They facilitate hydrolysis by xylanases and P-xylosidases and will improve the availability of monomeric sugars for utilisation when used in conjunction with other xylanolytic enzymes. Many of these side-chains are esters and they need to be removed through the action of esterases, either directly from the xylan backbone or from shorter xylo-oligomers. An existing genomic DNA library of Bacil/us pumilus in Escherichia coli was screened for the presence of an acetyl esterase encoding gene. Positive clones were identified by the formation of clearing zones on plates containing glucose pentaacetate. Plasmid DNA was isolated from a positive E. coli clone. The DNA insert was sequenced and found to contain two open reading frames, one of which encoded a novel esterase (estA). Using different primers the gene was amplified by polymerase chain reaction and inserted into an inducible expression vector (pKK223- 3) for expression in E. coli. The plasmid was introduced into E coli and the esterase activity determined, using the chromogenic substrate a-naphthyl acetate. Activity levels decreased shortly after induction with IPTG and therefore plasmid pAP4 was used for enzymatic assays. Cultures containing plasmid pAP4 produced extracellular activity of 2.5 nkatal/ml. The pH and temperature optima as well as temperature stability of the enzyme was determined. The enzyme exhibited optimal activity at pH 6 and 60°C and was stable at 60°C after 2 h. Enzyme assays on different substrates yielded activity on methylumbelliferyl butyrate and methylumbelliferyl acetate in addition to the glucose pentaacetate and a-naphthyl acetate. The estA gene was cloned into a yeast expression vector between the PGK promoter and terminator sequences for expression of the gene in Saccharomyces cerevisiae. The estA open reading frame was also fused to the MFa 1 secretion signal for secretion of the protein from S. cerevisiae. The expression vector was successfully transformed into S. cerevisiae, but no extracellular activity was detected. Only low intracellular activity of 0.260 nkatal/ml was detected in S. cerevisiae. / AFRIKAANSE OPSOMMING: Esterases speel 'n verskeidenheid van rolle in feitlik elke aspek van lewe, van sel metabolisme, sein transduksie tot verdedigingsmeganismes in plante. Een aspek van esterases wat al hoe meer aandag geniet, is die rol wat esterases in die afbraak van plant en plantmateriaal speel. Met olie- en steenkoolbronne wat na beraming oor 20 tot 30 jaar tot niet sal gaan, raak die rol wat hernubare bronne speel al hoe belangriker. Plantbiomassa bevat sellulose en hemisellulose wat tot die verskillende komponente afgebreek moet word voordat dit optimaal vir die vervaardiging van produkte aangewend kan word. Alhoewel die ensieme wat vir die hidrolise van die xilaanruggraat benodig word, (xilanases en ~-xulosidases) belangrik is, is die ensieme wat die sygroepe vanaf die polimeer verwyder ewe belangrik aangesien hulle die hidrolise deur xilanases en ~-xulosidases bevorder. Wanneer hulle saam met die ander xilanolitiese ensieme gebruik word, sal hulle die beskikbaarheid van monomeriese suikers vir fermentasie verhoog. Baie van hierdie sygroepe is esters en hulle word deur die aksie van esterases verwyder, of direk van die ruggraat, ofvanafkorter xilo-oligosakkariede. 'n Bestaande genoom DNA biblioteek van Bacillus pumilus in Escherichia coli is vir die teenwoordigheid van 'n asetielesterase-koderende geen gesif. Positiewe klone is deur die vorming van 'n sone op plate wat glukose pentaasetaat bevat, geïdentifiseer. Die DNA-invoeging van die positiewe E. coli-kloon se DNA-volgorde is bepaal en twee oopleesrame is gevind waarvan een vir 'n unieke esterase (estA) kodeer. Met behulp van verskillende inleiers is die geen met die polimerasekettingreaksie (PKR) geamplifiseer en in 'n induseerbare promotor vir uitdrukking in E. coli gekloneer. Die plasmied is getransformeer in E. coli en aktiwiteit is bepaal deur cc-naftielasetaatte gebruik. Vlakke van aktiwiteit het kort na induksie met IPTG weer gedaal en daarom was plasmied pAP4 vir ensiematiese toetse gebruik. E. coli-transformante met plasmied pAP4 het ekstrasellulêre aktiwiteit van 2.5 nkatal/ml gelewer. Die pH en temperatuur optima sowel as die temperatuurstabiliteit van die ensiem was bepaal. Die ensiem toon optimale aktiwiteit by pH 6 en 'n temperatuur van 60°C. Aktiwiteitstoetse op verskillende substrate het aktiwiteit op metielumbelliferielasetaat en metielumbelliferielbutiraat bo-en-behalwe die glukosepentaasetaat en c-naftielasetaar getoon. Die estA geen is in uitdrukkingskasette bevattende die PGKpromotor en-termineerder vir uitdrukking in Saccharomyces cerevisiae gekloneer. Dit is ook agter die MFal-sekresiesein gekoppel vir sekresie vanuit S. cerevisiae. Geen ekstrasellulêre aktiwiteit is gevind nie. Slegs intrasellulêre aktiwiteit van 0.26 nanokatal per mililiter was bepaal.

Anthony C.T. Liang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 73-76). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Introduction to antibiotics --- p.2 / Chapter 1.2 --- Use of antibiotics in antimicrobial chemotherapy --- p.3 / Chapter 1.3 --- Drug resistance in bacteria --- p.4 / Chapter 1.4 --- Genetic of infections drug resistance --- p.6 / Chapter 1.5 --- Clinical importance of drug resistance --- p.8 / Chapter 1.6 --- Resistance studies on a clinically- isolated Shigella Species --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Culture media for bacteria growth --- p.10 / Chapter 2.2 --- Large scale plasmid preparation by CsCl density gradient centrifugation --- p.12 / Chapter 2.3 --- Minipreps of plasmid DNA by the alkaline lysis method --- p.14 / Chapter 2.4 --- Elution of DNA using the Geneclean Kit --- p.16 / Chapter 2.5 --- Transformation of plasmid DNA into E.coli DH5α --- p.17 / Chapter 2.6 --- Antibiotic sensitivity test and screening of resistance colonies --- p.19 / Chapter 2.7 --- Agarose electrophoresis of DNA --- p.20 / Chapter 2.8 --- Restriction and ligation --- p.21 / Chapter 2.9 --- Protocol for studying substrate profiles of aminoglycoside-modifying enzyme (AME) --- p.23 / Chapter 2.10 --- DNA sequencing using the T7 sequencing Kit from Pharmacia --- p.26 / Chapter Chapter 3 --- Antibiotic resistance studies on multiple resistant Shigella spp / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Conjugation and transformation experiment --- p.35 / Chapter 3.3 --- Extraction of plasmid DNA from Shigella 2731and transconjugant 14R525(2731) --- p.37 / Chapter 3.4 --- Discussion --- p.39 / Chapter Chapter 4 --- Cloning and characterization of beta-lactamase gene of Shigella2731 / Chapter 4.1 --- Introduction --- p.40 / Chapter 4.2 --- Cloning of the beta-lactam gene in Shigella2731 --- p.42 / Chapter 4.3 --- "Resistance pattern of El, E2, and S1" --- p.43 / Chapter 4.4 --- "Plasmid DNA extraction of El, E2, and S1" --- p.44 / Chapter 4.5 --- Restriction mapping of the plasmid pSFlOO --- p.47 / Chapter 4.6 --- Discussion --- p.51 / Chapter Chapter 5 --- Cloning and characterization of the aminoglycoside resistance gene / Chapter 5.1 --- Introduction --- p.53 / Chapter 5.2 --- Cloning of the aminoglycoside resistance genes --- p.56 / Chapter 5.3 --- Substrate profile studies on aminoglycoside- modifying enzyme (AME) activity on transformant G and S --- p.57 / Chapter 5.4 --- Subcloning of plamsid DNA from transformant S --- p.60 / Chapter 5.5 --- "DNA sequencing of fragments A, B, and C" --- p.65 / Chapter 5.6 --- Discussion --- p.68 / Chapter Chapter 6 --- Conclusion --- p.79 / Chapter Chapter 7 --- Reference --- p.73

Mok Yu-Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (Leaves 195-201). / Abstract --- p.i / Acknowledgements --- p.iii / List of Abbreviations --- p.iv / Table of contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The need to increase the specificity and variety of restriction endonucleases --- p.1 / Chapter 1.2 --- Classification of methods used for increasing the specificity and variety of restriction endonculeases --- p.2 / Chapter 1.3 --- Isolation and characterization of restriction endonucleases from natural sources --- p.3 / Chapter 1.4 --- Modification of DNA substrate to produce new cleavage specificities --- p.6 / Chapter 1.4.1 --- Methylation of the DNA substrate --- p.6 / Chapter 1.4.1.1 --- Achilles' hell cleavage-The use of canonical methylation to produce novel specificities --- p.10 / Chapter 1.4.1.2 --- Cross protection-The use of non-canonical methylation to generate new cleavage specificity --- p.14 / Chapter 1.4.1.2.1 --- Recognition sequence of a restriction endonuclease and a methylase partially overlap --- p.14 / Chapter 1.4.1.2.2 --- Methylase recognizing a subset of the degenerate sequence of the restriction endonuclease --- p.16 / Chapter 1.4.1.2.3 --- Methylase-limited partial digestion --- p.16 / Chapter 1.4.1.3 --- The use of methylation dependent restriction endonucleases and methylases to generate new specificity --- p.17 / Chapter 1.4.1.4 --- Sequential double-methylation-A two step methylation procedure to generate new specificities --- p.20 / Chapter 1.4.2 --- The generation of a universal restriction endonuclease by combining a Type IIS restriction enzyme moiety and an oligonucleotide adaptor --- p.22 / Chapter 1.4.2.1 --- General principle for generating a universal restriction endonuclease --- p.22 / Chapter 1.4.2.2 --- Factors that affect the cleavage efficiency of universal restriction endonuclease --- p.25 / Chapter 1.4.2.3 --- Modifications and potential applications of the universal restriction endonuclease --- p.29 / Chapter 1.4.3 --- DNA triple helix formation-enhance restriction enzyme specificity by site-specific inhibition of restriction/modification enzymes --- p.32 / Chapter 1.5 --- Modification of the cleaving agent to produce new specificities --- p.36 / Chapter 1.5.1 --- Sequence-specific artificial endonucleases --- p.36 / Chapter 1.5.1.1 --- Oligonucleotides as sequence-specific ligand --- p.37 / Chapter 1.5.1.2 --- Protein or peptide as sequence-specific ligand --- p.40 / Chapter 1.5.1.3 --- General limitations and applications of artificial endonucleases --- p.42 / Chapter 1.5.2 --- Molecular cloning and protein engineering of the restriction-modification system of bacteria --- p.43 / Chapter 1.5.2.1 --- Molecular cloning of the bacterial restriction-modification systems --- p.43 / Chapter 1.5.2.1.1 --- The strategies used to clone and screen restriction-modification systems --- p.45 / Chapter 1.5.2.2 --- Protein engineering of the restriction-modification systems of bacteria --- p.50 / Chapter 1.5.2.2.1 --- Pre-requisites for protein engineering on the restriction-modification systems --- p.51 / Chapter 1.5.2.2.2 --- Effects of protein engineering on the activity and specificity of restriction endonuclease and methylase --- p.53 / Chapter 1.6 --- Variation of restriction endonuclease specificity by altering the reaction condition --- p.56 / Chapter 1.6.1 --- Effects of organic solvents --- p.57 / Chapter 1.6.2 --- Effects of pH and ionic environment on restriction endonuclease specificity --- p.58 / Chapter 1.6.3 --- Remarks on the use of star activity to introduce new specificity --- p.59 / Chapter 1.7 --- Aim of study --- p.59 / Chapter Chapter 2 --- Purification and characterization of thermophilic restriction endonucleases from soil Bacillus spp / Chapter 2.1 --- Materials and methods --- p.61 / Chapter 2.1.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.61 / Chapter 2.1.1.1 --- Preparation of crude enzyme extract --- p.61 / Chapter 2.1.1.2 --- Purification of BsiB I and BsiE 1 --- p.63 / Chapter 2.1.1.3 --- Purification of BsiY I --- p.63 / Chapter 2.1.1.4 --- Preparation of BsiG I and BsiU I --- p.64 / Chapter 2.1.1.5 --- Concentration and storage of the purified restriction endonucleases --- p.64 / Chapter 2.1.1.6 --- Regeneration of the columns --- p.64 / Chapter 2.1.2 --- Characterization of restriction endonucleases --- p.65 / Chapter 2.1.2.1 --- Assay for the working temperature and ionic requirement for the restriction enzymes --- p.65 / Chapter 2.1.2.2 --- Unit determination of the restriction endonucleases --- p.66 / Chapter 2.1.2.3 --- Assay for the purities of restriction endonucleases --- p.66 / Chapter 2.1.2.4 --- Determination of recognition specificity --- p.67 / Chapter 2.1.2.5 --- Determination of the restriction endonuclease's sensitivity to dam and dcm methylation --- p.68 / Chapter 2.1.2.6 --- Determination of the cleavage specificities of restriction endonucleases --- p.70 / Chapter 2.1.2.7 --- Sequencing using Deaza dGTP --- p.73 / Chapter 2.2 --- Results --- p.73 / Chapter 2.2.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.73 / Chapter 2.2.1.1 --- Strain identification --- p.74 / Chapter 2.2.1.2 --- Elution properties of the restriction endonucleases from columns --- p.74 / Chapter 2.2.1.2.1 --- BsiB I --- p.74 / Chapter 2.2.1.2.2 --- BsiE I --- p.77 / Chapter 2.2.1.2.3 --- BsiY 1 --- p.78 / Chapter 2.2.1.3 --- The working digestion temperature and ionic strength requirement --- p.81 / Chapter 2.2.1.4 --- Unit determination --- p.82 / Chapter 2.2.1.5 --- Purities of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6 --- Recognition sites of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6.1 --- BsiB I --- p.83 / Chapter 2.2.1.6.2 --- BsiE I --- p.85 / Chapter 2.2.1.6.3 --- BsiY 1 --- p.87 / Chapter 2.2.1.6.4 --- BsiU I and BsiG I --- p.88 / Chapter 2.2.1.7 --- Sensitivity of restriction endonucleases to dam and dcm methylation --- p.90 / Chapter 2.2.1.8 --- Cleavage specificities of the purified restriction endonucleases --- p.91 / Chapter 2.2.1.8.1 --- BsiB I --- p.91 / Chapter 2.2.1.8.2 --- BsiE I --- p.92 / Chapter 2.2.1.8.3 --- BsiY I --- p.93 / Chapter 2.2.1.9 --- Sequencing of a wrongly sequenced site in pACYC177 using Deaza-dGTP --- p.94 / Chapter Chapter 3 --- The use of Xcm I and BsiY I as an universal restriction endonuclease / Chapter 3.1 --- Materials and methods --- p.98 / Chapter 3.1.1 --- Assay of universal restriction endonuclease using ss DNAs --- p.98 / Chapter 3.1.1.1 --- Annealing reaction between adaptors and ss DNAs --- p.99 / Chapter 3.1.1.2 --- Digestion of the annealed DNA complex --- p.100 / Chapter 3.1.1.3 --- Assay of the digested ss DNA on alkaline denaturing agarose gel --- p.100 / Chapter 3.1.2 --- Assay system involving 5' end-labelled oligonucleotide --- p.101 / Chapter 3.1.2.1 --- Purification of oligonucleotides using preparative polyacrylamide gel electrophoresis --- p.102 / Chapter 3.1.2.2 --- 5'end-labelling of the oligonucleotide DNA substrate --- p.104 / Chapter 3.1.2.3 --- The annealing between adaptors and oligonucleotide DNA substrate and the digestion condition --- p.104 / Chapter 3.1.2.4 --- Assay of the labelled oligonucleotides in polyacrylamide gel after digestion --- p.105 / Chapter 3.2 --- Results --- p.106 / Chapter 3.2.1 --- Xcm I adaptors #2 and #4 --- p.106 / Chapter 3.2.1.1 --- Assay conditions used for the universal restriction endonucleases --- p.107 / Chapter 3.2.1.1.1 --- Conditions used for hybridization --- p.107 / Chapter 3.2.1.1.2 --- Conditions used for digestion --- p.108 / Chapter 3.2.1.2 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #4 --- p.110 / Chapter 3.2.1.2.1 --- Methods used to optimize the hybridization process --- p.110 / Chapter 3.2.1.2.2 --- Methods used to relax the secondary DNA structures --- p.112 / Chapter 3.2.1.2.2.1 --- Linearization of M13mp7 with BamH I befor annealing the adaptor --- p.113 / Chapter 3.2.1.2.2.2 --- Relaxation of secondary structure using boiling and NaOH denaturation --- p.114 / Chapter 3.2.1.2.3 --- Methods used to optimize the digestion process --- p.115 / Chapter 3.2.1.2.3.1 --- Addition of BSA --- p.115 / Chapter 3.2.1.2.3.2 --- Addition of the restriction endonuclease in separate batches --- p.115 / Chapter 3.2.1.3 --- Digestion of ss M13mpl8 and ssM13mpl9 DNA using Xcm I adaptor #2 and adaptor #4 --- p.116 / Chapter 3.2.2 --- Xcm I adaptor #1 and #3 --- p.118 / Chapter 3.2.2.1 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #1 and adaptor #3 --- p.119 / Chapter 3.2.2.1.1 --- Methods used to relax the secondary structure --- p.119 / Chapter 3.2.2.1.1.1 --- Linearization of M13mp7 with BamH I before the annealing reaction --- p.120 / Chapter 3.2.2.1.1.2 --- Relaxation of secondary structure by NaOH denaturation --- p.121 / Chapter 3.2.2.1.1.3 --- Relaxation of secondary structure by adding DMSO and urea --- p.122 / Chapter 3.2.2.1.2 --- Methods used to optimize the digestion and hybridization processes --- p.123 / Chapter 3.2.2.1.2.1 --- Annealing of M13mp7 with a different amount of adaptor #3 and digesting the DNA complex with Xcm I at different temperatures --- p.123 / Chapter 3.2.2.1.2.2 --- Optimization of digestion by adding Xcm I in separate batches --- p.124 / Chapter 3.2.3 --- BsiY I adaptor --- p.124 / Chapter 3.2.3.1 --- Methods used to optimize the cleavage of M13mp7-BsiY I adaptor complex with BsiY I --- p.126 / Chapter 3.2.3.1.1 --- Optimization of hybridization using various concentrations of NaCl during the annealing reaction --- p.126 / Chapter 3.2.3.1.2 --- Optimization of digestion by binding BsiY I to the BsiY I adaptor before annealing --- p.127 / Chapter 3.2.4 --- The use of 5' end-labelled oligonucleotide DNA substrates for digestion with universal restriction endonuclease --- p.128 / Chapter Chapter 4 --- Molecular cloning of the BsiY I restriction-modification system / Chapter 4.1 --- Materials and methods --- p.132 / Chapter 4.1.1 --- Preparation of chromosomal DNA from BsiY I producing Bacillus stearothermophilus --- p.132 / Chapter 4.1.1.1 --- Restriction digestion of the chromosomal DNA --- p.134 / Chapter 4.1.1.2 --- Southern hybridization to locate the position of the DNA fragment coding for the restriction-modification system --- p.135 / Chapter 4.1.1.2.1 --- Southern transfer of DNA fragments onto nitro-cellulose paper --- p.135 / Chapter 4.1.1.2.2 --- Labelling of the probes by Nick-translation --- p.136 / Chapter 4.1.1.2.3 --- Hybridization of the nick-translated probes onto the DNA fragments fixed on NC paper --- p.137 / Chapter 4.1.2 --- Large-scale preparation of the cloning vector --- p.137 / Chapter 4.1.2.1 --- Restriction endonuclease digestion and dephosphorylation of the vector ´Ø.… --- p.139 / Chapter 4.1.3 --- Ligation between vector and DNA inserts --- p.139 / Chapter 4.1.4 --- Transformation of the ligated DNA into competent cells --- p.140 / Chapter 4.1.4.1 --- Preparation of competent cells --- p.140 / Chapter 4.1.4.2 --- Transformation of the ligated vector and insert DNA into competent cells --- p.142 / Chapter 4.1.5 --- Rapid alkaline lysis method for screening transformants that contains an insert --- p.143 / Chapter 4.1.6 --- Preparation of the genomic library and its plasmid DNA --- p.144 / Chapter 4.1.7 --- Screening procedures used to clone the BsiY I restriction-modification system --- p.144 / Chapter 4.1.7.1 --- In vitro selection using Hungarian Trick --- p.145 / Chapter 4.1.7.2 --- In vivo selection using the host strain AP1-200 and AP1-200-9 --- p.145 / Chapter 4.1.7.2.1 --- Preparation of competent AP1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.7.2.2 --- Transformation of the genomic library plasmid into competent AP 1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.8 --- Assay of BsiY I restriction endonuclease and methylase activities in the suspecting clones --- p.147 / Chapter 4.1.8.1 --- Assay to BsiY I methylase activity - resistance of the plasmid to BsiY I digestion --- p.147 / Chapter 4.1.8.2 --- Assay of BsiY I methylase activity - ability to incorporate H3-methyl group from H3-SAM into DNA substrate molecules --- p.148 / Chapter 4.1.8.3 --- Assay of BsiY I restriction endonuclease activity - ability of crude enzyme extract to cleave DNA --- p.149 / Chapter 4.2 --- Results --- p.150 / Chapter 4.2.1 --- Construction of the BamH I genomic library --- p.150 / Chapter 4.2.1.1 --- Vector and insert used --- p.150 / Chapter 4.2.1.2 --- Optimization of the ligation and transformation process --- p.151 / Chapter 4.2.1.3 --- Preparation of the BamH I library --- p.153 / Chapter 4.2.1.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.155 / Chapter 4.2.1.4.1 --- The Hungarian Trick --- p.155 / Chapter 4.2.1.4.2 --- Screening of the restriction-modification system using the strains API-200 and AP1-200-9 --- p.159 / Chapter 4.2.2 --- Construction of the Hind III library --- p.161 / Chapter 4.2.2.1 --- Vector and insert used --- p.161 / Chapter 4.2.2.2 --- Optimization of the ligation and transformation process --- p.162 / Chapter 4.2.2.3 --- Preparation of the Hind III library --- p.164 / Chapter 4.2.2.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.165 / Chapter 4.2.2.4.1 --- The Hungarian Trick --- p.165 / Chapter 4.2.2.4.2 --- Screening of the restriction-modification system using the strain AP1-200 and AP1-200-9 --- p.168 / Chapter 4.2.2.5 --- Assay of methylase activity using H3-SAM --- p.170 / Chapter 4.2.3 --- The use of Southern blotting and hybridization to find if two available probes have homology to the BsiY I restriction-modification system --- p.173 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Purification and characterization of restriction endonucleases from Bacillus spp --- p.176 / Chapter 5.1.1 --- Methods used to purify the restriction endonuclease --- p.177 / Chapter 5.1.2 --- Characterization of the restriction endonucleases --- p.179 / Chapter 5.1.2.1 --- Determination of the purities of the purified restriction endonucleases --- p.179 / Chapter 5.1.2.2 --- Determination of the recognition site --- p.179 / Chapter 5.1.2.3 --- Determination of the cleavage site --- p.180 / Chapter 5.1.2.4 --- Sequencing using Deaza-dGTP --- p.181 / Chapter 5.2 --- The use of Xcm I and BsiY I as universal restriction endonucleases --- p.182 / Chapter 5.2.1 --- The adverse effects of hair-pin loop on the cleavage with universal restriction enzymes --- p.183 / Chapter 5.3 --- Molecular cloning of the BsiY I restriction-modification system --- p.187 / Chapter 5.3.1 --- Construction of the genomic library --- p.187 / Chapter 5.3.1.1 --- Preparation of the insert and vector --- p.188 / Chapter 5.3.1.2 --- Optimization of the ligation and transformation processes --- p.188 / Chapter 5.3.2 --- Screening strategies used to clone the BsiY I restriction-modification system --- p.189 / Chapter 5.3.2.1 --- The Hungarian Trick --- p.189 / Chapter 5.3.2.2 --- Screening using the strains AP1-200 and AP1-200-9 cells --- p.191 / Chapter 5.3.3 --- Assay of the gene products from the cloned restriction-modification system --- p.192 / Chapter 5.3.3.1 --- Methylase activity --- p.192 / Chapter 5.3.3.2 --- Restriction endonuclease activity --- p.193 / Chapter 5.4 --- Future prospects --- p.193 / References --- p.195 / Appendix --- p.201

Symbiotic relationships of bacteria with higher organisms are commonly observed in nature; however, the functional role of these relationships is only rarely understood. This is particularly evident in epibiotic bacterial associations in the marine environment where the bacteria are often a diverse ensemble of microorganisms, thus complicating the identification of the functionally important members. Classical microbiological techniques, relying primarily on culturing these organisms, have provided an incomplete picture of these relationships. Molecular genetic techniques, focusing on the analyses of bacterial 16S rRNA sequences cloned directly from natural microbial populations, are now available which allow a more thorough examination of these associated bacterial populations. This study sought to characterize the epibiotic bacterial population associated with a very unique organism, Alvinella pompejana, using such a molecular approach. Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise. This worm colonizes the walls of actively venting high temperature chimneys and is thought to be one of the most thermotolerant metazoans known. The chimney environment is characterized by high concentrations of sulfide and heavy metals in the vicinity of the worm colonies. A morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment; however, previous studies have been unable to confirm such a role. The primary goal of this study is to phylogenetically characterize the dominant epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of Alvinella pompejana. 16S rRNA genes were amplified with universal bacterial primers by the polymerase chain reaction (PCR). These genes were subsequently cloned and the resulting clone library was screened by restriction fragment length polymorphism (RFLP) analysis to identify unique clone types. Thirty-two distinct clone families were found in the library. Four of these families were clearly dominant, representing over 65% of the library. The main assumption in this study is that the numerical dominance of the phylotypes in the starting population will be reflected in the clone library. Thus, representative clones from the four most abundant clone families were chosen for complete gene sequencing and phylogenetic analysis. These gene sequences were analyzed using a variety of phylogenetic inference methods and were found to be related to the newly established epsilon subdivision of the Proteobacteria. In future studies, these gene sequences will be used to construct specific oligodeoxynucleotide probes which can be used to confirm the morphology of the clone types in the epibiont population. / Graduation date: 1995