Dissertations / Theses on the topic 'Bacillus (Bacteria) – Genetics'

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.

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.

Thesis (MScAgric)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source on earth. It is well established that efficient degradation of cellulose requires the synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that display broad substrate specificity with respect to hydrolysis of cellobiose and different aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate- dependent phosphotransferase systems (PTS) have been reported in various bacteria, including: Bacillus species. In this study, we have used a cellobiose chromophore analog, p-nitrophenyl- ~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose utilization genes that are functionally expressed in Escherichia coli. Cloning and sequencing of the most active clone with subsequent sequence analysis allowed the identification of four adjacent open reading frames. An operon of four genes (celBACH), encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence data. The amino acid sequence of the celH gene displayed good homology with ~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene product belongs to family 1 of the glycosyl hydrolases, which employ a retaining mechanism of enzymatic bond hydrolysis. In vivo PTS activity assays concluded that the optimal temperature and pH at which the recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively. Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a significant problem. Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was only achievable in the presence ofO.01% glucose. This phenomenon could be explained by the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated transport. Glucose supplementation induced the glycolytic pathway and subsequently the availability of PEP. Furthermore, it could be concluded that the general PTS components . (enzyme I and HPr) of E. coli must have complemented the CelBACH system from B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host.
AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases (CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem (PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies, beskryf. In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH) van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word. Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose, is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n baie vinnige tempo benut. Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat (PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik 'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk. Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die celBACH operon in E. coli toelaat.

Dissertation (PhD)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The acidophilic, chemolithoautotrophic bacterium, Acidothiobaci/lus ferrooxidans is one of a consortium of bacteria involved in biornining, including the recovery of gold from arsenopyrite ores. The genes conferring arsenic resistance to At. ferrooxidans were cloned and sequenced and shown to be chromosomally located. Homologues to the arsB (membrane located arsenite efflux pump), arsC (arsenate reductase) and arsH (unknown function) genes from known arsenic resistance (ars) operons were identified. A fourth gene was found to have weak homology to the ArsR-family of regulators. The arsenic resistance genes of At. ferrooxidans are arranged in an unusual manner, with the arsRC and arsBH genes divergently transcribed. This divergent arrangement was found to be conserved in all four of the At. ferrooxidans strains we tested. All of the At. ferrooxidans ars genes were expressed in Escherichia coli and the arsB and arsC genes conferred arsenite (and antimonite) and arsenate resistance, respectively, to an E. coli ars mutant (AW311 0). Analysis of the putative amino acid sequences of these ars genes revealed that the ArsB from At. ferrooxidans is closely related to the ArsB proteins from other Gram-negative bacteria. However, the ArsC protein is more closely related to the ArsC proteins from Gram-positive bacteria. Furthermore, a functional thioredoxin (trxA) gene was required for ArsC-mediated arsenate resistance in E. coli. This suggests that reduction of arsenate by At. ferrooxidans has a similar reaction mechanism as that by Gram-positive ArsC proteins. While arsH was expressed in an E. coli-derived in vitro transcription-translation system, the presence of this gene was not required for, nor enhanced, arsenite or arsenate resistance in E. coli. We predict that the function provided by this gene is not required in E. coli. While the putative ArsR from At. ferrooxidans does contain a potential DNA-binding helix-turn-helix (HTH) domain, it does not contain the arsenite binding motif (ELCVCDL), required for response to the presence of inducer. Instead, the ArsR-like protein from At. ferrooxidans is related to a group of unstudied ArsR-like proteins that have been associated with other ars-like genes identified during genome sequencing projects. Using arsB-lacZ, arsC-lacZ, and arsR-lacZ fusions, it has been shown that this atypical ArsR protein from At. ferrooxidans did repress expression from the arsBH and arsRC promoters and that this repression was relieved by the presence of either arsenite or arsenate. Deletion of 19 amino acids from the C-terminus of the ArsR protein did not affect regulation, while deletion of a further 28 amino acids inactivated ArsR. Northern blot hybridization confirmed that expression of the arsRC and arsBH transcripts is increased in the presence of either arsenite or arsenate. This study is the first to show that the ars genes from the acidophilic biorning bacterium At. ferrooxidans are able to be studied in the neutrophilic bacterium, E. coli. We have also shown that the atypical ArsR found in this ars operon is able to regulate expression of these genes in response to arsenic, despite not containing the arsenite binding domain, suggesting that this protein senses arsenic by a different mechanism to that used by the ArsR family members already studied.
AFRIKAANSE OPSOMMING: Acidothiobacillus ferrooxidans, 'n asidofiliese, chemolitotrofiese bakterium, is een van 'n konsortium bakterieë betrokke by biologiese ontgunnig ("biomining") asook by die herwinning van goud uit arsenopiriet erts. Die gene wat aan At. ferrooxidans weerstandbiedendheid teen arseen verleen, is gekloneer. Die DNA-volgorde van hierdie gene is bepaal en daar is bewys dat die gene op die chromosoom geleë is. Homoloë van die arsB (membraan geleë pomp wat arseniet uitpomp), arsC (arsenaat reduktase) en die arsH (funksie onbekend) gene is in bekende arseenweerstanbiedheidsoperons (arsoperons) geïdentifiseer. Verder is daar 'n vierde geen geïdentifiseer wat lae homologie met die ArsR-familie van reguleerders toon. At. ferrooxidans se ars gene is op 'n ongewone manier gerangskik met twee van die gene, arsRC en arsBH wat lil teenoorgestelde rigtings getranskribeer word. Hierdie rangskikking van gene IS waargeneem in al vier die At. ferrooxidans rasse wat getoets is. Al die At. ferrooxidans ars gene is in Escherichia coli uitgedruk. Die arsB en arsC gene het aan 'n E. coli ars mutant (AW311 0) weerstandbiedendheid teen aseniet, antimoniet en arseen verleen. Analiese van die afgeleide aminosuurvolgorde van die ars proteïene het getoon dat die At. ferrooxidans ArsB naby verwant aan die ArsB-proteïene van ander Gram negatiewe bakterieë is. In teenstelling hiermee, is gevind dat die ArsC-proteïene nader verwant aan die ArsC-proteïene van Gram positiewe bakterieë is. Daar is ook gevind dat 'n funksionele tioredoksien (trxA) geen vir ArsC-bemiddelde arsenaat weerstandbiedendheid in E.coli benodig word. Dit dui daarop dat die meganisme van arsenaatreduksie deur At. ferrooxidans soortgelyk is aan die ArsC-proteïen-meganisme van Gram positiewe bakteriee. In vitro studies met behulp van 'n E. coli gebaseerde transkripsie-translasie sisteem het getoon dat arsH nie nodig is vir arsenaat of aseniet weerstanbiedendheid in sensitiewe E.coli rasse nie en ook nie help om weerstand in hierdie rasse te verhoog nie. Daarom kan daar aangeneem word dat die funskie van die arsH geen nie deur E. coli benodig word nie. Die vermeende ArsR van At. ferrooxidans bevat 'n potensiële DNA-binding heliks-draaiheliks motief, maar nie die arsiniet binding motief (ELCVCDL) wat nodig is vir reaksie in die teenwoordigheid van 'n induseerder nie. Die ArsA-proteïen van At. ferrooxidans is soortgelyk aan 'n groep ArsA-proteïene wat tydens genoom DNA- volgordebepalingsprojekte geïdentifiseer is. Hierdie groep gene is egter nog nie verder bestudeer nie. Deur gebruik te maak van 'n stel fusie gene, arsB-IacZ, arsC-IacZ en arsRlacZ kon daar bewys word dat die ongewone ArsH-proteïen van At. ferrooxidans uitdrukking van arsBH en arsRC onderdruk en dat die onderdrukking deur arseniet of arsenaat opgehef kan word. Delesie van die eerste 19 aminosure vanaf die C-terminus van die ArsA-proteïen het geen uitwerking op die regulering van die proteïen nie, maar delesie van 'n vedere 28 aminosure het ArsR geïnaktiveer. Verhoogde vlakke van transkripsie van arsRC en arsBH in die teenwoordigheid van arseniet en arsenaat is met behulp van Noordelike kladanalise bewys. Hierdie is die eerste studie waarin daar bewys word dat die ars gene van die asidofiliese bakterium Atferrooxidans in die neutrofiliese bacterium E. coli bestudeer kan word. Daar is ook bewys dat ten spyte daarvan dat die ArsR in die ars operon nie 'n arseniet bindingsdomein het nie, dit die uitdrukking van die gene in hierdie operon reguleer in reaksie op arseen. Dit dui dus daarop dat hierdie proteïen op arseen in die omgewing reageer met behulp van 'n meganisme wat verskil van die ArsR-proteïene wat tot dusver bestudeer is.

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As proteínas Vip3Aa e Cry1Ia apresentam potencial no controle de lepidópteros-praga e surgem como alternativa promissora no manejo da resistência de pragas as proteínas Cry1A, que tem sido altamente utilizada na formulação de bioinseticidas comerciais à base de Bacillus thuringiensis (Bt) e em plantas transgênicas. Assim sendo, o presente trabalho teve como objetivo a clonagem e expressão das proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 em Escherichia coli, a fim de se analisar a correlação entre a união aos receptores por meio de análises de competição entre as diferentes toxinas Vip3Aa e a toxina Cry1Ia10, e a toxicidade a lepidópteros-praga, inferindo-se quais as combinações que poderiam ser utilizadas na produção de plantas transgênicas, contendo múltiplos genes, as quais vêm sendo empregadas para contornar a evolução da resistência dos insetos às toxinas Bt. Para tanto, os genes vip3Aa e cry1Ia10 foram clonados no vetor pET SUMO, expressos em E. coli e a toxicidade das proteínas foram testadas em bioensaios com lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalis e Heliothis virescens. As BBMVs (Brush Border Membrane Vesicles) foram preparadas a partir dos intestinos das três espécies e ensaios de competição homóloga e heteróloga foram realizados. As proteínas Vip3Aa42 e Vip3Aa43 apresentaram toxicidade para S. frugiperda e A. gemmatalis. Já a proteína Cry1Ia10 apresentou toxicidade apenas para A. gemmatalis e, as proteínas não se mostraram tóxicas para H. virescens. Os ensaios de ligação às BBMVs demonstraram que as proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 se unem aos receptores presentes no intestino médio de forma efetiva nas três espécies e que, portanto, houve correlação entre a toxicidade e a união aos receptores para as populações de S. frugiperda e A. gemmatalis, porém para H. virescens não houve relação entre a toxicidade e a união aos receptores. Sendo assim ...
Vip3Aa and Cry1Ia proteins have potential in control of Lepidopteran pest and emerge as a promising alternative in the pest resistance management the Cry1A proteins, which has been highly used in the formulation of commercial insecticides based on Bacillus thuringiensis (Bt) and in transgenic plants. Therefore, this study aimed to cloning and expression of Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins in Escherichia coli, in order to analyze the correlation between the binding to receptors through competition assays between the different Vip3Aa toxins and toxin Cry1Ia10, and toxicity to lepidopteran pests inferring up which combinations that could be used to produce transgenic plants containing multiple genes, which have been used to circumvent the development of insects resistance to Bt toxins. Therefore, vip3Aa and cry1Ia10 genes were cloned into the pET SUMO vector, expressed in E. coli and toxicity of proteins were tested in bioassays with neonate larvae of Spodoptera frugiperda, Anticarsia gemmatalis and Heliothis virescens. The BBMVs (Brush Border Membrane Vesicles) were prepared from the gut of the three species, and homologous and heterologous competition assays were performed. The Vip3Aa42 and Vip3Aa43 proteins were toxic to S. frugiperda and A. gemmatalis. Already Cry1Ia10 protein showed toxicity only for A. gemmatalis and proteins were not toxic to H. virescens. Binding assays to BBMVs demonstrated that Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins binding effectively to receptors present in the midgut in three species and, therefore, was correlation between toxicity and the binding to receptors for the populations of S. frugiperda and A. gemmatalis, but for H. virescens there was no relationship between toxicity and the binding to receptors. Thus, the combination of Cry1Ia10 and Vip3Aa42 or Vip3Aa43 proteins is indicated for the production of biological insecticidal, as well as for the production of transgenic plants to circumvent ...

The physiological role of the teichoic acid polymers found in Gram-positive bacterial cell walls is not known. Studies of Bacillus subtilis hybrid strains implicate a defined chromosomal region, which includes the tag-1 locus, as necessary for teichoic acid biosynthesis. A set of ten mutants carrying lesions in this region was identified from among forty-four temperature-sensitive (ts) mutants generated by nitrosoguanidine mutagenesis and bacteriophage 029 selection. This protocol gave a population enriched for ts, versus auxotrophic, mutants. For each of the ten mutants, the frequency of genetic reconstruction, or correction, of the ts phenotype indicated that it was due to change(s) in a single gene. Results of two-factor transformation crosses sorted the mutants into three complementation groups; all ten could complement tag-1. Mutants in two complementation groups were transformed to ts⁺ with cloned rodC DNA. The map order of the newly isolated ts markers was determined from the results of two factor crosses. Orientation with respect to the hisA marker was inferred from transduction experiments. The newly isolated strains were shown to be conditional rod⁻ mutants. Growth at 48°C resulted in reduced growth rates and spherically shaped cells. Additional phenotypes seen for some mutants, namely 029 phage resistance and ts spore outgrowth, appeared closely associated with the ts rod⁻ mutation. Wall phosphate content for two of the mutants, following growth at 48°C, was found to be reduced in comparison to the wild-type control. Taken together these results lend support to the argument that the tag-1 region of the chromosome, which most likely directs teichoic acid biosynthesis, is important for establishment and maintenance of the normal bacillary morphology seen for B. subtilis. The importance of other gene products to the organization of newly synthesized wall was examined using B. subtilis macrofibers. Left- and right-handed macrofibers were converted to spheroplasts and the multi-celled structures regenerated under the two sets of conditions conducive for production of the original, and inverse hand. The helix hands observed for the regenerated structures always corresponded to those expected on the basis of the parental genotype.

Bacteriophages (phages) are the smallest biological entity on the planet. They provide vast amounts of valuable knowledge to biologists. Phage genomes are relatively simple compared to the organisms they infect (prokaryotes) and yet continually point to the complexity surrounding molecular- and microbiological mechanisms of life. By studying phages we can learn of the systems of gene expression, protein interaction and DNA organization. Phages are useful not only from an academic perspective, but may also have useful clinical applications. In the face of the rise of antibiotic-resistant bacterial “super pathogens”, scientists and researchers turn to phages as alternative treatments to these types of infections. Phages are capable of infecting and killing even the deadliest of bacterial pathogens, such as carbapenem-resistant Enterobacteriaceae (CRE) or Bacillus anthracis, and may prove increasingly useful in the future for combatting harmful pathogens. This thesis looks at several aspects of phage biology—from the underlying genetics contributing to phage virulence, to the clinical application of phage therapy to treat infections. First, a look at CRE-Klebsiella pneumoniae isolates and phages capable of infecting some strains may reveal a potential therapeutic approach in the future. Additionally, genomic analysis reveals interesting features that may explain aspects of phage virulence and evolutionary history. Then, a collection of genetically diverse phages is used in infection assays on pathogenic strains of Bacillus anthracis to establish the first-reported phages capable of infecting these strains. Finally, the process of preparing phage samples for therapeutic application is explored in-depth to conclude with discussion of clinical application. During the course of these projects over 25 phages were isolated, as many phage genomes were assembled and annotated, resulting in the preparation of two genome announcements and near-completion of two publishable first-author papers (chapters II and III). In addition, participation in a variety of collaborative efforts may lead to a handful of co-author papers and on various topics, including phage biology and application.

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O presente trabalho objetivou avaliar a toxicidade de diferentes proteínas Cry1 e Vip3Aa de Bacillus thuringiensis em larvas de Diatraea saccharalis, verificar o modo de união dessas proteínas aos receptores do inseto alvo e analisar a interação dessas proteínas no controle larval, buscando informações para subsidiar o uso de plantas geneticamente modificadas com genes cry1 e vip3Aa de forma segura e duradoura. A análise de suscetibilidade larval revelou a proteína Cry1Ab como mais efetiva no controle, seguida das proteínas Cry1Ac, Vip3Aa, Cry1Ca e Cry1Fa. A população testada não foi suscetível à proteína Cry1Ea. A proteína Cry1Aa apresentou baixa toxicidade. Nos ensaios de união específica, as proteínas Cry1 ligaram-se a receptores presentes no intestino médio de D. saccharalis e um modelo com três diferentes receptores foi proposto com base nos ensaios de competição heteróloga. Um receptor comum para Cry1Aa, Cry1Ab e Cry1Ac, outro para Cry1Fa e Cry1Ab e um receptor diferente para a proteína Vip3Aa. Dentre as interações avaliadas por bioensaios, as combinações proteicas: Cry1Ab:Cry1Ca e Cry1Fa:Cry1Ca apresentaram efeito sinérgico; as demais combinações revelaram efeitos antagônicos
The aim of this research was to evaluate the toxicity of different Cry1 and Vip3A proteins from Bacillus thuringiensis to Diatraea saccharalis, verify the proteins binding to receptors of the target insect and to analyze the protein interactions in larval control, seeking information to support safe and sustainable the use of transgenic Bt plants. The most toxic protein assayed was Cry1Ab followed by Cry1Ac, Vip3Aa, Cry1Ca and Cry1Fa. The population tested was not susceptible to Cry1Ea protein. Cry1Aa showed very low toxicity. Biotinylated Cry1 proteins showed specific binding to the midgut brush border membrane vesicles of the larvae. Heterologous competitive binding assays suggested a model of three receptors, a common receptor for Cry1Aa and Cry1Ab another one for Cry1Fa and Cry1Ab. Vip3Aa did not compete for binding with any of the Cry proteins tested. Among interactions bioassays, the combinations between Cry1Ab:Cry1Ca and Cry1Fa:Cry1Ca showed synergistic effect, whereas the other combinations showed antagonistic effects

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2013.
Cataloged from PDF version of thesis. "September 2013."
Includes bibliographical references (pages 54-60).
DnaA is the bacterial replication initiator, which also functions as a transcription factor to regulate gene expression. In B. subtilis, DnaA has previously been shown to repress its own transcription and has also been implicated in directing part of the transcriptional response to replication stress. Because dnaA is essential, most of DnaA's potential effects on gene expression have been determined through indirect methods, which have implemented perturbations in replication and sequence analyses to predict direct effects of DnaA transcriptional regulation. Below, I take a more direct approach to assay DnaA's effect on gene expression and specific transcriptional regulatory networks by deleting dnaA in an oriN+ [delta]oriC strain background, which renders dnaA non-essential. Isogenic dnaA+ cells were constructed similarly and have dnaA constitutively expressed from an ectopic locus. In this background, DNA replication no longer depends on dnaA and is initiated instead by a plasmid replicon, oriN. The native origin of replication, oriC, is also deleted to eliminate differences in replication between [delta]dnaA and dnaA+ cells. Consequently, I can directly compare differences in gene expression due to the presence versus absence of dnaA. Deletion of dnaA results in approximately 463 significant differences in gene expression, most of which I show are due to DnaA direct activation of the gene sda. Many of these genes lie downstream of Sda activity and comprise several regulons, such as the Spo0A, AbrB, and SinR regulons. These regulons are known to become active during the transition from exponential growth to stationary phase. In addition to the many effects on gene expression, I show that deletion of dnaA results in lowered competence development. I also revisit the transcriptional response to replication stress and show that some of the previously predicted targets of DnaA respond to replication stress in a DnaA-dependent manner. Lastly, in collaboration with others, I have studied the relationship between a DnaA regulator, YabA and a nucleoid binding protein Rok. YabA and Rok associate at some of the same chromosomal regions, and at these regions YabA absolutely depends on Rok for its association. We are currently trying to understand the functional relationship between YabA, Rok, and DnaA.
by Tracy Washington.
Ph.D.

6-(p-Hydroxyphenylhydrazino) uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific DNA polymerase III (pol III) of Gram+ bacteria such as Bacillus subtilis. Although a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. The project described in this thesis dissertation involves the use of H2-HPUra-like inhibitors to probe the structure and function of the pol III active site. It consists of two separate problems which are summarized below. Production of a potent bona fide dGTP form of inhibitor. A method was devised to successfully convert the H2-HPUra inhibitor prototype to a bona fide purine, using N2-benzyl guanine as the basis. Structure-activity relationships of benzyl guanines carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl) guanine (DCBG) as a compound equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization. The latter experiment identified the site for the binding of the inhibitor's aryl moiety as a distinct site located at a distance of approximately 6-7 Å from the base-paired 2-NH group of a bound dGTP. Attempt to covalently label amino acid residue 1175, a putative participant in inhibitor binding. Azp-12, a point mutation of serine 1175, yields a form of pol III whose inhibitior sensitivity varies specifically as a function of the composition of the para substituent of the inhibitor's aryl ring. On the basis of the latter behavior, residue 1175 was hypothesized to be a residue directly involved in the binding of the inhibitor's aryl moiety. To test this hypothesis, residue 1175 was specifically mutated to either cysteine or lysine, each of which presents a side chain amenable to covalent bond formation with appropriately reactive inhibitor forms. Of the two mutant pol III forms, only the cysteine form (pol III-cys) was catalytically active. The kinetic properties and inhibitor sensitivity profile of pol III-cys identified it as a target suitable for potentially irreversible inhibitor forms containing the following groups in the meta position of the aryl ring: -CH2Br, -CH2C1, and -CH2SH. None of the several inhibitors tested selectively or irreversibly inactivated pol III-cys. Possible bases for the failure of this group of inhibitors and for the redesign of more useful covalently reactive inhibitor forms are considered.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xv, 139 p.; also includes graphics (some col.) Includes bibliographical references (p. 123-139).

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A caracterização molecular de 46 isolados de Bacillus thuringiensis foi realizada por meio da técnica de PCR com os oligonucleotídeos iniciadores gerais Gral-cry8(d)/Gral-cry8(r), resultando na identificação do isolado BT_I622 como portador do gene cry8 (coleóptero-específico). O fragmento obtido de aproximadamente 376 pares de base (pb), localizado na porção inicial do gene, foi clonado em vetor pGEM®-T Easy, sequenciado e analisado por bioinformática. A determinação da sequência completa do gene cry8 presente no isolado BT_I622, foi realizada pela estratégia de “primer walking” com a utilização de oligonucleotídeos específicos. Os iniciadores foram elaborados através do programa Primer3, com base nas sequências de genes cry8 depositadas no banco de dados público GenBank (NCBI) e, por meio da análise das sequências geradas com o par de oligonucleotídeos gerais utilizados inicialmente na caracterização dos isolados. As amplificações com diferentes combinações de iniciadores resultaram na obtenção de fragmentos em torno de 1420, 3302, 628, 1580, 508 e 510 pb, os quais foram clonados, sequenciados e analisados pelos programas Phred/Phrap/Consed visando a montagem do gene. A sequência contendo 3531 pb foi comparada ao GenBank pela ferramenta BLASTn, apresentando 98% de similaridade com o gene cry8Ka2. Paralelamente, a sequência de aminoácidos deduzida (1176 aa) revelou 75% de identidade com a proteína Cry8Ba1 na análise com o algoritmo BLASTp, demonstrando que o isolado BT_I622 é portador de um novo gene cry8. Os bioensaios indicaram que as larvas de Sphenophorus levis são sensíveis a nova proteína Cry8 de B. thuringiensis
The molecular characterization of 46 Bacillus thuringiensis isolates was carried out using the PCR technique with primers designed to detect cry8 genes (Gral-cry8(d)/Gral-cry8(r)), resulting in the identification of the isolate BT_1622 as a cry8 gene carrier (coleopteran specific). The ca. 376 base pairs (bp) fragment obtained was located in the initial part of the gene, which was cloned into the plasmid pGEM®-T Easy, sequenced and analyzed by bioinformatics. The complete sequence determination of this gene found within isolate BT_1622 was done using the “primer walking” strategy. The primers were designed using the software Primer3, comparing to previously sequenced cry8 genes found in the public database GenBank (NCBI) and analyzing the sequences generated with the pair of general primers used initially in the characterization of isolates. The amplifications with different primer combinations resulted other fragments of this gene with 1,420; 3,302; 628; 1,580; 508 and 510 bp, which were cloned, sequenced and analyzed using the software Phred/Phrap/Consed to obtain the full gene. The 3,531 bp sequence was compared to the GenBank using the BLASTn tool, and showed 98% of similarity with the gene cry8Ka2. Also, the deduced amino acid sequence (1,176 amino acids) showed 75% identity to the Cry8Ba1 using the algorithm BLASTp, demonstrating that the isolate BT_1622 carriers a new cry8 gene. The bioassay results indicated that the Sphenophorus levis larvae are sensitive to the new Cry8 protein of B. thuringiensis

Orientador: Manoel Victor Franco Lemos
Coorientador: Janete Apparecida Desidério
Banca: Ana Maria Guidelli Thuler
Banca: Fernando Hercos Valicente
Banca: Lucia Maria Carareto Alves
Banca: Odair Aparecido Fernandes
Resumo: A caracterização molecular de 46 isolados de Bacillus thuringiensis foi realizada por meio da técnica de PCR com os oligonucleotídeos iniciadores gerais Gral-cry8(d)/Gral-cry8(r), resultando na identificação do isolado BT_I622 como portador do gene cry8 (coleóptero-específico). O fragmento obtido de aproximadamente 376 pares de base (pb), localizado na porção inicial do gene, foi clonado em vetor pGEM®-T Easy, sequenciado e analisado por bioinformática. A determinação da sequência completa do gene cry8 presente no isolado BT_I622, foi realizada pela estratégia de "primer walking" com a utilização de oligonucleotídeos específicos. Os iniciadores foram elaborados através do programa Primer3, com base nas sequências de genes cry8 depositadas no banco de dados público GenBank (NCBI) e, por meio da análise das sequências geradas com o par de oligonucleotídeos gerais utilizados inicialmente na caracterização dos isolados. As amplificações com diferentes combinações de iniciadores resultaram na obtenção de fragmentos em torno de 1420, 3302, 628, 1580, 508 e 510 pb, os quais foram clonados, sequenciados e analisados pelos programas Phred/Phrap/Consed visando a montagem do gene. A sequência contendo 3531 pb foi comparada ao GenBank pela ferramenta BLASTn, apresentando 98% de similaridade com o gene cry8Ka2. Paralelamente, a sequência de aminoácidos deduzida (1176 aa) revelou 75% de identidade com a proteína Cry8Ba1 na análise com o algoritmo BLASTp, demonstrando que o isolado BT_I622 é portador de um novo gene cry8. Os bioensaios indicaram que as larvas de Sphenophorus levis são sensíveis a nova proteína Cry8 de B. thuringiensis
Abstract: The molecular characterization of 46 Bacillus thuringiensis isolates was carried out using the PCR technique with primers designed to detect cry8 genes (Gral-cry8(d)/Gral-cry8(r)), resulting in the identification of the isolate BT_1622 as a cry8 gene carrier (coleopteran specific). The ca. 376 base pairs (bp) fragment obtained was located in the initial part of the gene, which was cloned into the plasmid pGEM®-T Easy, sequenced and analyzed by bioinformatics. The complete sequence determination of this gene found within isolate BT_1622 was done using the "primer walking" strategy. The primers were designed using the software Primer3, comparing to previously sequenced cry8 genes found in the public database GenBank (NCBI) and analyzing the sequences generated with the pair of general primers used initially in the characterization of isolates. The amplifications with different primer combinations resulted other fragments of this gene with 1,420; 3,302; 628; 1,580; 508 and 510 bp, which were cloned, sequenced and analyzed using the software Phred/Phrap/Consed to obtain the full gene. The 3,531 bp sequence was compared to the GenBank using the BLASTn tool, and showed 98% of similarity with the gene cry8Ka2. Also, the deduced amino acid sequence (1,176 amino acids) showed 75% identity to the Cry8Ba1 using the algorithm BLASTp, demonstrating that the isolate BT_1622 carriers a new cry8 gene. The bioassay results indicated that the Sphenophorus levis larvae are sensitive to the new Cry8 protein of B. thuringiensis
Doutor

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvii, 176 p.; also includes graphics (some col.) Includes bibliographical references (p. 158-176). Available online via OhioLINK's ETD Center

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers.
Department of Biology

Over the past few years, the use of enzymes as catalysts for the preparation of novel organic molecules has received a steadily increasing amount of attention. Lipolytic enzymes are widely distributed in nature and attract great attention because of their biotechnological potential, as they catalyse the enantio- and regioselective hydrolysis and synthesis of a broad range of natural and non-natural esters. Bacteria produce different lipolytic enzymes, such as esterases (EC 3.1.1.1), which hydrolyse ester-containing molecules at least partly soluble in water, and lipases (EC 3.1.1.3), which hydrolyse water-insoluble long-chain triglycerides. In this study, a bacterial isolate, B. coagulans strain 81-11, isolated from popcorn seeds, was characterized with the specific aim of isolating and characterizing genes encoding novel lipolytic enzymes. A genomic library of B. coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4-kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723-bp open reading frame (ORF), designated estCl, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estCl gene exhibited significant amino acid sequence identity with carboxyl esterases and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrate confirmed the anticipated esterase activity. EstCl exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme was active in the pH range 7-9 and displayed activity at temperatures up to 55°C. Since bacterial esterases are potentially important for a variety of biotechnological applications, there is a considerable industrial interest to produce these enzymes at a larger scale. Among the many systems that are available for heterologous protein production, attempts were made to over express the newly identified B. coagulans estCI esterase¬encoding gene in different Gram-positive bacteria, as they are well known for their important contribution to food biotechnology and as production organisms for industrial enzymes. A recombinant expression vector was thus constructed (pMG36-EstCl) and introduced in Lactococcus lactis, Lactobacillus plantarum and Bacillus subtilis strains 154 and lA297. Of these different bacterial hosts, high levels of intracellular esterase activity were detected in B. subtilis lA297 only. In an attempt to increase extracellular expression of the B. coagulans EstCl esterase, a recombinant secretion plasmid (pNW-EstClaps) was constructed that contained an alkaline protease promoter and signal sequence from a Bacillus species. Following introduction of the construct in B. subtilis lA297, the derived recombinant strain displayed 2.3-fold higher extracellular esterase-activity levels than the parent B. coagulans strain, and the extracellular esterase activity represented 82% of the total esterase activity.
Dissertation (MSc (Microbiology))--University of Pretoria, 2006.
Microbiology and Plant Pathology
unrestricted

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