The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. Most of these metabolites are small peptides that have unusual components and chemical bonds and synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is one of the simplest peptide antibiotics known. It is a dipeptide with an N-terminal L-alanine and an unusual amino acid, L-anticapsin, at its C-terminal. Recently, ywfBCDEF operon of B. subtilis 168 was shown to carry bacilysin biosynthesis function, the genes of this operon were renamed as bacABCDE. The first member of bac operon, bacA gene was proved to encode the function of L-alanine &ndash / L-anticapsin amino acid ligation. Bacilysin production is regulated at different levels, negatively by GTP via the transcriptional regulator CodY and AbrB while positive regulation occurs by guanosine 5

Nous avons étudié une enzyme de Bacillus subtilis, YdiB, de fonction inconnue. Le gène, spécifiquement procaryote et décrit comme essentiel chez plusieurs espèces, fait de YdiB une cible de choix pour une future recherche d'antibiotiques. <br />Nous avons construit une souche délétée de ydiB dont la croissance est fortement réduite. Une faible activité ATPase a été mesurée, néanmoins spécifique de YdiB puisque la mutation d'un résidu conservé dans son site actif abolit quasiment toute l'activité. Différentes techniques ont révélé que YdiB était capable de former des oligomères, et des résultats similaires ont été observés pour YjeE, son homologue chez E. coli. L'addition de sels favorise le déplacement de l'équilibre vers le monomère, qui est plus actif que les multimères. Les formes dimériques ont été détectées par « cross-link » in vivo. Enfin, une recherche des partenaires cellulaires de YdiB suggère un rôle de la protéine lors d'une réponse à un stress, ou une interaction avec le ribosome.

A total of twenty-eight Bacillus spp. isolated from value-added surimi nuggets and their raw ingredients, were tested against each other and selected reference strains of Bacillus and Clostridium for their production of inhibitory substances using the deferred antagonism assay plating method. The isolated Bacillus strains showed inhibitory activity against all Bacillus strains, with the exception of the producer strain, as well as being effective against various strains of C. botulinum (type A, B and E). Subsequent studies showed that the inhibitory activity was detected in the culture supernatant in the late stationary phase of growth prior to sporulation. The inhibitory activity of two Bacillus strains (FN2A and FN33) were selected for further study. The inhibitory substances produced by these two strains were proteinaceous in nature, heat stable (100°C for 15min) and unaffected by organic solvents. A comprehensive study was conducted on the structural characterization of the inhibitor produced by B. subtilis FN2A using FPLC, FTIR, MS and MS/MS. Structural analysis of the inhibitor produced by B. subtilis FN2A showed that it was similar in structure to Surfactin. / Preliminary studies have shown that the Surfactin-like-compound from B. subtilis FN2A was produced in significant amounts during growth in bread with maximum production occurring in the late stationary phase (72h), at 30--35°C and at pH 6.5--7.0. Optimization studies on the production of the Surfactin-like-compound by B. subtilis FN2A in bread using a response surface methodology approach showed that temperature (33--36°C); autoclaving time (30 min); inoculum level (4%), alkali pre-treatment (0.16%), water activity (0.995) and pH 6.66 enhanced the production of the Surfactin-like-compound in bread. The compound produced under these optimal conditions also maintained its activity when subjected to various processing treatments (autoclaving, freezing and freeze drying). / Initial studies showed that low levels (1% w/w) of the Surfactin-like-compound inhibited the growth of B. cereus and proteolytic and non-proteolytic strains of C. botulinum in a model agar system. However, it had no effect on non-proteolytic strains of C. botulinum when bread, or methanol extracts of bread (1--20%), were added to formulated value-added sterile trout nuggets, with all nuggets being toxic after 28 days at 12°C. Furthermore, inoculation of B. subtilis FN2A directly into nuggets also failed to inhibit growth of non-proteolytic strains of C. botulinum. Omitting certain ingredients in the formulation failed to enhance the anti-botulinal effect of the bread or methanol extracts of the Surfactin-like-compound in the value-added nuggets. However, reducing the pH of the nuggets to ~5.5 enhanced the anti-botulinal effect of the Surfactin-like-compound. Further research is required to improve the dispersibility of the Surfactin-like-compound to inhibit the growth of C. botulinum in food systems.

Currently, most of the RNAs used in lab research are prepared by in vitro transcription or chemical synthesis, which can be costly. In vivo expression in bacterial cells is another approach to RNA preparation that allows large scale production at a lower cost. However, there are some obstacles in bacterial expression, including RNA degradation in host cell, as well as RNA extraction and purification. tRNA and 5S RNA have been reported as scaffolds to circumvent the degradation problem. These scaffolds can not only make the RNA product survive in the cell but also increase the stability after extraction. The packaging RNA (pRNA) of bacteriophage phi29 is a small non-coding RNA with a compact structure. The three-way junction (3WJ) region from pRNA is a thermodynamically stable RNA motif good for constructing therapeutic RNA nanoparticles. The 3WJ can not only integrate multiple RNA modules, but also stabilize them. Here I report a series of approaches made to express recombinant RNAs based on pRNA or 3WJ in bacteria, including 1) Investigating the mechanism of RNA folding in vitro and in vivo using 3WJ. 3WJ-based RNAs were expressed in E. coli using pET system. The results show that the folding of RNA is affected by both overall and regional energy landscape. 2) Expression of an RNA nanoparticle harboring multiple functional modules, a model of therapeutic RNA, in E. coli using a combination of tRNA scaffold and pRNA-3WJ. The expression was successful and all of the RNA modules were functional. 3) Expression of pRNA-based recombinant RNAs in B. subtilis. This is a novel system of expressing recombinant RNAs in Gram-positive bacteria.

Multi-drug resistance among bacterial pathogens can be mediated by a number of mechanisms, including multidrug efflux pumps. One such pump in Bacillus spp. is ykkCD, a heterodimer of the SMR family consisting of C and D subunits. Previous studies suggest that the expression of ykkCD is controlled by a putative riboswitch and that the antibiotic tetracycline binds to the riboswitch in vitro. Additional studies have shown that two derivatives of tetracycline also bind to the putative riboswitch. These findings now need to be validated by an in vivo study. In this study, the effects that tetracycline and its commercially available derivatives—doxycycline, minocycline, anhydrotetracycline, and oxytetracycline—have on the expression levels of the ykkCD gene in Bacillus subtilis were explored. The level of ykkCD expression was quantified using two different methods: (1) ykkCD protein levels was determined using a ykkCD RNA--galactosidase reporter gene construct and (2) ykkCD mRNA levels was quantified by quantitative RT-PCR. Although the findings from method (1) were inconclusive, upregulation was observed for tetracycline and minocycline, in agreement with the results of the previous binding studies. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry. Title from pdf file main screen (viewed on November 8, 2009). Includes bibliographical references.

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).

Thesis (M.S.)--Brigham Young University. Dept. of Microbiology and Molecular Biology, 2007. / Includes bibliographical references (p. 29-31).

RIBEIRO, Eliana Matos.Isolamento e caracterização parcial dos Genes beta-actina e miosina de cadeia pesada do Camarão rosa Farfantepenaeus subtilis. 2009. 107f. Dissertação(Mestrado em Engenharia de Pesca) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2009. / Submitted by Maria Naires Souza (marianaires@ufc.br) on 2011-12-02T23:19:34Z No. of bitstreams: 1 2009-dis-emribeiro.pdf: 1933705 bytes, checksum: a3df6513724ba614bd32cf3e837d1d16 (MD5) / Approved for entry into archive by Aline Nascimento(vieiraaline@yahoo.com.br) on 2011-12-08T12:07:33Z (GMT) No. of bitstreams: 1 2009-dis-emribeiro.pdf: 1933705 bytes, checksum: a3df6513724ba614bd32cf3e837d1d16 (MD5) / Made available in DSpace on 2011-12-08T12:07:33Z (GMT). No. of bitstreams: 1 2009-dis-emribeiro.pdf: 1933705 bytes, checksum: a3df6513724ba614bd32cf3e837d1d16 (MD5) Previous issue date: 2009-03-04 / The penaeid shrimp Farfantepenaeus subtilis is an important native species for fisheries industry in Brazil. Among marine shrimps of commercial importance, penaeids are recognized as a valuable resource for fishery and aquaculture in tropical and subtropical regions. However, data on these species is extremely reduced, especially concerning genetic elements involved in animal muscle growth. Therefore, aiming at identifying shrimp genes directly associated with muscle contraction in this research, beta-actin and myosin heavy chain genes of the pink shrimp F. subtilis were isolated from its muscular abdominal and partially sequenced. Shrimps collected from Pacoti estuary, Ceará, were first identified through taxonomy and, then, through DNA amplification followed by sequencing of Cytochrome Oxidase subunit I (COI) and 16S. From fresh shrimp tissues, total RNA was extracted and complementary cDNA was obtained. Based on specific primers designed after sequence alignments performed against sequences at GenBank/NCBI, genes were amplified from RT-PCR (reverse transcriptase - polimerase chain reaction) and sequenced. A 760bp partial F. subtilis beta-actin cDNA fragment was obtained, while the partial F. subtilis myosin heavy chain cDNA was 570bp long. Sequence analyses using the Basic Local Alignment Search Tool (BLAST) program indicated that F. subtilis beta-actin gene product is very similar to betaactin of other species of shrimps, while the myosin heavy chain protein is highly homologous to crustacean myosins heavy chain, confirming the identity of the isolated gene sequences. Alignment of these gene sequences with other sequences in GenBank showed high similarity with Penaeus monodon (93%) and Farfantepenaeus paulensis (88%). Results have showed the feasibility of partial gene identification as a means to identify genes of strategic interest. These data would help further attempts to elucidate the complete isolation of these genes, as well as the detection of other important genes, especially from shrimp species occurring at the Brazilian coast. Genes analyses involved with muscle growth might provide important genetic information on native species that are overexploited and may be viable for the shrimp cultivation. In addition, these data might also benefit the scientific community, improving a range of research areas such as physiology, phylogeny and evolution of penaeids / O camarão peneídeo Farfantepenaeus subtilis é uma importante espécie nativa do litoral nordestino que possui uma grande ocorrência na pesca. Dentre os camarões marinhos de importância comercial, os peneídeos se destacam por constituírem um valioso recurso para pesca e aqüicultura em regiões tropicais e subtropicais. Entretanto, a disponibilidade de informações sobre essas espécies é bastante escassa, principalmente em relação à estrutura genética que atua no crescimento muscular desses animais. Tendo como objetivo identificar genes envolvidos na contração muscular de camarões, neste trabalho foram parcialmente isolados e seqüenciados os genes de betaactina e miosina de cadeia pesada do camarão rosa F. subtilis, a partir do cDNA do músculo abdominal. Para tanto, camarões coletados no estuário do rio Pacoti, estado do Ceará, foram inicialmente identificados taxonomicamente e, depois através de amplificação de DNA seguida por sequenciamento das regiões citocromo oxidase subunidade I (COI) e 16S. Utilizando-se os tecidos frescos dos camarões, foi extraído o RNA total e foram obtidos os respectivos DNAs complementares (cDNAs). Baseado na construção de primers específicos a partir do alinhamento entre sequências descritas no Genbank/NCBI, os genes foram isolados por meio de RT-PCR (Reação em Cadeia da Polimerase através da transcriptase reversa) e seqüenciados. Foi obtido um fragmento parcial de 760 pares de base para o cDNA de beta-actina e para o cDNA de miosina de cadeia pesada foi obtido um fragmento de 570 pares de base. Análises das sequências realizadas pela ferramenta BLAST (Basic Local Alignment Search Tool) revelaram alta similaridade com outras beta-actinas e miosinas de camarões, confirmando a identidade das sequências genéticas isoladas. Como resultado do alinhamento pareado entre as sequências desses genes obtidos no trabalho com as de outras espécies presentes no GenBank, pôde-se observar que as maiores similaridades foram com Penaeus monodon (93%) e com Farfantepenaeus paulensis (88%). Os resultados obtidos neste estudo demonstraram a viabilidade da metodologia utilizada na identificação de genes relacionados com características importantes. Esses dados irão facilitar o isolamento completo das sequências desses genes, além de contribuir para incentivar a identificação de outros genes importantes em camarões, principalmente os nativos do Brasil. Análise de genes que atuam desenvolvimento do tecido muscular do animal poderá fornecer informações genéticas importantes acerca de uma espécie nativa que está sendo superexplorada e que poderá ser viável para cultivo. Outrossim, esses dados beneficiarão a comunidade científica, servindo como base para estudos de fisiologia, filogenia e evolução em peneídeos