DivIVA is a Gram-positive cell division protein involved in chromosome segregation, midcell placement of the cell division machinery, complete septum closure, and polar growth and morphogenesis. Although well conserved across various Gram-positive species, DivIVA is believed to be relatively species specific. One similarity among DivIVA homologues is the ability to oligomerize through coiled-coil interaction into complexes comprising 10-12 monomers. To date, the importance of DivIVA oligomerization and the N-terminal coiled-coil for its proper function in bacterial cell division has not been reported. This study examined the biological significance of DivIVA oligomerization and the N-terminal coiled-coil in bacterial cell division. This research provides evidence that the N-terminal coiled-coil and oligomerization is essential for the proper biological function of DivIVA_Ef in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i>_Ef known to disrupt either the N-terminal coiled-coil or the two central coiled-coils, involved in oligomerization, were found to be lethal unless rescued by <i>in trans</i> expression of wild type DivIVA_Ef. Using this rescue method, the N-terminal <i>divIVA</i>_Ef mutant strain, <i>E. faecalis</i> MWMR5, and the mutant strain with partial disruption of oligomerization, <i>E. faecalis</i> MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of <i>divIVA</i>_Ef mutant strains <i>E. faecalis</i> MWMR5 and MWMR10. Both these strains showed asymmetrical division, loss of normal lancet shape, and irregular chains. Full disruption of oligomerization with point mutations in both central coiled-coils resulted in a dominant lethal phenotype. These results demonstrate the essentiality of the N-terminal coiled-coil and oligomerization of DivIVA_Ef for its proper biological function in <i>E. faecalis</i> cell division.<p> Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVA_Ef interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVA_Ef and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The <i>in vitro</i> and <i>in vivo</i> results thus confirm interaction between DivIVA_Ef and MLJD1.<p> Another objective of this study was to determine the localization of DivIVA and MLJD1 in <i>E. faecalis</i>. Localization of DivIVA_Ef in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA_Ef was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA_Ef migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA_Ef in <i>E. faecalis</i>, further implicating MLJD1 as a bacterial cell division protein.<p> Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVA_Ef and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA_Ef is involved in chromosome replication. This model is similar to the role of RacA in sporulation in <i>B. subtilis</i> where RacA directs the chromosome during sporulation through direct interaction with DivIVA_Bs and Spo0J.<p> This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.

Metabolic were used to evaluate Enterococcus as an indicator of faecal pollution. Enterococci were isolated using m-Enterococcus agar and speciated using conventional biochemical tests. Forty percent of the isolates were identified and metabolically characterized by the automated Biolog system. The biochemical test scheme recognized 16 enterococcal species, while Biolog recognized nine. Both methods identified E. faecalis at the greatest frequency. Overall species frequencies varied between the two methods. Biolog was unable to identify 31% of the isolates; 7% of the isolates were unidentified by the biochemical test scheme. Of the identified isolates, metabolic profiling with Biolog achieved speciation with 60 substrates. Unique profiles were obtained for 89% of the isolates. Isolates also demonstrated inter-trial differntial metabolism of substrates. This and the large number of unidentified isolates suggest great diversity among enterococci. Diversity and inter-trial metabolic inconsistencies will complicate use of enterococcal metabolic profiles as a source-tracking tool. / xxiii, 264 leaves ; 29 cm.

Orientador: Carla Raquel Fontana / Banca: Maurício da Silva Baptista / Banca: Ana Claudia Pavarina / Banca: Eduardo Maffud Cilli / Banca: Denise Maria Zezell / Resumo: Nos últimos anos, a Organização Mundial da Saúde vem alertando que a era pós-antibióticos é uma ameaça cada vez mais real. Além da resistência individual das células bacterianas, essas podem se tornar ainda mais tolerantes aos agentes antimicrobianos ao crescerem em biofilmes. A maior tolerância aos antibióticos observada nos biofilmes baseia-se principalmente na proteção das bactérias pela matriz polimérica extracelular autoproduzida, e nos diversos fenótipos de crescimento encontrados na estrutura, que podem ser refratários à ação os fármacos convencionais. Nesse cenário de crescente e disseminada resistência a antimicrobianos, a busca por novos fármacos e terapias alternativas se tornou crucial, especialmente aqueles que sejam capazes de eliminar os microrganismos resistentes, impedir o desenvolvimento de novas formas de resistência, e serem ativos contra biofilmes. A terapia fotodinâmica antimicrobiana (aPDT, do inglês antimicrobial photodynamic therapy) e os peptídeos antimicrobianos (PAM) ganham destaque nesse contexto, em especial para o tratamento de infecções localizadas. Entretanto, ambas as abordagens apresentam limitações terapêuticas quando empregadas individualmente. Dessa forma, este trabalho teve por objetivo estudar os efeitos e mecanismos da combinação dos PAMs aureína 1.2 (AU) e seu dímero (AU)2K com a aPDT mediada pelos fotossensibilizadores (FS) azul de metileno (AM), clorina-e6 (Ce6) ou curcumina (CUR), usando como modelo a bactéria Enterococcus faecalis... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the past few years, the World Health Organization has been warning that the post-antibiotic era is an increasingly real threat. In addition to the individual resistance of bacterial cells, they may be even more tolerant to antimicrobial agents by growing in biofilms. The higher tolerance to antibiotics observed in biofilms is mainly based on the protection of bacteria by the self-produced extracellular polymeric matrix, and in the various growth phenotypes found in the structure, which may be refractory to the action of conventional drugs. In this scenario of increasing and widespread antimicrobial resistance, the search for new drugs and alternative therapies has become crucial, especially for those that are capable of eliminating resistant microorganisms, preventing the development of new forms of resistance, and are active against biofilms. Antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides (AMP) are highlighted in this context, especially for the treatment of localized infections. However, both approaches have therapeutic limitations when used individually. Therefore, the aim of this study was to evaluate the effects and mechanisms of the combination of the AMPs aurein 1.2 (AU) and its dimer (AU)2K with aPDT mediated by the photosensitizers (PS) methylene blue (MB), chlorine-e6 (Ce6) or curcumin CUR), using the bacteria Enterococcus faecalis as a model, in planktonic phase and biofilm. The dimer (AU)2K did not present antibacterial activity in plankto... (Complete abstract click electronic access below) / Doutor

Evalúa la eficacia antimicrobiana del hipoclorito de calcio al 2,5% y el hipoclorito de sodio al 2,5% sobre un biofilm conformado por Enterococcus faecalis y Candida albicans ,donde primero se reactivan las cepas de Enterococcus faecalis y Cándida albicans luego se inoculan las colonias por separado en la escala de Mc Farland en el estándar de turbidez n°0,5 para asegurar una cantidad de 108 (UFC/ml) y luego son combinadas en 5 ml de solución salina, seguidamente se siembra este biofilm formado en Agar BHI, para lo cual se usan 10 placas Petri con 4 discos estériles en cada placa embebidas por separado en las soluciones de hipoclorito de sodio al 2,5% e hipoclorito de calcio al 2,5%, se usa como control positivo al hipoclorito de sodio al 5.25% y agua destilada para el control negativo. Luego de su incubación a 37°C por 24 horas se realiza la medición de los halos de inhibición correspondientes. / Tesis

Made available in DSpace on 2014-06-11T19:31:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-29Bitstream added on 2014-06-13T18:42:17Z : No. of bitstreams: 1 maekawa_le_dr_sjc.pdf: 1187305 bytes, checksum: 2d80ab639c955945003b165b56fe5fd6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A proposta deste trabalho foi avaliar a efetividade dos extratos glicólicos de própolis e de gengibre, hidróxido de cálcio, clorexidina gel e associações como medicação intracanal (MIC) sobre Candida albicans, Enterococcus faecalis, Escherichia coli e endotoxinas em canais radiculares. Foram utilizadas 96 raízes de dentes unirradiculados que tiveram seus canais contaminados com suspensões dos microrganismos por 28 dias. Após a coleta de confirmação, os canais foram instrumentados com solução salina e divididas de acordo com a medicação intracanal utilizada: hidróxido de cálcio [Ca(OH)2] + solução salina; clorexidina gel 2% (CLX); Ca(OH)2 + CLX; própolis (PRO); PRO + Ca(OH)2; gengibre (GENG); GENG + Ca(OH)2; solução salina. Foram realizadas as seguintes coletas do conteúdo do canal radicular: coleta de confirmação (após 28 dias de contaminação), 1a coleta (imediatamente após a instrumentação), 2ª coleta (imediatamente após 14 dias da ação da MIC), 3ª coleta (7 dias após remoção da MIC). Para todas as coletas foram avaliadas: a) atividade antimicrobiana; b) quantificação de endotoxinas pelo teste cromogênico do lisado de amebócitos de Limulus. Os resultados foram submetidos aos testes estatísticos Kruskal-Wallis e Dunn (5%). Verificou-se que todas as MIC foram capazes de eliminar os microrganismos dos canais radiculares, entretanto, o Ca(OH)2 não foi capaz de eliminar completamente C. albicans e E. faecalis. Verificou-se que as MIC contendo Ca(OH)2 foram capazes de diminuir significativamente as endotoxinas dos canais radiculares, sendo semelhantes entre si e diferentes do grupo salina. As medicações CLX, PRO, GENG e salina foram semelhantes entre si. Conclui-se que todas as MIC foram capazes de eliminar os microrganismos do canal radicular e reduzir a quantidade de endotoxinas dos canais radiculares, entretanto, as MIC à base de hidróxido... / The purpose of this study was to evaluate the effectiveness of glycolic propolis and ginger extracts, calcium hydroxide, chlorhexidine gel and associations as intracanal medication (ICM) on Candida albicans, Enterococcus faecalis, Escherichia coli and endotoxins in root canals. Ninety-six roots of single-rooted teeth were contaminated with suspensions of the microorganisms for 28 days. After the confirmation collection, the canals were instrumented with saline and divided according to the intracanal medication used: calcium hydroxide [Ca(OH)2] + saline; 2% chlorhexidine gel (CLX); Ca(OH)2 + CLX; propolis (PRO); PRO + Ca(OH)2; ginger (GENG); GENG + Ca(OH)2; saline. Samples of the root canal content were performed in the following periods: confirmation collection (after 28 days of contamination), 1st collection (immediately after instrumentation), 2nd collection (immediately after 14 days of the action of ICM), 3rd collection (7 days after removal of the ICM). All samples were analyzed according to the following parameters: a) antimicrobial activity, b) quantification of endotoxins by the chromogenic Limulus amoebocyte lysate assay. The results were statistically analyzed by the Kruskal-Wallis and Dunn tests (5%). It was verified that all ICM were able to eliminate the microorganisms from the root canals; however, the Ca(OH)2 was not able to completely eliminate C. albicans and E. faecalis. It was verified that ICM containing Ca(OH)2 were able to significantly decrease endotoxins from the root canals, being similar to each other and to the saline group. The medications CLX, PRO, GENG and saline were similar to each other. It was concluded that all ICM were able to eliminate the microorganisms from the root canal and reduce the amount of endotoxins from the root canals. However, the ICM containing calcium hydroxide were more effective in neutralizing endotoxins

Made available in DSpace on 2014-06-11T19:27:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-04Bitstream added on 2014-06-13T20:36:15Z : No. of bitstreams: 1 sangalli_j_me_araca.pdf: 740058 bytes, checksum: ace9f134bfbf4ab2d5bb78eb6e273e42 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Extratos da folha de araçá (Psidium cattleianum) apresentam biocompatibilidade e atividade inibitória frente a microrganismos bucais. O objetivo do estudo foi avaliar a atividade antimicrobiana in vitro de associações de extratos de araçá (Psidium cattleianum) associados ao hidróxido de cálcio (Ca(OH)2 frente a biofilme multiespécie de Enterococcus faecalis e Candida albicans. Tubos de dentina de incisivos bovinos foram infectados in vitro durante 14 dias com cepas de Enterococcus faecalis ATCC 29212 e Candida albicans ATCC 10231. A luz dos canais radiculares foi preenchida com pasta de extrato etanólico de Psidium cattleianum com Ca(OH)2, extrato propilenoglicólico de Psidium cattleianum com Ca(OH)2 e hidróxido de cálcio com água destilada. Como controle foi empregado o soro fisiológico. Os períodos experimentais foram de 24 horas, 3, 7 e 14 dias. Após cada período realizou-se irrigação com solução salina para remoção dos medicamentos e secagem com cones de papel estéril. Brocas de diâmetros crescentes foram utilizadas para a coleta de pó de dentina. Essas amostras eram replicadas em placas ágar contendo caldo infuso de cérebro e coração (BHI), e levadas em contador digital de colônias. Para Enterococcus faecalis as associações de hidróxido de cálcio com extrato de araçá etanólico e propilenoglicólico apresentaram maior propriedade antimicrobiana que o hidróxido de cálcio associado à água destilada (p<0,01). O extrato etanólico exibiu em 24h atividade antibacteriana que o extrato propilenoglicólico e água levaram de 7 a 14 dias para atingir. Para Candida albicans todos os medicamentos foram efetivos em reduzir significativamente o número de unidade formadora de colônias em todos os períodos de tempo. Nas condições experimentais empregadas pode ser concluído que o extrato etanólico de Psidium... / Araça (Psidium cattleianum) extracts exhibit biocompatibility and inhibitory activity against oral microorganisms. The aim of this estudy was in vitro evaluation of antimicrobial activity of Psidium cattleianum extracts associated with calcium hydroxide Ca(OH)2 against multispecies biofilm of Enterococcus faecalis and Candida albicans. Dentin tubes of extracted bovine maxillary central incisors were infected for 14 days with Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 10231. The root canals were filled with paste of ethanolic extract of Psidium cattleianum with Ca(OH)2, propyleneglycolic extract of Psidium cattleianum with Ca(OH)2 and calcium hydroxide with distilled water. Saline was used as control. The experimental periods were 24 hours, 3, 7 and 14 days. After each period, irrigation with sterile saline to remove the medicament was performed and the canals were dried with sterile paper points. Burs of increasing diameters were used to collect dentin chips. The collected samples were replicated in agar plates with BHI, and taken into digital counter of colonies. The associations of calcium hydroxide with ethanolic extract and with propyleneglycolic extract of Psidium cattleianum presented higher antimicrobial property than calcium hydroxide associated with distilled water (p<0.01). The ethanolic extract showed the same antibacterial activity in 24 hours than that of Ca(OH)2 associated to propyleneglycolic extract or water in 7 days of activity. All drugs were effective to significantly reduce the number of CFU in all periods of time for Candida albicans. In the experimental conditions used it can be concluded that ethanolic extract of Psidium cattleianum associated with Ca(OH)2 was faster and more effective against E. faecalis than Ca(OH)2 associated with propyleneglycol extract or distilled water. The... (Complete abstract, click electronic address below)

Resumo: Extratos da folha de araçá (Psidium cattleianum) apresentam biocompatibilidade e atividade inibitória frente a microrganismos bucais. O objetivo do estudo foi avaliar a atividade antimicrobiana in vitro de associações de extratos de araçá (Psidium cattleianum) associados ao hidróxido de cálcio (Ca(OH)2 frente a biofilme multiespécie de Enterococcus faecalis e Candida albicans. Tubos de dentina de incisivos bovinos foram infectados in vitro durante 14 dias com cepas de Enterococcus faecalis ATCC 29212 e Candida albicans ATCC 10231. A luz dos canais radiculares foi preenchida com pasta de extrato etanólico de Psidium cattleianum com Ca(OH)2, extrato propilenoglicólico de Psidium cattleianum com Ca(OH)2 e hidróxido de cálcio com água destilada. Como controle foi empregado o soro fisiológico. Os períodos experimentais foram de 24 horas, 3, 7 e 14 dias. Após cada período realizou-se irrigação com solução salina para remoção dos medicamentos e secagem com cones de papel estéril. Brocas de diâmetros crescentes foram utilizadas para a coleta de pó de dentina. Essas amostras eram replicadas em placas ágar contendo caldo infuso de cérebro e coração (BHI), e levadas em contador digital de colônias. Para Enterococcus faecalis as associações de hidróxido de cálcio com extrato de araçá etanólico e propilenoglicólico apresentaram maior propriedade antimicrobiana que o hidróxido de cálcio associado à água destilada (p<0,01). O extrato etanólico exibiu em 24h atividade antibacteriana que o extrato propilenoglicólico e água levaram de 7 a 14 dias para atingir. Para Candida albicans todos os medicamentos foram efetivos em reduzir significativamente o número de unidade formadora de colônias em todos os períodos de tempo. Nas condições experimentais empregadas pode ser concluído que o extrato etanólico de Psidium... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Araça (Psidium cattleianum) extracts exhibit biocompatibility and inhibitory activity against oral microorganisms. The aim of this estudy was in vitro evaluation of antimicrobial activity of Psidium cattleianum extracts associated with calcium hydroxide Ca(OH)2 against multispecies biofilm of Enterococcus faecalis and Candida albicans. Dentin tubes of extracted bovine maxillary central incisors were infected for 14 days with Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 10231. The root canals were filled with paste of ethanolic extract of Psidium cattleianum with Ca(OH)2, propyleneglycolic extract of Psidium cattleianum with Ca(OH)2 and calcium hydroxide with distilled water. Saline was used as control. The experimental periods were 24 hours, 3, 7 and 14 days. After each period, irrigation with sterile saline to remove the medicament was performed and the canals were dried with sterile paper points. Burs of increasing diameters were used to collect dentin chips. The collected samples were replicated in agar plates with BHI, and taken into digital counter of colonies. The associations of calcium hydroxide with ethanolic extract and with propyleneglycolic extract of Psidium cattleianum presented higher antimicrobial property than calcium hydroxide associated with distilled water (p<0.01). The ethanolic extract showed the same antibacterial activity in 24 hours than that of Ca(OH)2 associated to propyleneglycolic extract or water in 7 days of activity. All drugs were effective to significantly reduce the number of CFU in all periods of time for Candida albicans. In the experimental conditions used it can be concluded that ethanolic extract of Psidium cattleianum associated with Ca(OH)2 was faster and more effective against E. faecalis than Ca(OH)2 associated with propyleneglycol extract or distilled water. The... (Complete abstract, click electronic address below) / Orientador: Elói Dezan Júnior / Coorientador: Élerson Gaetti Jardim Júnior / Banca: Marco Antonio Húngaro Duarte / Banca: Cristiane Yumi Koga Ito / Mestre

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present study evaluated different formulations of methylene blue (MB) with the aim to improve the antimicrobial effect of photodynamic therapy. Photophysical and photochemical properties and antimicrobial effect of the MB dissolved in different formulations: water (MB/water); 20% ethyl alcohol (MB/ethanol); 0,1% chitosan solution (MB/chitosan); and in mixture of water, ethanol and chitosan (50:20:30 v:v) (MB/new formulation), was evaluated. Molecular aggregation of MB, evaluated by monomer-to-dimer ratio, was dependent on MB concentration and the solvents used. The MB/water and MB/chitosan formulations showed higher aggregation than MB/ethanol and MB/new formulation. In substrate oxidation models (Uric acid and N-acetyl-l-tryptofanamide) the formulation MB/new formulation showed the greatest ability to form singlet oxygen and greater photo-oxidation kinetics. In vitro biofilms of Enterococcus faecalis (Gram positive) and Escherichia coli (Gram negative) was used to evaluated the antimicrobial effect of MB different formulations. No difference was observed when biofilms was treated with MB/water (Light +) and MB/water (Light -). The MB/new formulation group (Light +) showed significant decrease to MB/water (Light -) for both microorganisms. The MB formulation dissolved in water, ethanol and chitosan showed promising photochemical, photophysical, and antimicrobial results. Our results demonstrated that different solvents can enhanced the antimicrobial effect of photodynamic therapy. / Diferentes formulações de azul de metileno (AM) com objetivo de potencializar o efeito antimicrobiano da terapia fotodinâmica foram avaliadas. Propriedades fotofísicas, fotoquímicas e antimicrobianas do MB dissolvido em: água (MB/água); álcool etílico 20% (MB/etanol); quitosana 0,1% em solução de ácido acético 1%/água (30:70 v:v) (MB/quitosana); e sistema água, etanol e quitosana (50:20:30 v:v) (MB/nova formulação), foram avaliadas. Agregação molecular do AM, mensurada pela razão entre a proporção de monômeros e dímeros, foi dependente da concentração do AM e dos solventes. Formulações AM/água e AM/quitosana apresentaram maior agregação molecular do que AM/etanol e AM/nova formulação. Utilizando os modelos de oxidação de substrato (Ácido Úrico e NATA), AM/nova formulação apresentou maior capacidade para formação de oxigênio singleto e maior cinética de foto-oxidação. O efeito antimicrobiano foi avaliado sobre biofilmes in vitro de Enterococcus faecalis (Gram positiva) e Escherichia coli (Gram negativa). Nenhuma diferença estatística foi observada quando biofilmes foram tratados com AM/água com foto-ativação (L+) ou sem foto-ativação (L-). O grupo tratado com AM/nova formulação (L+) apresentou redução significativa em relação ao controle (AM/água L-) para ambos os micro-organimos. A utilização da formulação de AM em água, etanol e quitosana apresentou resultados fotoquímicos e antimicrobianos promissores, demonstrando que diferentes solventes podem potencializar o efeito antimicrobiano da terapia fotodinâmica.

Indiana University-Purdue University Indianapolis (IUPUI) / Root canal therapy is a recommended treatment for apical periodontitis. Root canal failure can occur as a result of microbial leakage. Resilon, a resin based root canal obturating cone material introduced in 2004 attempts to minimize leakage by a unique bonding method of the resin sealer to both the core material and to the dentin of the canal walls. Resilon has no bactericidal or antimicrobial effect15. Furthermore, it has been shown that Resilon is susceptible to alkaline and enzymatic hydrolysis as well as bacterial degradation.73, 184-186 It has been suggested that Resilon may be susceptible to degradation by microorganisms found in the infected root canal space. This work focuses on the susceptibility of root canal obturating materials to be degraded by endodontic pathogens seen in root canal treated teeth with apical periodontitis. The aim of this study was to determine if Resilon could be degraded by selected pathogenic bacteria found in the infected root canal system, and if this degradation is more severe than with gutta-percha, a conventional obturating material. P. intermedia, E. faecalis and P. aeruginosa, known endodontic pathogens were inoculated on discs of obturating material (Resilon or gutta-percha) mounted on a platform and placed in wells containing TSB incubated at 37°C under aerobic conditions. The discs were polished, examined by SEM, profilometry, and elemental analysis prior to inoculation to establish a baseline, and were then re-examined by these methods one month after inoculation. The overall results were inconclusive; and using these methods it cannot be determined that the selected bacteria can degrade Resilon. An ideal future study would utilize SEM with gold coated samples as well as atomic force microscopy to evaluate for changes in topographical features of these obturating materials. A notable finding was that Resilon turns black when exposed to bacteria, and the significance of this finding should be addressed in future studies.

A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.