Dissertations / Theses on the topic 'Bacillus proteus'

Orientador: Hélia Harumi Sato
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Proteases bacterianas são enzimas de elevada importância comercial, amplamente aplicadas em diversas áreas como nas indústrias de detergentes, de alimentos, farmacêutica e têxtil. Este trabalho teve como principais objetivos selecionar entre 59 linhagens de Bacillus sp., da coleção de culturas do Laboratório de Bioquímica de Alimentos da FEA, aquelas que apresentam potencial de maior produção de proteases com características tais como estabilidade em diferentes condições de temperatura, pH, detergentes e solventes orgânicos, atividade em ampla faixa de pH e capacidade de lisar células de Xanthomonas campestris. Em seguida, visou-se otimizar a produção de proteases pela linhagem selecionada, determinar as características bioquímicas da protease parcialmente purificada e estudar a aplicação do extrato enzimático bruto e preparação parcialmente purificada. Entre as cinquenta e nove linhagens de Bacillus sp. testadas foram selecionadas nove linhagens que produziram maior atividade de proteases. A produção de protease pelas nove linhagens foi testada em frascos agitados contendo o meio de cultura nº 1 (10g/L de caseína, 1g/L de extrato de levedura e sais), meio nº 2 (35 g/L de melaço de cana de açúcar, 20g/L de água de maceração de milho, 3g/L de extrato de levedura Prodex-Lac SD® e 20g/L de soro de queijo), e por fermentação em meio sólido nº 3 (farelo de trigo e água, na proporção 1:1, m:m). As linhagens de Bacillus sp. LBA 07, Bacillus sp. LBA 46 e Bacillus sp. LBA 08 fermentadas nos meios de cultura nº 1, nº 2 e nº 3 produziram 222 U/mL, 548 U/mL e 13480 U/grama de substrato seco (gss) respectivamente. As proteases dos extratos enzimáticos brutos obtidos das nove linhagens fermentadas nos três meios de cultura apresentaram atividade ótima na faixa de pH 7 a 9 e 60° C, estabilidade na faixa de pH 5 a 9 por 24h a 4º C , e após 1 h de tratamento a 50° C. Entre os extratos enzimáticos brutos de proteases testados, aqueles obtidas da fermentação de Bacillus sp. LBA 46 nos três meios de cultura foram as mais estáveis em detergente Ariel®. Quando incubadas em solventes orgânicos alguns extratos enzimáticos brutos proteases mantiveram mais de 60% de atividade residual após 24h em acetona (Bacillus sp. LBA 8 e 44), hexano (Bacillus sp. LBA 19, 29, 44, 46 e 60), clorofórmio (Bacillus sp. LBA 44 and 60) e etanol (Bacillus sp. LBA 60). Os extratos enzimáticos brutos de proteases obtidos do cultivo da linhagem de Bacillus sp. LBA 46 nos meios n° 2 e n° 3 foram as mais eficientes na lise de células de Xanthomonas campestris, aumentando cerca de 30% a transmitância a 620 nm (Trans 620nm) do meio fermentado de goma xantana. A linhagem de Bacillus sp. LBA 46 foi selecionada como melhor produtora de protease e estudos preliminares de identificação biomolecular indicam que se trata de uma linhagem de Bacillus licheniformis. Utilizando-se a linhagem de Bacillus sp LBA 46 e o meio de cultura otimizado (meio n° 4) por metodologia de superfície de resposta (MSR), composto de 40g/L de melaço de cana de açúcar, 6g/L de água de maceração de milho, 2g/L de extrato de levedura Prodex-Lac SD® e 20g/L de soro de queijo, foi obtido 3000 U/mL de protease após 96h de fermentação a 30° C e 200 rpm. No estudo da aplicação da enzima para a remoção de manchas de tecidos de algodão foram obtidos melhores resultados de remoção de manchas de sangue e molho de tomate com carne moída, utilizando-se a combinação de extrato bruto de protease (100 ou 1000U) com o detergente Omo®. O extrato enzimático bruto da linhagem de Bacillus sp. LBA 46 foi parcialmente purificado por fracionamento com sulfato de amônio (80% de saturação), diálise e cromatografia de filtração em gel (Sephadex G100), resultando em fator de purificação de 3,69. Após caracterização com MSR observou-se que a protease da preparação parcialmente purificada apresentou atividade ótima a 55° C e pH 7,5 e considerável estabilidade (95% de atividade residual) na faixa de pH 5,7 ¿ 9,3 após 1h de incubação a 30 ¿ 36° C, e acima de 78,9% quando incubadas por 1h em pH 7,5 e 50° C. A condição ótima de lise das células de X. campestris do meio fermentado de goma xantana utilizando-se o extrato enzimático bruto de protease e a preparação parcialmente purificada de proteases, foi observada utilizando 42 U de protease /mL de suspensão celular de X. campestris a 60° C, resultando em aumento de mais de 20% da Trans 620nm do meio fermentado de goma xantana. Um aumento de quase 40% de Trans 600nm foi observado após 2h de reação utilizando extrato enzimático bruto de protease (42 U de protease/mL de suspensão celular de X. campestris) a 65° C. A produção de proteases de Bacillus sp. LBA 46 por fermentação em estado sólido foi otimizada utilizando MSR, sendo obtido 5000 U/grama de substrato seco utilizando-se meio de cultura composto de farelo de trigo e água (60%:40%) após 96h de fermentação a 30° C
Abstract: Proteases are commercially relevant enzymes widely applied in several industrial areas, such as in detergent, food, pharmaceutical and textile industries. Proteases from Bacillus sp. can present advantages compared to the proteases from other sources, including better thermostability, stability in pH range from slightly acid to alkaline pH values and stability in organic solvents. The aims of this work were selecting Bacillus sp. strains with capability of producing proteases with better biochemical properties, such as stability in different conditions of temperature, pH, detergents and organic solvents, activity in a wide range of pH and capability of lysing cells of Xanthomonas campestris. Afterwards, it was aimed the optimization of the production of proteases by the selected Bacillus sp. strain and the determination of the biochemical characteristics of the partially purified protease and the application of the crude and partially purified protease. Nine Bacillus sp. strains were selected as the best protease producers among fifty nine Bacillus sp. strains tested. The protease production by the nine strains was carried out in Erlenmeyer flasks containing medium no. 1 (10g /L of casein, 1g/L of yeast extract and salts), medium no. 2 (35 g/L of sugar cane molasses, 20g/L corn steep liquor, 3g/L of yeast extract Prodex-Lac SD® and 20g/L of dried whey), e by fermentation using solid substrate medium no. 3 (wheat bran and water, 1:1, m:m). The strains Bacillus sp. LBA 07, Bacillus sp. LBA 46 and Bacillus sp. LBA 08 when fermented in medium no. 1, no. 2 e no. 3 produced 222 U/mL, 545 U/mL and 13480 U/gram of dried substrate (gds) respectively. Proteases from the crude enzymatic extracts obtained from the fermentation of the nine Bacillus sp. strains in the three media showed optimal activity in pH range 7-9 and 60° C, stability in pH range 5-9 for 24 hours at 4° C and after 1h at 50° C. The protease preparations from the fermentation of Bacillus sp. LBA 46 in the three media were the most stable when incubated in detergent Ariel®, among the proteases tested from the Bacillus sp. strains. In addition, some proteases presented more than 60% residual activity after 24h in the organic solvents acetone (Bacillus sp. LBA 8 and 44), hexane (Bacillus sp. LBA 19, 29, 44, 46 and 60), chloroform (Bacillus sp. LBA 44 and 60) and ethanol (Bacillus sp. LBA 60). The protease preparations obtained from the cultivation of Bacillus sp. LBA 46 in medium no. 2 and no. 3 presented the best results on the lysis of Xanthomonas campestris cells, resulting in an increase of approximately 30% in transmittance at 620 nm (Trans 620nm) of the fermented broth of xanthan. Bacillus sp. LBA 46 strain was selected as the best protease producer and after preliminary biomolecular analysis of identification, the results indicate that this microorganism correspond to a Bacillus licheniformis strain. Protease preparation containing 3000 U/mL was obtained from Bacillus sp. LBA 46 cultivated in Erlenmeyer flasks containing medium no. 4 composed of 40g/L of sugar cane molasses, 6g/L of corn steep liquor, 2g/L of yeast extract Prodex-Lac SD® and 20 g/L of dried whey after 96h of fermentation at 30° C and 200 rpm, optimized with response surface methodology (RSM). In the the washing tests, the best results of the removal of blood and tomato sauce with ground beef stains from cotton fabrics were observed using the combination of crude extract of protease (100 or 1000U) with detergent Omo®. Crude protease extract of the Bacillus sp. LBA strain was partially purified by ammonium sulfate fractionation (80% saturation), dialysis and gel filtration chromatography (Sephadex G100), resulting in the purification fold of 3.69. After characterization with RSM it was observed that the crude protease extract and partially purified proteases presented optimal activity at 55° C and pH 7.5 and considerable stability (95% of residual activity) in pH range 5.7 ¿ 9.3 after 1h incubation at 30-36° C and more than 78.9% when incubated at pH 7.5 and 50 °C for 1h. The optimal conditions of the lysis of X. campestris cells contained in the fermentation broth using crude and partially purified protease preparations were observed using 42 U of protease/mL of cell suspension of X. campestris at 60° C, resulting in a increase of more than 20% in Trans 620 nm of the fermented broth of xanthan. It was observed an increase of almost 40% in Trans 620 nm after 2h reaction using crude protease (42 U de protease/mL of cell suspension of X. campestris) at 65° C. The production of proteases by Bacillus sp. LBA 46 under solid state fermentation was optimized using RSM, resulting in 5000 U/gram of dry substrate utilizing wheat bran and water (6g:4g) after 96h of fermentation at 30° C
Doutorado
Ciência de Alimentos
Doutor em Ciência de Alimentos

Thesis (Ph. D.)--University of Oregon, 2003.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-114). Also available for download via the World Wide Web; free to University of Oregon users.

Les propriétés insecticides du Bacillus thuringiensis, découvert par ShigentaneIshiwatari, ont été utilisées pendant des décennies comme biopesticides et cette utilisation a augmenté rapidement en raison de préoccupations au sujet des effets environnementaux négatifs des pesticides chimiques. Actuellement, la toxine Bt dans la forme de biopesticides et des plantes transgéniques Bt peut compléter ou remplacer les pesticides chimiques. Il y a peu d’indication que la toxine Bt a un effet nocif pour l'environnement ou la santé humaine. Néanmoins, il ya des préoccupations que les cultures transgéniques commerciales peuvent avoir des effets néfastes sur l'environnement. Après son introduction dans le sol l'exsudation racinaire et la dégradation des résidus végétaux, la toxine Bt interagit avec les particules de sol. Les interactions de la toxine Bt avec des particules de sol influencent sa mobilité, sa biodisponibilité, sa persistance et sa toxicité.Dans cette étude, nous visons à établir l'importance relative des facteurs biologiques et physico-chimiques dans la détermination de la dynamique des protéines Cry détectables dans les sols, de clarifier si la protéine adsorbée conserve ses propriétés insecticides et d'identifier les propriétés du sol qui déterminent le devenir des protéines Cry dans le sol. Les résultats montrent que les protéines Cry ont une forte affinité sur la surface du sol. Cependant, il y avait peu de relation entre l'affinité pour le sol ou le rendement d'extraction et les propriétés du sol, y compris la teneur en argile, teneur en carbone organique et le pH du sol. Il y avait peu de rapport entre l'affinité et le rendement d'extraction. Les protéines diffèrent à la fois dans leur affinité pour les sols et leurs rendements d'extraction.Une évaluation du rôle du sol et des facteurs environnementaux dans le sort des protéines Cry de la formulation de biopesticides commerciale a montré un déclin rapide de la protéine Cry détectable soumise aux rayons du soleil sous la condition de laboratoire, alors que peu d'effet a été observé dans des conditions de terrain. La demi-vie des protéines dans le sol dans des conditions naturelles était d'environ 1 semaine. Des effets de la température forts ont été observés, mais ils diffèrent pour les biopesticides et la protéine purifiée, indiquant différentes étapes limitantes. Pour le biopesticide, la baisse observée était ralenties par des facteurs biologiques, y compris éventuellement sporulation. En revanche pour des protéines purifiées, augmentation de la température améliorée des changements conformationnels de la protéine adsorbée du sol, conduisant à une fixation et, par conséquent diminué efficacité d'extraction qui a diminué avec le temps. En outre, l'étude de la persistance de diverses protéines Cry dans les sols contrastés a été réalisée par immuno-détection et dosage biologique a montré que la toxine extractible diminue avec incubation allant jusqu'à quatre semaines. L'activité insecticide était toujours maintenue à l'état adsorbé, mais a disparue après deux semaines d'incubation à 25°C. La baisse de la protéine extractible et la toxicité était beaucoup plus faible à 4°C à 25°C. La stérilisation du sol n'a pas eu d'effet significatif sur la persistance de la toxine Cry indiquant que le déclin observé était provoqué par la fixation en fonction du temps de la protéine adsorbée ce qui diminue la quantité de toxine Cry extractable, la dégradation de la protéine par l’activité microbienne jouant un rôle plus mineur.L’exposition des insectes aux protéines Cry sous la forme adsorbé pourrait avoir un impact significatif sur les insectes cibles et même les insectes non cibles, et devrait être plus étudiée afin de déterminer son impact potentiel
The insecticidal properties of Bacillus thuringiensis, discovered by Shigentane Ishiwatari, have been used for decades as biopesticides and this use has been increasing rapidly because of concerns about the negative environmental effects of chemical pesticides. Currently, Bt toxin in the form of both biopesticides and Bt transgenic plantsmay supplement or replace chemical pesticide. There is little evidence to demonstrate that Bt toxin has any harmful effect to the environment or to human health. Nevertheless, there are concerns that commercial transgenic crops may have harmful impacts on the environment. After release into soil via root exudation and breakdown of plant residues, Bt toxin interacts with soil particles. The interactions of Bt toxin with soil particles influence its mobility, its bioavailability, its persistence and its toxicity. In this study, we aim to establish the relative importance of biological and physicochemical factors in the determination of the dynamics of detectable Cry proteins in soils, to clarify if adsorbed protein maintains its insecticidal properties and to identify the soil properties that determine the fate of Cry proteins in soil. The results show that Cry proteins have strong affinity on soil surface. However, there was little relationship between affinity for soil or the extraction yield and soil properties including clay content, organic carbon content and soil pH. There was little relationship between the affinity and the extraction yield. The proteins differ in both their affinity for soil and their extraction yields.An assessment of role of soil and environmental factors in the fate of Cry protein from commercial biopesticide formulation showed a rapid decline of detectable Cry protein subjected to direct sunlight under the laboratory condition, whereas, little effect was observed under field conditions. The half-life of proteins in soil under natural conditions was about one week. Strong temperature effects were observed, but theydiffered for biopesticide and purified protein, indicating different limiting steps. For biopesticide, the observed decline was due to biological factors, possibly including sporulation. In contrast for purified proteins, increased temperature enhanced conformationalchanges of the soil-adsorbed protein, leading to fixation and hence extraction efficiency decreased that decreased with time. Moreover, the study of persistence of various Cry proteins in contrasting soils was carried out by immuno-detection and bioassay showed that extractable toxin decreased with incubation of up to four weeks. Insecticidal activity was still retained in the adsorbed state, but lost after two weeks of incubation at 25°C. The decline in extractable protein and toxicity was much lower at 4°C than 25°C. There was no significant effect of soil sterilization to persistence of Cry toxin indicating that decrease in detectable Cry toxin in soil may be time-dependent fixation of adsorbed protein as well as decreasing solubilization in larva midgut, but not microbial breakdown.Exposition to Cry in the adsorbed form could have a significant impact on target and even non target insects and should be investigation to determine the potential impact

Peptidoglycan not only protects bacterial cells against intracellular pressure but also provides the cells with a defined morphology. Penicillin-binding proteins (PBPs) catalyze the polymerization of the peptidoglycan in Bacillus subtilis. PBP2a and PBP2b are class B PBPs which have been known to have transpeptidase activities and they localize at different positions on the cell membrane. PBP2a spreads around the cylindrical wall as well as some at the septum, and PPB2b localizes exclusively to the septum and some at the cell poles. Both PBP2a and PBP2b are composed of four domains: S, N, P, and C domains from the N- to C- terminus. A FLAG epitope was tagged to the C-terminal ends of PBP2a and PBP2b. Cells with FLAG tagged PBP2a or PBP2b grow as well as wild type strain. Expression of PBP2a-FLAG and PBP2b-FLAG can be detected by western blotting using anti FLAG antibody. The expression of wild type PBP2a/PBP2b in these strains was tightly controlled by a xylose promoter. The FLAG fusion didnâ t influence the normal membrane localizations of PBP2a or PBP2b. PBP2a/2b mutant strains with the S and/or N domains switched between PBP2a and PBP2b were constructed. All these domain-switch proteins were tagged with a FLAG at the C-terminus. The expression of these recombinant proteins can be detected by western blotting. None of these domain-switch proteins was able to complement the wild type PBP2a and PBP2b and cells with only these recombinant proteins but no wild type proteins were non-viable. Cellular localization of these domain switch proteins were visualized using immunofluorescence microscopy. Proteins containing the PBP2a S domain had the same localization patterns as wild type PBP2a. Proteins that have the PBP2b S domain localized specifically at the septum and cell poles, which is similar to the wild type PBP2b. These results indicate that the S domain is the determinant to direct PBP2a and PBP2b to their cellular destinations.
Master of Science

For many years the Gram positive bacterium Bacillus subtilis has been a model organism for prokaryotic cell and molecular biology. The asymmetric cell division which B. subtilis undergoes during sporulation is a simple system by which to study the process of cell differentiation. This process is governed by a series of genetic, temporal and spatial controls. Gene regulation, brought about by a series of a factors and transcriptional regulators, is coupled to key morphological stages or checkpoints.

Many spore forming bacteria are the causative agents of severe disease, such as Bacillus anthracis and anthrax. In these cases, the spore often acts as the infectious agent. Spores boast extreme resistance to chemical and UV damage among other bactericidal conditions. This is problematic due to the difficulty and economic costs of decontaminating exposure sites. The present work focuses on identifying and characterizing proteins active within spore germination, with a focus towards understanding the triggering of the major stages of germination. Understanding how each stage is initiated could allow for development of methods that induce these processes to efficiently germinate spores, thus facilitating cheap and effective decontamination. Sequencing of a spore transposon insertion library after exposure to germinants led to the identification of 42 genes with previously uncharacterized roles in spore germination. Fourteen of the genes, encoding proteins associated with the inner spore membrane, were further characterized. Mutants lacking these genes portrayed phenotypes consistent with failure of a GerA receptor-mediated germination response, and these genes affect the earliest stages of germination. Chemical cross-linking was used to characterize protein interactions important for stage II of spore germination. Site-directed in vivo crosslinking indicated that YpeB may exist as a multimer within the dormant spore. Further investigation of individual protein domains using bacterial two-hybrid analysis suggested that both N- and C-terminal domains of YpeB contribute to the formation of a multimer. In addition, the uncharacterized YpeB N-terminal domain was demonstrated to have strong self-association and may mediate self-association within the dormant spore. Additional genes that contribute to efficient initiation of spore germination in a GerA-dependent manner were identified via TnSeq. Chemical cross-linking of dormant spores was implemented to characterize protein interactions leading to stabilization and activation of an important enzyme that contributes to cortex degradation in stage II of germination. The presented studies employed a variety of techniques to provide additional insight into both stages of spore germination with a goal of furthering understanding of specific events that contribute to a loss of spore dormancy.
Doctor of Philosophy
Few bacterial species can undergo a specialized division process leading to the generation of a bacterial endospore. Endospores are dormant cells that boast resistance to a variety of environmental conditions that would otherwise cause bacterial cell death. These resistance traits make endospores immune to traditional bactericidal methods, making decontamination a nontrivial task. Further complicating the matter, spores are often the infectious particle of the associated disease, including hospital acquired diarrhea, infant botulism, anthrax, and many others. Presented work focuses on furthering understanding the process by which a dormant spore returns to a typical growing bacteria cell. Comprehension of major steps in this process may lead to novel methods for spore cleanup in which mechanisms within the spore are subverted to force a return to a typical bacterial cell state.

Le cytosquelette bactérien des homologues d'actine (protéines de la famille MreB) joue un rôle majeur dans la morphogénèse cellulaire. Des homologues de MreB sont retrouvés chez la plupart des espèces bactériennes non sphériques, où ils sont essentiels pour la viabilité cellulaire. Les bactéries à Gram-positif ont généralement plusieurs isoformes. L'organisme modèle Bacillus subtilis en possède trois : MreB, Mbl et MreBH, tous trois impliqués dans la détermination de la forme de la cellule. Le postulat actuel est une organisation, des complexes de synthèse du peptidoglycane, le long des parois latérales par les filaments hélicoïdaux des MreB-like. Cependant, les mécanismes moléculaires et les protéines effectrices impliqués dans cette fonction ne sont pas encore élucidés. Par analogie avec les rôles de l'actine eucaryote, des implications dans d'autres processus cellulaires cruciaux et la présence de partenaires protéiques sont également attendus pour les actines procaryotes. Afin d'explorer les rôles des protéines MreB chez B. subtilis nous avons généré, par des criblages génomiques double hybride chez la levure, un réseau d'interaction protéine-protéine centré sur MreB, Mbl et MreBH. Une vérification systématique et drastique de toutes les interactions obtenues lors des criblages a été réalisée afin d'éliminer les faux positifs. Les interactions identifiées révèlent des liens entre les protéines MreB-like et seize protéines issues de catégories fonctionnelles variées ou de fonction inconnue. Une étude exploratoire a été menée pour huit des protéines partenaires par des approches in silico et in vivo et nous a permis de sélectionner une seule interaction à caractériser plus en détail. Nous nous sommes principalement intéressés à l'interaction physique et directe entre MreB et DapL, une protéine essentielle vraisemblablement impliquée dans la voie de biosynthèse des précurseurs du peptidoglycane, par analogie à DapE d'E. coli. La caractérisation approfondie de DapL a confirmé son essentialité dans la synthèse du peptidoglycane. Bien que l'interaction MreB-DapL ait été confirmée biochimiquement, son rôle biologique exact n'a pas été élucidé. Cependant, nous avons mis en évidence d'autres interactions entre MreB et DapG, LysA et MurE, des enzymes également impliquées dans les étapes précoces de la synthèse du peptidoglycane. L'existence de telles interactions renforce le rôle du cytosquelette MreB de B. subtilis dans l'orchestration des machineries de synthèse de la paroi cellulaire.

The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5&rsquo
end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through PCR. The resulting hybrid gene pre(subC)::skc was cloned into the pUC19 plasmid. Then, the hybrid gene was sub-cloned to pMK4 plasmid which is an E. coli-Bacillus shuttle vector with high copy number and high stability. Recombinant plasmid pMK4::pre(subC)::skc was finally transferred into B. subtilis (npr- apr-) and B. licheniformis 749/C (ATCC 25972) species. Streptokinase production capacities of these two recombinant Bacillus species were compared. The highest production was observed in recombinant B. lichenifomis 749/C (ATCC 25972) strain in a defined medium which was optimized in terms of carbon and nitrogen sources by a statistical approach, namely Response Surface Methodology (RSM). RSM evaluated the streptokinase concentration as the response and the medium components as the independent variables. The highest recombinant streptokinase concentration was found as 0.0237 kgm-3 at glucose and (NH4)2HPO4 concentrations of 4.530 and 4.838 kgm-3 respectively. The fermentation and oxygen transfer characteristics of the streptokinase production were investigated in a 3 dm3 pilot scale batch bioreactor (Braun CT2-2) equipped with temperature, pH, foam, air inlet and agitation rate controls having a working volume of VR=1.65 dm3 using the production medium optimized for the recombinant B. lichenifomis 749/C (ATCC 25972) strain. Streptokinase and &
#946
-lactamase activities, cell, glucose and organic acid concentrations, dissolved oxygen, pH, oxygen uptake rate, overall liquid phase mass transfer coefficient for oxygen, maintenance coefficient for oxygen, specific cell growth rate and yield coefficients were determined through the bioprocess. The bioprocess of recombinant streptokinase production was performed at uncontrolled pH of these bioreactor operation conditions: air inlet rate of Q0/VR=0.5 vvm, and the agitation rate of N=400min-1. The resulting streptokinase volumetric activity reached its maximum as 1.16 PUml-1 (0.0026 g/l streptokinase) at t=20 h.

Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn2+ or Ca2+ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination.
Ph. D.

The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.

Anthrax, a fatal bacterial infection is caused by Bacillus anthracis, a gram-positive, spore forming, capsulated, rod shaped organism. Centers for Disease Control (CDC) lists anthrax as Category A biological agent due to its severity of impact on human health, high mortality rate, acuteness of the disease and potential for delivery as a biological weapon. The currently available human vaccine in the United States (AVA anthrax vaccine adsorbed) is prepared from Alum adsorbed formalin treated supernatant culture of toxigenic, non-encapsulated strain of Bacillus anthracis with the principle component being protective antigen (PA83). Evaluation of anthrax vaccine given to nearly 400,000 US military personnel by Vaccine Adverse Event Reporting System (VAERS) showed adverse effects such as flu-like symptoms, local pain, large degree of inflammation, edema, malaise, rash, arthralgia, and headache following vaccination. All the adverse reactions are attributed to the composition of vaccine components. These vaccine preparations contain trace contaminants of lethal and edema factors that contribute to the adverse side effects. Also, the current method of vaccine manufacture has limited production capacity.The production of PA83, in plants through chloroplast genetic engineering might eliminate the concerns of adverse side effects and the levels of expression would ensure the availability of vaccine for the human population in an environmentally friendly approach. The primary concern is whether the PA83 purified from transgenic chloroplasts is as immunogenic as the PA83 in the AVA. For this, PA83 has been expressed in transgenic chloroplasts of Nicotiana tabacum var. petit Havana, by inserting the pag (2205 bp) with the N-terminal 6X histidine tag, into the chloroplast genome by homologous recombination. Chloroplast integration of the pag was confirmed by PCR and Southern analysis. The PA83 protein was detected in transgenic chloroplasts by immunoblot analysis using anti-PA83 antibodies. Maximum expression levels of PA83 (14.17% TSP) were observed in mature leaves upon continuous illumination, due to the presence psbA 5'UTR, a light and developmentally regulated translation enhancer sequence. The PA83 has been purified by affinity chromatography using Ni resin columns. Chloroplast derived PA83 was functional in vitro and was able to lyse the mouse macrophages when combined with the lethal factor. The in vitro assays showed that the crude extracts contained up to 20ug/ml of functional PA83.The immunization studies of PA83 on Balb/c mice, revealed highly immunogenic IgG titers. Subcutaneous immunization with purified chloroplast derived PA83 with adjuvant yielded IgG titers up to 1:320,000, similar to that of the group immunized with PA83 derived from Bacillus anthracis. Immunization of groups with PA83 combined with alhydrogel adjuvant showed four - eight times higher immune response than the groups without adjuvant. The higher expression levels of PA83 in transgenic chloroplasts might ensure the availability of anthrax vaccine to the general public and the high immune response observed in the mouse model would enable the replacement of the current AVA with a cleaner and safer vaccine.
M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.

During sporulation, Baccilus thuringiensis subsp. kurstaki produces a crystalline inclusion body which is toxic upon ingestion by susceptible Lepidopteran larvae. The major component of crystals from Lepidopteran-specific subspecies of B. thuringiensis is a 130-kDa protein, protoxin. Following ingestion by susceptible larvae, protoxin is proteolyzed to yield a 58-70 kDa toxic fragment, toxin. In the present study, a simplified procedure was used for isolating and purifying toxin generated by the tryptic digestion of protoxin from B. thuringiensis subsp. kurstaki HD-73. Characterization of this toxin showed that it is derived from the N-terminal half of the protoxin molecule. The toxin is insoluble at neutral pH values but is moderately soluble at alkaline values above pH 9. Application of several spectroscopic and theoretical procedures to the purified toxin showed that the protein is composed of approximately equal amounts of a $\alpha$-helix, $\beta$-sheet and random coil structures. The tertiary structure of toxin was shown to be comprised of two primary domains; these domains correspond to the toxic and specificity (or binding) domains predicted from analysis of protoxin gene nucleotide sequences. Evidence was obtained that at least one additional domain is present as a structural component of the C-terminal specificity domain. Both the toxic moiety within the protoxin molecule and free toxin were found to be unusually resistant to unfolding by chemical denaturants and to proteolysis. In contrast, the C-terminal half of protoxin could be readily unfolded and was extremely susceptible to proteolytic digestion. The unfolded protoxin and unfolded toxin were shown to refold rapidly into their native and biologically active conformations. Evidence was obtained that the conformation of the toxic moiety of protoxin is very similar to the conformation of toxin. Chemical modification of the cysteine and lysine residues in the protoxin did not affect the biological activity of the protein. However, the introduction of positive, negative or neutral groups onto these residues had a large effect on the solubility of the protein. These results, along with the results obtained from the unfolding/folding, studies, strongly indicate that the primary function of the C-terminal half of the protoxin molecule is to promote the formation of a stable crystal.

Bacillus thuringiensis produces a crystalline inclusion body composed of a 130-kDa protein which is rendered toxic upon ingestion by lepidoteran larvae. It was shown that proteinases adsorb on the surface of the crystalline body lead to proteolysis of the protein crystal especially on solubilization in alkali. Extensive washing of the protein crystal was shown to remove these proteinases and give a stable preparation. Exposure of the protein crystal to simulated sunlight results in a loss of toxicity and in the destruction of the side-chains of tryptophan, histidine, tyrosine and methionine. Destruction of amino acid side-chains is not the primary cause of the photo-inactivation of the protein crystal. The finding that all the disulfide linkages in the protein crystal are interchain and symmetrical accounts for its alkaline lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies. (Abstract shortened by UMI.)

Bacillus subtilis ist einer der bislang am besten untersuchten Organismen. Es gilt als Modellbakterium, an welchem stoffwechselphysiologische und genetische Phänomene grundlegend aufgeklärt wurden. Daneben gehört B. subtilis zu den generell unbedenklichen Mikroorganismen und wird von der U.S. Food and Drug Administration (FDA) als ‚GRAS‘ (Generally Regarded As Safe) eingestuft. Dementsprechend wird B. subtilis in der biotechnologischen Industrie zur Herstellung vielfältiger Produkte, z.B. in der Lebensmittelindustrie, eingesetzt. Als Gram-positives Bakterium eignet sich B. subtilis vor allem zur Produktion extrazellulärer Enzyme wie Proteasen und Amylasen. Die Untersuchung der Sekretion dieser Proteine ist sowohl für die grundlagen- als auch für die anwendungsorientierte Forschung von wichtiger Bedeutung. Vorarbeiten der Arbeitsgruppe von Prof. Wiegert haben gezeigt, dass eine Deletionsmutante im Gen der Protease RasP einen vollständigen Defekt der Sekretion einer α-Amylase verursacht. In meiner Promotionsarbeit soll untersucht werden, welche Rolle RasP bei der Sekretion dieser α-Amylase spielt, und welchen Einfluss diese Protease auf die Proteinsekretion allgemein ausübt. Zudem soll, u.a. am Beispiel von Signalpeptiden, der Mechanismus der Substraterkennung durch RasP untersucht werden. Ich erhoffe, mit dieser Arbeit einen entscheidenden Beitrag zum Verständnis der Funktion der Protease RasP zu leisten und damit eine weitere gezieltere Optimierung von B. subtilis Produktionsstämmen in der biotechnologischen Industrie zu ermöglichen.

Members of the Bacillus genus, when faced with unfavorable environmental conditions such as depletion of nutrients, undergo an asymmetric division process ultimately leading to the formation of an endospore. In some instances, the spore serves as the infectious agent of an associated disease; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of unfavorable environmental conditions including traditional decontamination techniques. Spore resistance is due to the formation of specialized structures that contribute to spore dormancy through several mechanisms, including maintenance of the dehydrated state of the spore core. Spore germination is a rapid process resulting in the irrevocable transformation of the non-metabolizing dehydrated spore into a vegetative outgrowing bacterium. The exact mechanism by which individual proteins function in the germination pathway remains unknown. In this study, we have focused on the roles of putative ion transporters and other germination-active proteins in affecting spore formation and germination. Metal ions can activate enzymes during the sporulation process and/or be factors in spore resistance properties. In B. subtilis, six proteins within the spore membrane proteome (ChaA, YcnL,YflS, YloB, YugS, ZnuA) are similar to components of known cation transport systems. These proteins may play roles in the accumulation of ions during sporulation and/or the release of ions during germination. Multiple mutants altered in the putative ion transporter genes were generated, and the effects of these mutations were analyzed. All strains containing a yloB deletion showed a decrease in heat resistant cfu/ml, and >40% of the spores appeared phase dark during microscopy, indicating the formation of unstable spores. Studies were conducted to quantify the amounts of individual ions in phase-bright spores using atomic emission spectroscopy and to analyze the rate at which ions are released from germinating spores. The transport of Ca2+ from mother cell to forespore during sporulation seems to be affected in the yloB deletion mutant. This Ca2+ deficit apparently renders the spores unstable, heat sensitive, and partially germination defective, suggesting that a high-affinity transporter for Ca2+ is nonfunctional. To better understand the underlying mechanisms of germination, a high-throughput genetic screening method called transposon sequencing was used. This analysis identified genes that had not been previously implicated in germination. To investigate their functions, a number of functional assays of all the Ger mutant strains were performed that indicated a delay in stage I of germination. The mutant strains showed significant reduction in germination efficiency with L-valine: about 50% of the population failed to initiate germination suggesting a defect in the GerA-mediated response. The expression of gerA was studied using a lacZ transcriptional fusion followed by quantitative western blot analyses to determine abundance of GerA in mutant strains. The mutants were classified based upon normal or decreased gerA transcription and normal or reduced GerA protein. Further work involves understanding the functions of the identified genes and their correlation to the GerA receptor. Insight into ion transporters of spore-forming bacteria and understanding the germination apparatus may lead to promising new applications, detection methods, or therapeutics for spores, and may allow the development of better spore decontamination procedures.
Doctor of Philosophy
Bacillus subtilis is an ubiquitous bacterium that is capable of forming endospores when faced with unfavorable environmental conditions. Spores are highly resistant to heat, radiation, lack of nutrients, desiccation and oxygen deprivation. They lack metabolism, which effectively keeps them in a state of suspended animation until germinated. They may remain stable and viable in this state for extremely long periods of time. Several important pathogenic bacteria are spore formers. This leads to difficulty in their environmental eradication and the treatment of patients. Germination allows spores to resume metabolism and reestablish a vegetative state. Certain key molecules activate the germination process. Each species of spore-forming bacteria has a specific set of these molecules called germinants that will enable the spore to exit its dormant state. The work presented focuses on the understanding of the germination apparatus of Bacillus subtilis, which may provide a model to understand the germination of other spore formers and help to improve methods of decontamination.

Orientador: Ranulfo Monte Alegre
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Consideráveis pesquisas têm sido conduzidas nos últimos anos para descobrir novas enzimas que possam ser utilizadas no tratamento de resíduos. Devido ao aumento de poluentes no meio ambiente, tornando-se difícil a sua remoção por processos convencionais, o desenvolvimento de pesquisas com enzimas busca um processo alternativo de tratamento, de menor custo, mais rápido, mais simples e mais confiável. As proteases são a classe mais importante das enzimas industriais e compreendem cerca de 25% de todas as enzimas comerciais no mundo. A maior aplicação destas enzimas é nas indústrias de alimentos, farmacêuticas e de detergentes. A finalidade do presente trabalho consistiu num estudo para determinar as melhores condições para produção de protease, bem como sua eficiência na hidrólise enzimática sobre a etapa biológica no tratamento de efluente líquido. Para a produção da protease foi testada uma cepa de Bacillus subtilis. O inóculo foi preparado em frascos Erlenmeyers (250 mL) contendo 50 mL de meio BHI , previamente esterilizado (121ºC, 15 min). Os frascos foram incubados a 30ºC durante 24 h com agitação de 200 rpm. Para a produção de protease foi utilizado um Planejamento Experimental Fatorial Fracionário 24-1, com triplicata no ponto central, tendose como variáveis independentes as concentrações de lactose, cloreto de sódio, caseína e pH. Como base foi utilizado o meio descrito por KEMBHAVI et al. (1993). Baseado nos resultados do planejamento experimental, foi realizado um ensaio cinético em fermentador do tipo tanque agitado contendo lactose (5,0 g.L-1) e caseína (5,0 g.L-1) a pH 6, 30OC e 300 rpm durante 30 h. O meio foi inoculado numa proporção de 10% (v/v) de inóculo. As amostras foram retiradas em intervalos de 1h, centrifugadas e o sobrenadante armazenado para análise. A atividade da protease foi medida pelo Método de KUNITZ (1947), utilizando caseína como substrato. A produção de 1124 U. mL-1 de atividade proteolítica foi obtida após 24 horas de fermentação. Com o objetivo de utilizar os próprios rejeitos industriais disponíveis em grande quantidade e sem custo como fonte de nitrogênio para o enriquecimento do meio de cultivo foi empregada água residuária do abatedouro de aves Pena Branca suplementada com caseína e MgSO4 na produção da enzima. O emprego do extrato enzimático, contendo protease, em uma das etapas do tratamento de efluente foi avaliado em um sistema de lodo ativado em reator tipo batelada, variando-se a água residuária e o lodo ativado. Nos primeiros ensaios foram colocados nos reatores o lodo proveniente de um reator do abatedouro de aves Pena Branca, o efluente bruto, a cultura de Bacillus subtilis ou o extrato enzimático. Em ensaios posteriores utilizou-se o lodo aclimatado, água residuária sintética, a cultura de Bacillus subtilis ou o extrato enzimático. Comparando os resultados dos tratamentos das águas residuárias do abatedouro de aves Pena Branca e sintética, as taxas de remoção de DQO da água residuária sintética foram em média 4 vezes maiores do que as taxas de remoção de DQO da água residuária do abatedouro de aves Pena Branca. A atividade do extrato enzimático B utilizado na água residuária sintética foi maior do que a do extrato enzimático produzido pela água residuária do abatedouro de aves Pena Branca, 1.124 U.mL-1 e 730 U.mL-1, respectivamente
Abstract: Considerable researches were carried out in the last years to discover new enzymes for treatment of residues. Due to the increase of pollutants in the environment, becoming difficult its removal for conventional processes, the development of these researches seek a alternative treatment process, lower cost, faster, simpler and more trustworthy. Proteases are the most important class of industrial enzymes and comprise about 25% of commercial enzymes on the world. The major applications of these enzymes are in the food, pharmaceutical and detergent industries. The purpose of this work consisted of a study to determine the best conditions for production of protease, as well as its efficiency in enzymatic hydrolysis on the biological stage in the liquid effluent treatment. Bacillus subtilis was tested for protease production. Inoculum was prepared in 250-mL Erlenmeyer flasks containing 50 mL BHI strain, previously sterilized (121ºC, 15 minutes). The Erlenmeyer flasks were incubated at 30ºC for 24 h under agitation at 200 rpm. A Fractionary Factorial Experimental Planning was carried out in 24-1 with triplicate in the central point for protease production, independent variables were lactose, sodium chloride and casein concentration and pH. Strain described by KEMBHAVI et al (1993) was used as basis medium. Based in the results of the experimental planning a kinetic trial was carried out in fermentor type agitated tank containing lactose (5.0 g/L), casein (5.0 g/L), pH 6, 30ºC and 300 rpm for 30 h. The medium was inoculated in 10% (v/v) of inoculum proportion. The samples were removed in intervals of 1h, centrifuged and the supernatant stored for analysis. Protease activity was measured by the method of KUNITZ (1947) using casein as substrate. The production of 1,124 Um.L-1 proteolytic activity was obtained after 24 h of fermentation. With the objective to use the proper industrial wastes available in great amount and no cost as nitrogen source for the enrichment of the culture medium were carried out using wastewater from poultry slaughter Pena Branca supplemented with casein and MgSO4 for enzyme production. Using the enzymatic extract, contained protease, in one of the stages of the effluent treatment was evaluated in a batch reactor with activated sludge, varying the wastewater and the activated sludge. In the first assays were placed into the reactors the sludge (from poultry slaugther Pena Branca reactor), the crude effluent, Bacillus subtilis strain or enzymatic extract. Posterior assays were carried out with acclimatized sludge, synthetic wastewater, Bacillus subtilis strain or enzymatic extract. Comparing the results of the wastewater from poultry slaughter Pena Branca and synthetic wastewater treatments, the COD removal rates from synthetic wastewater were 4-fold higher than the wastewater from poultry slaughter Pena Branca. The enzymatic activity of B extract used in the synthetic wastewater was higher than the enzymatic extract produced by wastewater from poultry slaughter Pena Branca, 1,124 U.mL-1 and 730 U.mL-1, respectively
Doutorado
Doutor em Engenharia de Alimentos

Chez Bacillus subtilis nous avons identifié un complexe protéique membranaire impliquant une protéine inconnue, YteJ, et une autre protéine membranaire, SppA, une signal peptide peptidase également impliquée dans la résistance aux peptides antibactériens de la famille des lantibiotiques. Après délétion des gènes correspondant, nous avons montré que les deux protéines sont impliquées dans cette résistance. Dans la souche ΔsppA, la surexpression ectopique de SppA a non seulement restauré la résistance, mais elle a également induit la formation de cellules allongées, un phénotype supprimé par la surexpression simultanée de YteJ. L'expression de versions tronquées de YteJ a mis en évidence le rôle inhibiteur d'un domaine spécifique de YteJ. Enfin, des études biochimiques in vitro ont confirmé que l'activité de la protéase SppA était fortement réduite par la présence de YteJ, confirmant l'hypothèse d'une inhibition par YteJ. Nos études in vivo et in vitro ont montré que YteJ, via l'un de ses domaines, agit comme régulateur négatif de l'activité protéase de SppA dans ce complexe. En conclusion, nous avons montré que le complexe SppA/YteJ est impliqué dans la résistance aux lantibiotiques à travers l’activité protéase de SppA, elle-même régulée par YteJ
We have identified a membrane protein complex of Bacillus subtilis involving an unknown protein, YteJ, and SppA, a membrane protein first described as a signal peptide peptidase and later shown to be also involved in the resistance to antibacterial peptides of the lantibiotic family. Using deletion mutant strains, we showed that both proteins are involved in this resistance. In the ΔsppA strain, the ectopic overexpression of SppA not only restored the resistance, it also induced the formation of elongated cells, a phenotype suppressed by the simultaneous overexpression of YteJ. Furthermore, the expression of truncated versions of YteJ pinpointed the inhibitory role of a specific domain of YteJ. Finally, in vitro biochemical studies showed that SppA protease activity was strongly reduced by the presence of YteJ, supporting the hypothesis of an inhibition by YteJ. Our in vivo and in vitro studies showed that YteJ, via one of its domain, acts as a negative regulator of the protease activity of SppA in this complex. In conclusion, we have shown that SppA/YteJ complex is involved in lantibiotic resistance through the protease activity of SppA, which is regulated by YteJ