La spore bactérienne est une forme microbienne multicouche extrêmement résistante aux perturbations environnementales. Cette résistance est notamment liée à sa structure unique qui est particulièrement peu perméable et compacte. Ce travail de thèse a pour but d’identifier et de caractériser les structures sporales impliquées dans ces propriétés. Des méthodes d’investigations globales comme la RMN ou l’anisotropie de fluorescence ont permis de montrer que le cortex des spores de Bacillus subtilis est modifié par la température, pour des valeurs proches de celle de l’activation de la germination. Ceci aura pour conséquence de modifier l’accès à la membrane interne. Un outil d’étude à l’échelle de la spore, l’imagerie en temps de vie de fluorescence (FLIM) couplé à l’utilisation d’un rotor moléculaire, a également été mis au point. Cet outil a permis de mettre en évidence que la membrane interne de B. subtilis possède une très forte viscosité, environ deux fois plus importante que celle de la membrane d’une cellule végétative. Cette viscosité n’est modifiée par la température qu’au-delà de 65 °C, correspondant également à l’activation de la germination. Une perturbation connue pour modifier l’intégrité de la structure de la spore a également été étudiée : l’éthanol couplé à une température importante (65 ou 70°C). Ce traitement est responsable d’une perméabilisation et d’une inactivation des spores. L’éthanol conduit notamment à l’altération de la membrane interne, dont la viscosité et la perméabilité sont modifiées. Ces résultats apportent de nouvelles données pour la compréhension des mécanismes responsables de l’inactivation des spores. Ils permettent d’envisager des applications, pour lesquelles une maitrise des modifications structurales est nécessaire, comme la microencapsulation. / The bacterial spore is a multilayer microbial form which is extremely resistant to environmental perturbations. This resistance is especially due to its unique structure which is particularly compact and weakly permeable. This work aims to identify and characterize the spore structures involved in these properties. Overall investigation methods, such as NMR and fluorescence anisotropy, have shown that the cortex of Bacillus subtilis spores is modified by temperature for level similar to that of the activation of germination. This will result in changes to the access to the inner membrane. A tool at the spore’s scale, the fluorescence lifetime imaging microscopy (FLIM) in conjunction with the use of a molecular rotor, has been set up. This tool allowed demonstrating that inner membrane of B. subtilis has a very high viscosity, about two times greater than that of the membrane of a vegetative cell. This viscosity is changed by temperature near 65 °C, which corresponds to activation of germination. A stress known to modify the structural integrity of the spore has also been studied: ethanol combined with significant temperature (65 ou 70 °C). This treatment is responsible for inactivation of spores in parallel with their permeabilization. Ethanol especially leads to alteration of the inner membrane for which the viscosity and permeability are changed. These results provide new understanding of mechanisms implicated in spores’ destruction. They allow considering some new applications, for which it is necessary to control structural changing, for example microencapsulation.

Les probiotiques sont des « microorganismes vivants qui, lorsqu’ils sont ingérés en quantité suffisante, exercent un effet positif sur la santé de l’hôte ». Ils agissent en modulant le système immunitaire, en empêchant l’adhésion et/ou la croissance des bactéries pathogènes, en renforçant la barrière intestinale et en stabilisant sa microflore. Cependant, les mécanismes d’action de ces bactéries restent encore peu connus.Ce travail de thèse propose d’élucider les modes d’action de deux souches de Bacillus probiotiques humains : Bacillus clausii O/C et de Bacillus subtilis CU1.L’adhésion des probiotiques aux surfaces intestinales est un facteur important pour leur persistance dans l’organisme, l’immunomodulation et la compétition envers les agents pathogènes. B. clausii et B. subtilis présentent de fortes capacités d’adhésion sous forme de spores grâce à leurs protéines de surface préférentiellement impliquées dans les interactions avec l’hôte. En effet, celles-ci jouent un rôle prépondérant dans la stimulation de l’expression des gènes codant les cytokines dans les cellules Caco-2 et la production de cytokines par les cellules immunitaires, induite par les souches probiotiques, via la liaison avec des récepteurs de l’hôte. Des protéines S-layers, protéines ribosomales et protéases ont été identifiées à la surface de ces souches, ainsi qu’une grande quantité de flagelline à la surface de B. subtilis.Par ailleurs, les composés sécrétés par les souches stimulent également la production de cytokines chimiotactiques et anti-inflammatoires. B. clausii sécrète une protéase capable de neutraliser plusieurs types de toxines dont celles sécrétées par C. difficile et B. cereus. B. subtilis n’a montré aucune propension pour l’inhibition de l’adhésion des agents pathogènes testés, mais un essai clinique a démontré sa capacité à moduler le système immunitaire et la composition du microbiote intestinal. / Probiotic are ”live microorganisms, which when administered in adequate amounts confer a health benefit on the host”. They act by modulating the immune system, preventing the adhesion and / or growth of pathogenic bacteria, reinforcing the intestinal barrier and stabilizing the microbiota.However, the mechanisms of action of these bacteria are still poorly understood.This thesis proposes to elucidate the mode of action of two human probiotic strains of Bacillus : Bacillus clausii O / C and Bacillus subtilis CU1.The adhesion of probiotics to intestinal surfaces is an important factor for their persistence in the host, immunomodulation and competition with pathogens. B. clausii and B. subtilis have strong abilities to adhere as spores, through their surface-associated proteins which are preferentially involved in interactions with the host. Indeed, they play a key role in the up-regulation of gene expression encoding cytokines in Caco-2 cells and in the stimulation of cytokine production by immune cells, induced by probiotic strains, through binding with host receptors. Some S-layers proteins, ribosomal proteins and proteases have been identified on the surface of these strains, and a large quantity of flagellin on the surface of B. subtilis.In addition, secreted compounds of these probiotics also stimulate the production of chemotactic and anti-inflammatory cytokines. B. clausii secretes a protease able to neutralize several types of toxins, including those secreted by C. difficile and B. cereus. B. subtilis has not predisposition to compete with the adhesion of pathogens tested, but a clinical trial has demonstrated its ability to modulate the immune system and the composition of the intestinal microbiota.

Made available in DSpace on 2017-06-01T18:20:37Z (GMT). No. of bitstreams: 1 dissertacao_joao_caitano_alves_neto.pdf: 777326 bytes, checksum: 2e15b6afc4e315a3f94d61e55ec5b9ce (MD5) Previous issue date: 2012-09-04 / The reuse of agro-industrial waste as sources of carbon and nitrogen has been investigated in biotechnology for the production of enzymes by microorganisms. Among the microbial enzymes imported into Brazil, the proteases are applied in technological processes in the fields of detergents, pharmaceuticals, cosmetics, food, among others. The aim of this work was to produce proteases by submerged cultivation in the presence of agro-industrial waste. The determination of proteolytic activity was in the presence of 0.2 % azocasein. Submerged cultivation of Bacillus sp. strains isolated were carried out in Erlermeyer flasks. The maximum protease activity was determined in the presence of corn steep liquor. The concentration of the liquid metabolic by ultrafiltration of the metabolic liquid with proteolytic activity retained 80% of the activity. A factorial experimental design was carried out to investigate the stability of the metabolic liquid. The maximum proteolitic activity of the liquid metabolic cell-free (196 U/mL) was determined in the presence of 0.5 % sodium sorbate, 0.5 % calcium chloride, and 7.5 % of glycerol and polyethyleneglycol-200. The enzyme extract formulated retained 68 % of the proteolytic activity after 10 days at storage at room temperature (28 ˚C). The retentate with proteolytic activity showed optimum pH 9 and 11 and retention 90 - 100 % of the activity for 90 min at optimum pH; the optimum temperature was 50 ˚C e the maximum thermal stability was at 40 ˚C for 30 min at pH 11. The formulation of metabolic liquid concentrate with proteolytic activity which has thermal stability at alkaline pH is a bioproduct which can be used as an additive in detergents. / O reaproveitamento de resíduos agroindustriais como fontes de carbono e de nitrogênio tem sido investigado na área de biotecnologia para produção de enzimas por micro-organismos. Dentre as enzimas microbianas importadas no Brasil, as proteases são aplicadas no processamento tecnológico de detergentes, fármacos, cosméticos, alimentos, dentre outros. O objetivo deste trabalho foi produzir proteases por cultivo submerso utilizando resíduos agroindustriais. A determinação da atividade proteolítica ocorreu na presença de azocaseína a 0,2 %. Cultivos submersos de culturas isoladas de Bacillus sp. foram realizados em frascos de Erlermeyer. A atividade máxima de proteases foi determinada na presença de milhocina. A concentração do líquido metabólico com atividade proteolítica por ultrafiltração reteve cerca de 80 % da atividade inicial. Um planejamento experimental fatorial foi realizado para investigar a estabilidade do líquido metabólico. A maior atividade proteolítica média do líquido metabólico livre de células (196 U/mL) foi determinada na presença de sorbato de sódio a 0,5 %, cloreto de cálcio a 0,5 %, glicerol a 7,5 % e polietilenoglicol-200 a 0,5 %. O extrato enzimático formulado reteve 68 % da atividade proteolítica com 10 dias de armazenamento à temperatura ambiente (28 ˚C). O retentado com atividade proteolítica apresentou pH ótimo 9 e 11 e retenção de 90 - 100 % da atividade durante 90 min em pH ótimo; a temperatura ótima foi 50 ˚C e a estabilidade térmica máxima a 40 ˚C durante 30 min a pH 11. A formulação de líquido metabólico concentrado com atividade proteolítica que apresenta estabilidade térmica em pH alcalino é um bioproduto que pode ser utilizado como aditivo em detergentes.

A citocinese bacteriana é controlada por diversas proteínas que se agrupam em um complexo chamado divisomo. O cerne do divisomo é constituído por FtsZ, uma proteína homóloga à tubulina eucariótica, que se auto-associa formando uma estrutura chamada anel Z. O anel Z serve como arcabouço e recruta diversas outras proteínas componentes do divisomo para o sítio onde o septo será sintetizado na célula. A formação do anel Z é modulada por proteínas que se ligam diretamente a FtsZ e regulam a sua auto-associação, tanto induzindo como inibindo a sua polimerização. Apesar de muitos destes moduladores de FtsZ já serem conhecidos, muito pouco se sabe sobre o mecanismo pelo qual eles controlam a estruturação do anel Z in vivo. O objetivo do presente trabalho foi estudar a interação entre FtsZ e um modulador de divisão, a proteína ZapA, da bactéria gram-positiva Bacillus subtilis. Para isso construímos uma biblioteca de mutantes de ftsZ por \"Error Prone PCR\", com aproximadamente 1 substituição por cópia de ftsZ e contendo um total de 1x105 clones. A partir dessa biblioteca, utilizamos duas triagens genéticas para identificar mutantes incapazes de interagir com ZapA. Na primeira estratégia, selecionamos 12 mutantes de FtsZ resistentes à superexpressão de uma forma tóxica de ZapA, que bloqueia a divisão, causando filamentação e morte das células. Surpreendentemente, apesar destes mutantes serem insensíveis ao efeito de ZapA, ensaios citológicos mostraram que nenhum deles perdeu a interação com ZapA. Como as mutações foram mapeadas nas vizinhanças do sítio catalítico e de polimerização de FtsZ, e como a maioria delas confere resistência cruzada aos efeitos de outros moduladores de FtsZ, suspeitamos que elas afetassem a estabilidade do polímero de FtsZ e, consequentemente, o comportamento do anel Z. Essas suspeitas foram confirmadas em ensaios de FRAP e cálculos de proporção de FtsZ no anel Z, indicando que os mutantes formam um anel Z mais estável que o normal. Como não obtivemos mutantes que perderam a interação com ZapA na primeira triagem, aplicamos a biblioteca em uma segunda estratégia de triagem genética, procurando um mutante de FtsZ que voltasse a interagir com um mutante de ZapA que não se liga mais a FtsZ (ZapAN62A). Esta estratégia de ganho de função identificou um candidato, FtsZE91V , que, tanto por critérios genéticos como citológicos, voltou a interagir com ZapAN62A. Apesar do mutante FtsZE91V mostrar-se capaz de restaurar a interação com ZapAN62A, ele não afetou a interação com ZapA selvagem, segundo nossos ensaios de microscopia de fluorescência e viabilidade. O mutante FtsZE91V, mapeia na hélice H3 de FtsZ. Esta hélice está exposta na superfície de FtsZ (compõe um dos lados da molécula de FtsZ) de uma maneira compatível com a idéia de que ela seria importante para interações laterais entre polímeros de FtsZ. Nossos resultados apontam, portanto, que a hélice H3 deve ser o sítio de interação para ZapA em FtsZ. / The bacterial cytokinesis is ruled by a number of proteins that constitute the divisome complex. FtsZ, a homologue of eukaryotic tubulin, is the main component of the divisome and self-associates in a structure named Z ring. The Z ring works as a scaffold and recruits the other components of divisome, establishing itself where the septum will be synthesized in the cell. Some of these proteins interact directly with FtsZ and control self-association, promoting polymerization or preventing it. Although there have been discovered many of FtsZ modulators, little is known about the mechanisms that control the formation of the Z ring in vivo. The aim of this work was study de interaction between FtsZ e one of its division modulators, ZapA protein, on Bacillus subtilis grampositive bacteria. We created a mutagenized ftsZ plasmid library by error prone PCR, which contained 1,0x105 transformants and exhibited a mutation rate of one substitution per ftsZ copy. The library was transformed into a modified Bacillus subtilis strain and we performed two genetic screenings to select cells with FtsZ mutants incapable of interacting with ZapA. In first strategy, we selected 12 resistant ftsZ mutants for a toxic ZapA overexpression, that blocked division and caused filamentation and cell death. Surprisingly, although these mutants were insensitive to ZapA effect, cytological assays showed that none of them lost interaction with ZapA. As the substitutions were mapped around the catalytic and interaction site of FtsZ structure and showed resistance to other modulators, we suspected that the mutations were affecting the polymer stability of FtsZ and, consequently, the behavior of Z ring. This hypothesis was confirmed by FRAP experiments and by calculations of FtsZ proportions in Z ring, pointing out that the mutants form more stable Z rings. As we didnt\' find mutants that lost their ZapA´s interaction, we applied our library in a second genetic screen, looking for mutants that return to interact with a ZapA mutant (ZapAN62A) that doesn´t bind to FtsZ anymore. This gain of function strategy identified one candidate, FtsZE91V, which returns to interact with ZapAN62A in our genetic and cytological assays. Although the mutant FtsZE91V showed itself capable to interact with ZapAN62A, that didn´t affect the interaction with wild type ZapA by our fluorescent microscopy and viability assays. The substitution E91V was mapped on H3 helix of FtsZ structure. This helix is exposed on FtsZ surfaces (on FtsZ´s lateral side), being compatible with the idea that lateral interaction is important in FtsZ polymers. So, we concluded that helix H3 is the binding site of ZapA in FtsZ.

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.

Poly-gamma-glutamic acid (γPGA), which is a biodegradable, non-immunogenic and unusual anionic amino-acid polymer consist of D- and L-glutamic acid units, was exploited for a wide array of useful applications. Bacillus are well known cellular system important for fermentation to synthesize γPGA, which is used as thickener, drugs carrier, cryoprotectant, humectant, biological adhesive, flocculants, or heavy metal absorbent. This study focused on the isolation of Bacillus spp. that is possible to produce γ-PGA from different soil samples from different places in Vietnam. Study the effect of precursors, temperature, carbon sources, times and pH on γ-PGA production. From 31 soil samples and 4 straws samples, strain 20.2 which produced the highest γ-PGA yields (riches 15.2 mg/ml), was identified as Bacillus sp. 20.2 by molecular biology method. The suitable conditions for growing of Bacillus sp. 20.2 strain to produce γ-PGA are at 37°C, pH 7 after 72 hours. Citric acid instead of glucose in a GSP medium is better for producing γ-PGA by strain Bacillus sp. 20.2. / Poly-gamma-glutamic acid (γ-PGA) là một polymer amino-acid gồm D và L-glutamic acid, có khả năng phân hủy sinh học, không gây miễn dịch, đã được ứng dụng rộng rãi trong công nghiệp, y học. Bacillus subtilis được biết đến là hệ thống tế bào ý nghĩa quan trọng trong quá trình lên men để tổng hợp γ-PGA. γ-PGA hòa tan trong nước, phân hủy sinh học và không độc đối với con người và môi trường. γ-PGA ổn định với nhiều protease vì các protease thường không nhận acid γ- glutamic (Obst et al., 2004). γ-PGA có cấu trúc đồng phân đơn giản, không gây miễn dịch. Do đó, γ-PGA đã được quan tâm ứng dụng trong các lĩnh vực như y học, công nghiệp thực phẩm, mỹ phẩm và đặc biệt là xử lý nước nhiễm kim loại nặng. Trong nghiên cứu này chúng tôi tập trung phân lập, tuyển chọn các chủng Bacillus có khả năng sinh tổng hợp PGA cao. Sau đó định danh và đánh giá khả năng sinh tổng hợp PGA từ chủng đã phân lập được. Kết quả cho thấy từ 34 mẫu rơm và đất, chúng tôi đã phân lập được chủng với mã số 20.2 có khả năng sinh PGA cao nhất đạt 15.2 mg/ml. Chủng này đã được định danh bằng phân tích trình tự gene 16S rRNA và thuộc loài Bacillus sp. Môi trường thích hợp sinh tổng hợp PGA là GSP ở điều kiện 37oC pH7 sau 72 giờ nuôi cấy.

Antifungal proteins were isolated from the crude bacterial supernatant using ammonium sulfate salt precipitation followed by passage over DEAE -cellulose and Biogel P100 columns. The purified protein had an apparent molecular mass of 14 kDa. Its antifungal activity was retained even at 100°C, for 60 min. The results of protein identification using MALDI -TOF/TOF mass spectrometer suggested that the purified protein is indeed a chitin binding protein that has 206 acid amine containing chitin -bind -3 region with a relative molecular mass of 22230 Da. / Protein có hoạt tính kháng nấm được tinh sạch từ dịch ngoại bào chủng vi khuẩn Bacillus subtilis sau khi qua ba bước tinh sạch: tủa muối ammonium sulphate 30-70%, qua cột sắc ký trao đổi ion DEAE – cellulose và cột săc ký lọc gel Biogel P100. Protein tinh sạch có khối lượng phân tử đạt 22 kDa trên điện di SDS-PAGE. Hoạt tính kháng nấm của protein tinh sạch vẫn còn duy trì khi ủ ở 100°C trong 60 phút. Kết quả nhận dạng bằng khối phổ MALDI -TOF/TOF đã chỉ ra rằng protein bền nhiệt này là chitin binding protein được mã hóa bởi 206 acid amin cùng với khối lượng phân tử là 22230 Da. trị an toàn đối với Al và Atrazie trong môi trường nước tự nhiên về khía cạnh bảo vệ sức khỏe sinh thái.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Antagonism of antimicrobial action represents an alternative survival strategy for cohabiting soil organisms. Under competitive conditions, our group previously showed that surfactin (Srf) produced by Bacillus subtilis acts antagonistically toward gramicidin S (GS) from a cohabiting bacillus, Aneurinibacillus migulanus, causing the loss the antimicrobial activity of GS. This antagonism appeared to be caused by inactive complex formation. This study aimed to elucidate whether the previously observed antagonism of GS activity by Srf is a general resistance mechanism that also extends to related peptides such as the tyrocidines (Trcs) and linear gramicidins (Grcs) from Bacillus aneurinolyticus. Molecular interaction between the antagonistic peptide pairs was investigated using biophysical analytical methods such as electrospray mass spectrometry (ESMS), circular dichroism (CD), fluorescence spectroscopy (FS) and nuclear magnetic resonance (NMR). Results from this study corroborated the previous findings, namely that Srf antagonised the activity of GS towards Gram positive bacteria. However, for Micrococcus luteus synergism of GS action was observed at low Srf concentrations, while antagonism only occurred at Srf concentrations above the critical micelle concentration (CMC) of Srf when the bacteria were pre-incubated with Srf. This result and an ultra-performance liquid chromatography massspectrometry (UPLC-MS) study indicated that Srf pre-absorbed to cells, as well as Srf micelles interacted with GS, preventing GS from reaching the membrane target. Antagonism of GS action by Srf was also observed towards the Srf producer B. subtilis ATCC21332 and B. subtilis OKB120, a non-producer. The Srf producer was less sensitive than the nonproducer towards GS, possibly due to Srf production. Pre-incubation of Srf at different concentrations caused a dose-dependent antagonism, from as low as 0.9 μM Srf of GS activity towards B. subtilis OKB120. This antagonism at the low Srf concentration may be related to the induction of more resistant biofilms by Srf in B. subtilis. It was also found that Srf significantly improved the survival of B. subtilis OKB120 above that of M luteus in a mixed culture. In addition, the Srf producer B. subtilis ATCC21332 grew in the inhibition zone of the GS producer A. migulanus ATCC9999 during co-culturing, while B. subtilis OKB120 growth was inhibited. Srf induced biofilm formation in B. subtilis may be important in protecting the bacteria in solution, but not on solid phase such as on or in agar plates. Also, the protection of various cell types (previous studies by our group) by Srf from GS indicated a directed antagonistic Srf mode of action. Srf formed complexes that are visible and stable under ESMS conditions with GS, with the peptide bonds in the Val-Orn-Leu-D-Phe moiety of GS and the Val-Asp- D-Leu-Leu moiety of Srf protected from fragmentation. 1H-NMR titration studies strongly indicated that the molecular interaction of Srf and GS involved the re-orientation of the DPhe4,9 and Orn2,7 residues in GS. From CD spectra it was observed that Srf induced a concentration dependent decrease in the β-turn component and increase in β-sheet structures of the GS-Srf mixture. Diffusion orientated NMR (DOSY) indicated that Srf and GS formed homo-oligomers with the Srf-GS mixture having a slightly higher diffusion coefficient indicating the formation of smaller homo-oligomers or more compact hetero-oligomers. These hetero-oligomers involve intermolecular interaction at <5Å between the Orn2,7 residue of GS with Asp residue of Srf, as observed with ROESY-NMR. These results strongly indicate that inactive complex formation between Srf and GS is part of the antagonistic mechanism of action of Srf towards GS. Two high performance liquid chromatography (HPLC) methods was developed to purify peptides from the tyrothricin complex, namely the Trcs (contains one GS Val-Orn-Leu-DPhe- Pro moiety) and Grcs. These peptides were used to assess if Srf has an antagonistic activity beyond that of GS. Srf indeed showed antagonistic action against the antimicrobial activity of Trcs towards B. subtilis ATCC21332 and OKB120, with the tyrocidine C (TrcC) being more sensitive to antagonism than tyrocidine B (TrcB). Srf had an ambiguous effect on the linear gramicidin A (GA) that is co-produced with Trcs in tyrothricin. GA acted synergistically with Srf at low GA concentrations, but slight antagonism was observed at high GA concentrations. In contrast, GA showed pronounced synergism with TrcB towards the M. luteus. However, Srf at 30 μM, antagonised the synergistic action of a lethal mixture of 25 μM GA and TrcB. The Srf producer was also able to withstand and grow in the presence of the tyrothricin producer B. aneurinolyticus ATCC10068, indicating that antagonism of peptide action may allow different organisms to cohabit. Basic NMR and ESMS studies failed to show complex formation between Srf and the Trcs. However, CD presented clear evidence of Srf induced changes in secondary structures and/or higher order self-assembled structures of the Trcs-Srf mixture. FS also provided evidence of the reorientation/exposure of the Trp6 residue of the Trcs in the presence of Srf. These results corroborated the previous findings that complexation between Srf and GS or peptides analogous to GS may be part of the mechanism of Srf antagonistic action. In conclusion, this study showed that the antagonism of GS activity by Srf, conferred in part by inactive complex formation, is a putative resistance mechanism that also extends to other peptides containing the Val-Orn-Leu-D-Phe-Pro moiety such as the Trcs from B. aneurinolyticus. / AFRIKAANSE OPSOMMING: Antagonisme van antimikrobiese aksie verteenwoordig ʼn alternatiewe oorlewingstrategie vir grondorganismes wat in dieselfde habitat gevestig is. Ons groep het gewys dat surfaktien (Srf), geproduseer deur Bacillus subtilis, antagonistiese werking teenoor gramisidien S (GS) vanaf die bacillus Aneurinibacillus migulanus, onder kompeterende kondisies, toon. Die antagonistiese werking, wat moontlik veroorsaak word deur vorming van onaktiewe komplekse, lei tot die verlies van die antimikrobiese aktiwiteit van GS. Hierdie studie se doel was die ontrafeling van die moontlikheid dat die antagonisme van GS aktiwiteit deur Srf, soos deur vorige studies uitgewys, ʼn algemene weerstandsmeganisme is wat moontlik ook verwante peptiede soos die tirosidiene (Trcs) en lineêre gramisidiene (Grcs), afkomstig vanaf Bacillus aneurinolyticus, insluit. In hierdie studie is die molekulêre interaksie tussen antagonistiese peptiedpare ondersoek met biofisiese analitiese metodes wat elektrosproeimassaspektroskopie (ESMS), sirkulêre dichroïsme (SD), fluoressensie-spektroskopie (FS) en kernmagnetiese resonansspektroskopie (KMR) insluit. Die resultate wat tydens hierdie studie verkry is, het gewys dat Srf die werking van GS teenoor Gram-positiewe bakterie teenwerk, en het die vorige waarnemings ondersteun. Daar is egter sinergisme tussen Srf en GS werking by lae Srf-konsentrasies teenoor Micrococcus luteus waargeneem, terwyl antagonisme slegs waargeneem is by Srf-konsentrasies hoër as die kritiese miselêre Srf konsentrasie wanneer bakterieë vooraf met Srf met inkubeer is. Hierdie resultaat, tesame met ʼn ultra-hoë verrigting vloeistofchromatografie gekoppelde massaspektroskopie (UPLC-MS) studie, het daarop gedui dat Srf wat voorheen op selle geabsorbeer het, sowel as Srf-miselle in die media, met GS interaksie het en sodanig kan voorkom dat GS die membraanteiken bereik. Antagonisme deur Srf op die GS aktiwiteit is ook waargeneem teenoor die Srf-produseerder B. subtilis ATCC21332 en B. subtilis OKB120, ʼn nie-produseerder. Hierdie tipe antagonisme by ʼn lae konsentrasie van Srf mag verwant wees aan die induksie van meer weerstandige biofilms deur Srf in B. subtilis. Dit is ook gevind dat Srf die oorlewing van B. subtilis OKB120 aansienlik verhoog teenoor dié van M luteus in ʼn gemengde kultuur. Daar is verder bevind dat die Srf-produseerder, B. subtilis ATCC21332, in die inhibisiesone van die GS-produseerder, A. migulanus ATCC9999, gegroei het tydens kokultivering, terwyl die groei van B. subtilis OKB120 geïnhibeer is. Srf induseer biofilm-vorming in B. subtilis wat moontlik belangrik kan wees om die bakterieë in suspensie te beskerm, maar nie op soliede fase soos byvoorbeeld agar plate nie. Verder dui die beskerming van ʼn verskeidenheid sel-tipes (vorige studies deur ons groep) deur Srf teen GS, ʼn direkte antagonistiese aksie van Srf. Sigbare en stabiele komplekse tussen Srf en GS is waargeneem onder ESMS kondisies, waar die peptiedbindings in die Val-Orn-Leu-D-Phe-Pro eenheid van GS en die Val-Asp-Leu-D-Leu eenheid van Srf beskerm is teen fragmentering in die komplese. 1H-KMR titrasiestudies het duidelik aangetoon dat die molekulêre interaksie van Srf en GS die D-Phe4,9 en Om2, 7 residue in GS heroriënteer. SD-spektra van GS-Srf mengsels het daarop gedui dat Srf ʼn konsentrasieafhanklike vermindering in die β-draai komponente van die mengsel veroorsaak, maar dat β- plaat komponent van die mengsel vermeerder. Diffusie-georiënteerde KMR spektrometrie (DOSY) toon dat Srf en GS homo-oligomere vorm, maar ʼn hoër diffusie koeffisiënt vir die mengsel het aangedui dat die Srf-GS mengsel kleiner of meer kompakte hetero-oligomere. ROESY-KMR toon dat hierdie oligomere intermolekulêre interaksie(s) van <5Å tussen die Om2, 7 residue van GS en die Asp residu van Srf het. Die resultate gee ʼn sterk aanduiding dat die onaktiewe kompleks-vorming tussen Srf en GS deelneem in die antagonistiese werking van Srf teenoor GS. Twee hoë verrigting vloeistofchromatografie metodes is ontwikkel om peptiede uit die tirotrisienkompleks, naamlik die Trcs (bevat een GS Val-Om-Leu-D-Phe-Pro eenheid) en die gramisidiene (Grcs), te suiwer. Hierdie peptiede is gebruik om te bepaal of Srf antagonistiese aktiwiteit het wat verder strek as net dié van GS. Dit was inderdaad die geval en daar is gevind dat Srf antagonisties is teenoor die antimikrobiese aktiwiteit van Trcs met B. subtilis ATCC21332 en OKB120 as teikens, met tirosidien C (TrcC) wat meer sensitief vir antagonistiese werking van Srf was as tyrosidien B (TrcB). Srf het ʼn gemengde effek getoon teenoor lineêre gramisidien A (GA) wat saam met die Trcs in tirotrisien gekoproduseer word. GA het sinergisties met Srf gewerk by lae GA konsentrasies, maar milde antagonistiese werking getoon by hoë GA konsentrasies. Daarteenoor het GA en TrcB uitgesproke sinergisme getoon teenoor M. luteus. In teenstelling het Srf by 30 μM die sinergistiese aksie van die dodelike mengsel van 25 μM GA en TrcB elk geantagoniseer. Die Srf produseerder was ook bestand en kon in die teenwoordigheid van die tirotrisien produseerder B. aneurinolyticus ATCC10068 groei wat aangedui het dat die antagonisme van antibiotiese peptiedaktiwiteit die kohabitasie van organismes toelaat. Basiese KMR en ESMS studies kon nie kompleksvorming tussen Srf en die Trcs aantoon nie, terwyl SD duidelike bewyse gelewer het dat Srf verandering geïnduseer het in die sekondêre strukture en/of hoër orde/self-geassosieerde strukture van die Trc-Srf mengsel. FS het ook bewyse gelewer van die reoriëntasie/blootstelling van die Trp6 residu in die Trcs in die teenwoordigheid van Srf. Hierdie resultate ondersteun die vorige bevindinge dat kompleksvorming tussen Srf en GS of GS-peptiedanaloë deel van die meganisme van Srf se antagonistiese aksie uitmaak. Samevattend het hierdie studie getoon dat die antagonisme van GS aktiwiteit deur Srf deels toegeken kan word aan onaktiewe kompleksvorming tussen die twee peptiede en dat die voorgestelde weerstandsmeganisme ook ander peptiede wat die Val-Orn-Leu-D-Phe-Pro eenheid, soos die Trcs van B. aneurinolyticus, insluit.

Les grands programmes de séquençage des génomes ont révélé l'existence de nombreux gènes de fonction inconnue. L'invalidation systématique de ces gènes chez les bactéries a permis de révéler le caractère essentiel de certains d'entre eux. L'étude des protéines issues de ces gènes s'est amplifiée ces dernières années car elles sont des cibles potentiellement intéressantes pour le développement de nouvelles molécules antibactériennes. YphC est une GTPase de Bacillus subtilis qui répond à ces critères. Elle est très conservée au sein des bactéries mais n'est pas retrouvée chez les organismes eucaryotes ou les archaebactéries, ce qui fait d'elle une cible de choix pour le développement de nouvelles molécules antibactériennes. YphC a la particularité de posséder deux domaines GTPases en tandem. Unique en son genre, nous avons voulu étudier cette protéine sous son aspect biochimique afin de mieux comprendre son mécanisme de fonctionnement. Nous avons donc mis au point la production et la purification de YphC et généré des mutations ponctuelles ou des délétions. Nous avons ainsi pu mesurer les constantes enzymatiques de cette protéine et caractériser l'effet d'activation du potassium sur son activité d'hydrolyse du GTP. Nous avons ainsi montré la forte activité GTPase de la protéine portée par le premier domaine GTPase et le rôle régulateur du deuxième domaine GTPase. Nous avons également étudié le rôle de YphC par une approche in vitro. Nous avons pu ainsi montrer que YphC est capable d'interagir avec les ribosomes de façon nucléotide dépendante suggérant un rôle de la protéine dans les processus de biogenèse du ribosome.

Spores of foodborne pathogens such as Clostridium botulinum, Clostridium perfringens and Bacillus cereus are widely distributed in nature. Presence of those spores in food products, particularly C. botulinum spores in vacuum packed, ready-to-eat low-acid products, is a great safety concern. The research here described is a first effort towards understanding the role of the spore coat proteins in the inactivation of bacterial spore using high pressure processing. This study proposes a coat protein solubilization methodology using non-ionic detergents minimizing protein damage and compatible with spectroscopy methods. The methodology developed here was compared with approaches proposed in the literature with respect to protein yield, protein fractions identified, amino acid composition and suitability with spectroscopy techniques for the further analysis of coat proteins. Bacillus subtilis ATCC 6633 spore coat proteins were solubilized (n=3) using octyl-β-D-glucopyranoside (OGP) at room temperature and urea/sodium dodecyl sulphate (UDS) at 37C and 70C. Analysis of variance (ANOVA) showed no significant (95% confidence) differences between the three repetitions of the three spore coat protein solubilization methods. Protein yield was significantly larger (95% confidence) when using UDS at 70C as compared to UDS at 37C. OGP gave the lowest protein yield but allowed circular dichroism (CD) analysis of the spore coat protein solution with minimum blank signal. SDS-PAGE revealed that the UDS-70C coat protein solutions consisted of five major and six minor proteins ranging 6 to 65 kD while the OGP solution appear to consist of four major and nine minor bands in the same mw range. Amino acid analysis of the protein extracted by the OGP method was conducted using reverse phase HPLC (RP-HPLC) and compared with published information. The OGP spore coat protein solution showed a higher proportion of aspartate, glutamate, alanine and tyrosine. Pressure, heat and time effects were studied on spore coat proteins obtained from untreated and pressure-treated B. subtilis ATCC 6633 spores. Pressure treatments of spores, and of extracted spore coat protein solutions, at 50 kpsi (345 mPa) and 85 kpsi (586 mPa) for 10 and 30 min at constant 85C along with appropriate heat- and pressure-only controls and untreated sample, were used to study the effect of pressure, heat and time on spore coat proteins. Both spore coat protein solubilization procedures showed a significant reduction in protein yield for pressure-only, heat-only and pressure/heat treated spores when compared with untreated spores. When OGP-solubilized proteins from untreated spores were pressure treated, SDS-PAGE profile showed an increasing overall band intensity with increasing pressure and time. In the case of protein solution obtained from pressure-treated spores the electrophoretic pattern showed the loss of higher molecular weight proteins. The significance of this study is that for the first time we have observed extensive changes on spore coat proteins caused by pressure, as well as heat treatments. Future studies will examine what is the probable physiological role of the proteins damaged by these physical treatments. An advantage of the protein solubilization here developed will allow the application of spectroscopy techniques to characterize changes in spore coat proteins. / Graduation date: 2003