Propionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram-positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudil’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129¿slpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhési / Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti-inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probioticinteraction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129¿slpBa mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129¿slpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, th

Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23952号 / 農博第2501号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5387(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.