Self-assembling protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and many additional processes, which makes them appealing targets for therapeutics and biotechnological applications, including live vaccines, liposome drug delivery and biosensors. Unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of specialized S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Characterization of this interaction in the Gram-positive model organism Paenibacillus alvei CCM 2051T reveals that, remarkably, binding-site switches can occur between two distinct SLH-domain SCWP receptor-site grooves in the S-layer protein SpaA, possibly as part of a mechanism to alleviate strain in the S-layer. To date, however, analysis of this novel mechanism has been limited to the terminal SCWP monosaccharide and the internal SCWP repeat disaccharide ligand analogues, leaving open the role of subsequent SCWP sugar residues in binding, as well as whether the two receptor sites are also suited to accommodate longer SCWP ligands that better approximate the biological target at the surface of P. alvei. To address this, the objective of this work aims to uncover and characterize the details of the SpaA SLH-domain (SpaASLH¬) SCWP-interaction by determining the co-crystal structures of SpaASLH¬, and single (SpaASLH/G109A) and the corresponding double (SpaASLH/G46A/G109A) mutants in complex with synthetic terminal disaccharide and trisaccharide analogues of the P. alvei CCM 2051T SCWP target. These structural characterizations have been supplemented with disaccharide and trisaccharide binding data, which was obtained through thermodynamic ITC analyses carried out by collaborators. The co-crystal structures of P. alvei SpaASLH with synthetic, terminal SCWP disaccharide and trisaccharide analogues, together with previously published monosaccharide-bound SpaASLH structures, reveal that while the SLH trimer accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly-exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations for longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division. In addition, to further clarify the steps involved in SCWP biosynthesis, we present a crystal structure of the unliganded UDP-GlcNAc 2-epimerase enzyme MnaA, which catalyzes the interconversion of UDP-GlcNAc into UDP-ManNAc—an essential building block of the P. alvei SCWP target. The P. alvei MnaA epimerase adopts a GT-B fold that is consistent with the architecture of previously published structures of other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerase enzymes for which substrate binding is observed in the cleft located between the two domains. Characterization of this structure, coupled with an analysis of the sequence of the MnaA protein, reveals the presence of conserved residues that define the catalytic and allosteric sites in homologous enzymes from different organisms. These residues are positioned to accommodate substrate within the MnaA binding cleft in much the same manner as the published enzyme homologues, suggesting that allosteric regulation as a mechanism for enzyme regulation is conserved in P. alvei MnaA. These investigations are part of a greater effort toward understanding SLH domain-mediated SCWP-interactions in Gram-positive organisms, and provide insight into the structure and putative function of this SCWP biosynthetic enzyme. By understanding these processes, this knowledge may contribute to providing a platform for the rational design of Gram-positive inhibitors. Such inhibitors could selectively target, for example, the bacterial S-layer SCWP-binding interaction, or perhaps the essential biosynthetic enzymes involved in producing the exclusive targets that these S-layer proteins recognize and bind, and would thus represent a new class of antimicrobial therapeutics. / Graduate

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.