A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.     

High-affinity interaction between the S-layer protein SbsC and the secondary cell wall polymer of Geobacillus stearothermophilus ATCC 12980 determined by surface plasmon resonance technology

Surface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC(31-270)] and rSbsC(31-443)) and the secondary cell wall polymer (SCWP) isolated from Geobacillus stearothermophilus ATCC 12980 confirmed the exclusive responsibility of the N-terminal region comprising amino acids 31 to 270 for SCWP binding. Quantitative analyses indicated binding behavior demonstrating low, medium, and high affinities.     

Isolation,gene structure,and comparative analysis of the S-layer gene <Emphasis Type="Italic">sslA</Emphasis> of <Emphasis Type="Italic">Sporosarcina ureae</Emphasis> ATCC 13881

The surface (S)-layer of Sporosarcina ureae strain ATCC 13881, a periodic ordered structure with p4 square type symmetry, was recently reported to be an excellent biotemplate for the formation of highly ordered metal clusters. The S-layer is formed by self-assembly of a single subunit, the 116 kDa SslA protein. Here we report on the isolation and sequence analysis of the sslA gene. The protein sequence reveals a high degree of similarity to the sequences of other S-layer proteins that form self-assembly lattices with the p4 square type symmetry, especially to those of Bacillus sphaericus. Two conserved surface layer homology (SLH) domains in the extreme aminoterminal portion are likely to mediate attachment of the protein to secondary cell wall polymers. A central HisXXXHis motif and a cysteine residue in the carboxyl-terminal part of the protein, both extremely rare in S-layer proteins, may contribute to the high affinity for metal ions. The strong bias in the codon usage may explain that heterologous expression of SslA in E. coli is not very intense. With respect to the regulatory region we notice several features that are also present in 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.     

Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980.

During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.     

Designing a Cell Surface Display System of Protein Domains in Lactobacilli Based on S-Layer Proteins of <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> ATCC 367

S-layer proteins of lactobacilli may be utilized for developing a surface display system in these bacteria. In this study, S-layer proteins of Lactobacillus brevis ATCC 367 were identified for the first time. Using the peptide fingerprint method, it was shown that the main protein of the S-layer of this strain, SlpE, having a mass of 52 kDa is the product of translation of the consecutive open reading frames LVIS_2086 and LVIS_2085. Repeated sequencing of a genome region of L. brevis ATCC 367, containing LVIS_2086 and LVIS_2085 loci, has showed that the LVIS_2086 sequence contains the TGG tryptophan codon instead of the TAG stop codon. Thus, LVIS_2085 and LVIS_2086 form a single slpE gene, the nucleotide sequence we deposited in the Genbank database under No. KY273133. The translation product of the slpE gene consists of 465 amino acids and has a calculated mass of 51.6 kDa, which corresponds to the experimentally obtained value. An S-layer protein with a mass of 56 kDa, identified as a form of the SlpE, is probably formed during the posttranslational modification. The concomitant 48 kDa S-protein was proven to be product of the LVIS- 2083 gene. The N-terminal domains of LVIS_2083 and SlpE have 70.7 and 96.5%, respectively, identity to the anchoring N-terminal domain of SlpA from L. brevis ATCC 8287, which is responsible for attachment to the cell wall. In this work, fusion proteins consisting of N-terminal domains of Lvis_2083 and SlpA proteins and the eGFP marker protein were obtained. The ability of fusion proteins SlpA_eGFP and Lvis_2083_eGFP, as well as the recombinant Lvis_2083 protein, to be specifically sorbed on the cell wall of L. brevis ATCC 8287, ATCC 367, and L. acidophilus ATCC 4356 strains has been demonstrated. It was shown that in the chimeric Lvis_2083_eGFP construction the N-terminal domain Lvis_2083 is responsible for an attachment to the cell wall and provides display of the functionally active eGFP protein on its surface. Thus, the N-terminal domain Lvis_2083 can be used as a basis of the protein display system on the cell surface of L. brevis strains in vitro.     

The high-molecular-mass amylase (HMMA) of Geobacillus stearothermophilus ATCC 12980 interacts with the cell wall components by virtue of three specific binding regions

The complete nucleotide sequence encoding the high-molecular-mass amylase (HMMA) of Geobacillus stearothermophilus ATCC 12980 was established by PCR techniques. Based on the hmma gene sequence, the full-length rHMMA, four N- or C-terminal rHMMA truncations as well as three C-terminal rHMMA fragments were cloned and heterologously expressed in Escherichia coli . Purified rHMMA forms were used either for affinity studies with the recombinant (r) S-layer protein SbsC (rSbsC), peptidoglycan-containing sacculi (PGS) and pure peptidoglycan (PG) devoid of the secondary cell wall polymer (SCWP), or for surface plasmon resonance (SPR) studies using rSbsC and isolated SCWP. In the C-terminal part of the HMMA, three specific binding regions, one for each cell wall component (rSbsC, SCWP and PG), could be identified. The functionality of the PG-binding domain could be confirmed by replacing the main part of the SCWP-binding domain of an S-layer protein by the PG-binding domain of the HMMA. The present work describes a completely new and highly economic strategy for cell adhesion of an exoenzyme.     

The S-Layer Proteins of Two Bacillus stearothermophilus Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition

Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.     

Molecular and biochemical properties of the S-layer protein from the wine bacterium <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> B706

Different strains of the genus Lactobacillus can be regularly isolated from must and wine samples. By various physiological activities, they can improve or reduce the wine quality. Lactobacillus hilgardii that is known to survive under harsh wine conditions is classified as a spoilage bacterium, e.g. due to the production of histamine. Many lactobacilli form an S-layer as the outermost cell wall component which has been found to facilitate the colonization of special ecological niches. A detailed understanding of the properties related to their S-layer proteins is necessary to improve the knowledge of the interactions between different bacterial cells and with the surrounding environments. The S-layer protein from the wine-related L. hilgardii strain B706 has been isolated and its gene sequence determined. The deduced amino acid sequence corresponds to a 41 kDa protein with an isoelectric point of 9.6 without additional posttranslational modifications after splitting off the leader peptide. The complete protein is organized in a 32 amino acids signal sequence for membrane translocation, a positively charged N-terminal domain that binds to the cell wall and a negatively charged C-terminal domain. When the S-layer was removed, the corresponding L. hilgardii B706 cells became more sensitive to bacteriolytic enzymes and some wine-related stress conditions. From a practical point of view, the S-layer may be considered as a target for the inhibition of food-spoiling lactobacilli.     

Expression and Assembly of Recombinant Surface Layer Proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Most bacterial surface layers (SLs) are formed by self-assembly of a single type of protein. Native and recombinant surface layer monomers are capable to self-assemble on solid substrates and in solution to highly regular nanosized arrays which make them attractive for nanobiotechnological applications. In this study, we expressed the surface layer protein SbsC of Bacillus stearothermophilus ATTC 12980, tagged with Enhanced Green Fluorescent Protein, in the yeast Saccharomyces cerevisiae. We observed a network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis, SL structures were formed de novo in the buds. SL assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point.     

<Emphasis Type="Italic">CbLEA</Emphasis>, a Novel<Emphasis Type="Italic">LEA</Emphasis> Gene from<Emphasis Type="Italic">Chorispora bungeana</Emphasis>, Confers Cold Tolerance in Transgenic Tobacco

A novel late embryogenesis abundant (LEA) gene (AY804193), namedCbLEA, has now been isolated fromChorispora bungeana. This rare alpine subnival plant can survive sudden snowstorms and low temperatures. The full-lengthCbLEA is 842 bp, with an open reading frame encoding 169 ami no acids. The putative molecular weight ofCbLEA protein is 17.9 kDa, with an estimatedpl of 6.45. To investigate the functioning of thisCbLEA protein in cold-stress tolerance,CbLEA was introduced into tobacco under the control of the CaMV35S promoter. Second-generation (R₁) transgenic tobacco plants exhibited significantly increased tolerance to cold. These transgenics maintained lower malondialdehyde (MDA) contents and electrolyte leakage (EL) but their relative water content (RWC) was significantly higher compared with non-transgenic plants under chilling stress. Further experimental results showed that non-transgenic plants had severe freezing damage after exposure to -2°C for 1 h, whereas the transgenics suffered only slight injury under the same conditions. Moreover, survival was longer in the latter genotype at that temperature. The extent of increased cold tolerance was positive correlated with the level ofCbLEA protein accumulation, and was also reflected by the delayed development of damage symptoms. This indicates thatCbLEA is an excellent stress tolerance gene, and holds considerable potential as a new molecular tool for engineering improved plant genetics.     

<Emphasis Type="Italic">In Silico</Emphasis> sequence analysis and molecular modeling of the three-dimensional structure of DAHP synthase from <Emphasis Type="Italic">Pseudomonas fragi</Emphasis>

The shikimate pathway is involved in production of aromatic amino acids in microorganisms and plants. The enzymes of this biosynthetic pathway are a potential target for the design of antimicrobial compounds and herbicides. 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS) catalyzes the first step of the pathway. The gene encoding DAHPS was cloned and sequenced from Pseudomonas fragi, the bacterium responsible for spoilage of milk, dairy products and meat. Amino acid sequence deduced from the nucleotide sequence revealed that P. fragi DAHPS (Pf-DAHPS) consists of 448 amino acids with calculated molecular weight of ∼50 kDa and isoelectric point of 5.81. Primary sequence analysis of Pf-DAHPS shows that it has more than 84% identity with DAHPS of other Pseudomonas species, 46% identity with Mycobacterium tuberculosis DAHPS (Mt-DAHPS), the type II DAHPS and less than 11% sequence identity with the type I DAHPS. The three-dimensional structure of Pf-DAHPS was predicted by homology modeling based on the crystal structure of Mt-DAHPS. Pf-DAHPS model contains a (β/α)₈ TIM barrel structure. Sequence alignment, phylogenetic analysis and 3D structure model classifies Pf-DAHPS as a type II DAHPS. Sequence analysis revealed the presence of DAHPS signature motif DxxHxN in Pf-DAHPS. Highly conserved sequence motif RxxxxxxKPRT(S/T) and xGxR present in type II DAHPS were also identified in Pf-DAHPS sequence. High sequence homology of DAHPS within Pseudomonas species points to the option of designing a broad spectrum drug for the genus. Pf-DAHPS 3D model provides molecular insights that may be beneficial in rationale inhibitor design for developing effective food preservative against P. fragi.     

Isolation and characterization of a new phycoerythrin from the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)₆ subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.     

Molecular characterization of the S-layer gene,sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.     

A scytalone dehydratase gene from<Emphasis Type="Italic"> Ophiostoma floccosum</Emphasis> restores the melanization and pathogenicity phenotypes of a melanin-deficient<Emphasis Type="Italic"> Colletotrichum lagenarium</Emphasis> mutant

Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin. We have isolated and characterized OSD1, a gene encoding scytalone dehydratase from the sap-staining fungus Ophiostoma floccosum by PCR-based cloning. Sequence analysis suggests that the OSD1 gene encodes a protein of 216 amino acids with a molecular weight of 24.2 kDa that shows 51-70% sequence identity to other scytalone dehydratases. The cloned OSD1 contains two introns of 76 bp and 63 bp in length, and is the longest scytalone dehydratase gene sequence so far reported. Transformation of a DHN melanin-deficient, non-pathogenic, mutant of Colletotrichum lagenarium with the OSD1 gene restored melanin production and pathogenicity. The ability of the mutant to produce the OSD1 gene product was confirmed by RT-PCR analysis. These data show that the cloned OSD1 gene product can function in the DHN melanin biosynthetic pathway in C. lagenarium.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of Arginine Deiminase from <Emphasis Type="Italic">Pseudomonas plecoglossicida</Emphasis> CGMCC2039 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and Its Anti-Tumor Activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent in clinical trials for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. The arcA gene encoding ADI was cloned from a recently isolated strain Pseudomonas plecoglossicida CGMCC2039. The nucleotide sequence of ADI comprises an ORF of 1,254 bp encoding 417 amino acids. The deduced ADI protein sequence has a calculated molecular weight of 46.5 kDa and shows 97% and 85% identity to ADIs from P. putida and P. aeruginosa, respectively. The arcA from P. plecoglossicida CGMCC2039 was expressed in Escherichia coli BL21 with a N-terminal His₆-tag, and purified to homogeneity. A molecular mass of approximate 49 kDa was confirmed by SDS-PAGE analysis and specific activity was determined to be 4.76 U/mg (pH 6.0 and 37°C). In vivo activity study showed that the rADI could effectively inhibit H22 tumor growth at a total dose of 5 U/mouse over a 2-week dosing period.