Comparison of Membrane ATPases from Extreme Halophiles Isolated from Ancient Salt Deposits

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to sub unit composition. enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest

Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy

<p>Abstract</p> <p>Background</p> <p>Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host <it>E. coli </it>had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism <it>Bacillus subtilis </it>1012.</p> <p>Results</p> <p>The chimaeric gene encoding the S-layer protein SbpA of <it>Lysinibacillus sphaericus </it>CCM 2177 as well as Bet v1 was cloned and expressed in <it>B. subtilis </it>1012. For that purpose, the <it>E. coli-B. subtilis </it>shuttle vectors pHT01 for expression in the <it>B. subtilis </it>cytoplasm and pHT43 for secretion of the recombinant fusion protein into the culture medium were used. As shown by western blot analysis, immediately after induction of expression, <it>B. subtilis </it>1012 was able to secret rSbpA/Bet v1 mediated by the signal peptide amyQ of <it>Bacillus amyloliquefaciens</it>. Electron microscopical investigation of the culture medium revealed that the secreted fusion protein was able to form self-assembly products in suspension but did not recrystallize on the surface of the <it>B. subtilis </it>cells. The specific binding mechanism between the N-terminus of the S-layer protein and a secondary cell wall polymer (SCWP), located in the peptidoglycan-containing sacculi of <it>Ly. sphaericus </it>CCM 2177, could be used for isolation and purification of the secreted fusion protein from the culture medium. Immune reactivity of rSbpA/Bet v1 could be demonstrated in immunoblotting experiments with Bet v1 specific IgE containing serum samples from patients suffering birch pollen allergy.</p> <p>Conclusions</p> <p>The impact of this study can be seen in the usage of a gram-positive organism for the production of pyrogen-free self-assembling recombinant S-layer/allergen fusion protein with great relevance for the development of vaccines for immunotherapy of atopic allergy.</p

Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted α-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium

Biogenesis and functions of bacterial S-layers.

The outer surface of many archaea and bacteria is coated with a proteinaceous surface layer (known as an S-layer), which is formed by the self-assembly of monomeric proteins into a regularly spaced, two-dimensional array. Bacteria possess dedicated pathways for the secretion and anchoring of the S-layer to the cell wall, and some Gram-positive species have large S-layer-associated gene families. S-layers have important roles in growth and survival, and their many functions include the maintenance of cell integrity, enzyme display and, in pathogens and commensals, interaction with the host and its immune system. In this Review, we discuss our current knowledge of S-layer and related proteins, including their structures, mechanisms of secretion and anchoring and their diverse functions

Molecular and functional analysis indicates a mosaic structure of Salmonella pathogenicity island 2

Our study allowed us to define genes in SPI2 important for virulence further and indicated that this locus has a complex mosaic structure