Search PubMed⌕ Search

Biomedical subjects

M Sarvas

Publications and source records attributed to M Sarvas.

At least 73 records · Page 4Linked to original sources
PubMed1986

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis.

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.

Amino Acid Sequence↗
PubMed1985

Secretion of Semliki Forest virus membrane glycoprotein E1 from Bacillus subtilis.

The gene coding for the Semliki Forest virus (SFV) membrane protein E1 was joined to a secretion vector containing the promoter and signal sequence regions of the alpha-amylase gene from Bacillus amyloliquefaciens. To facilitate secretion, the regions coding for the N-terminal signal peptide (the 6K protein) and the C-terminal hydrophobic transmembrane domain of the E1 gene were deleted. After transformation into B. Subtilis, E1 was shown by immunoblotting to be expressed at a low level (about 0.5-1 mg/1). Contrary to what was expected, most of the E1 remained cell-associated. Deletion of a residual 7 C-terminal amino acids from the 6K region neither increased the level of expression nor significantly improved the secretion. Immunofluorescence microscopy of protoplasts prepared from B. subtilis cells expressing E1 suggested that the cell-associated E1 was located at the outer surface of the bacterial membrane. Addition of protease inhibitors to the culture medium somewhat increased the amount of extracellular E1, suggesting that proteolytic degradation of the foreign gene product may be one reason for the low level of expression. This conclusion was also supported by experiments carried out in Bacillus minicells, which indicated that the expression of the E1 gene in the absence of synthesis of bacterial proteases was about the same as that of alpha-amylase expressed from the cloned gene using the same promoter and signal sequence.

Bacillus subtilis↗
PubMed1985

Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector.

Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.

Bacillus subtilis↗
PubMed1982

Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence.

We describe a secretion vector system for introducing foreign genes into Bacillus subtilis. We constructed secretion vectors from the plasmid pUB110 and the promoter and signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens. Foreign structural genes can be inserted into the various vectors after the signal sequence region of the alpha-amylase gene. Demonstrating secretion of a foreign gene product from Bacillus, we here report that the Escherichia coli beta-lactamase gene, devoid of its own signal sequence coding region, can be expressed in B. subtilis by the aid of the secretion vectors so that greater than 95% of the enzyme activity is secreted to the growth medium. Efficient secretion of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is observed if the complete signal sequence coding region of the alpha-amylase gene precedes the beta-lactamase structural gene. However, an incomplete alpha-amylase signal peptide lacking the six carboxy-terminal amino acid residues does not promote secretion of the fused beta-lactamase, which remains unprocessed and cell-associated.

Amino Acid Sequence↗
PubMed1981

Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens.

We have isolated and partially sequenced the gene coding for alpha-amylase (EC 3.2.1.1) from Bacillus amyloliquefaciens by molecular cloning in the plasmid pUB110 using Bacillus subtilis as a host. The nucleotide sequence of the NH2-terminal region of the cloned gene was determined and found to contain a 31-residue-long stretch of amino acids preceding the NH2-terminal sequence of the extracellular alpha-amylase. Within this sequence there is a 15-residue-long stretch of uncharged amino acids similar to that found at the NH2 terminus of other precursors to exported proteins. This "signal sequence" is probably removed in conjunction with the translocation of alpha-amylase through the cytoplasmic membrane. In vitro labeling of alpha-amylase with radioactive amino acids in a coupled transcription-translation system followed by partial sequencing established the exact location of the NH2 terminus of the alpha-amylase gene. The nucleotide sequence preceding the NH2 terminus has properties resembling the RNA-polymerase- and ribosome-binding sites found at the 5' terminus of many prokaryotic genes.

Amylases↗
PubMed1981

Bacillus licheniformis penicillinase synthesized in Escherichia coli contains covalently linked fatty acid and glyceride.

DNA sequence analysis of the structural gene for Bacillus licheniformis penicillinase has revealed a tetrapeptide sequence of Leu-Ala-Gly-Cys within the NH2-terminal part of the precursor form of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6). The same tetrapeptide occurs in the signal sequence of the prolipoprotein of Escherichia coli, and the cysteine residue in the tetrapeptide of prolipoprotein is modified to form glyceride-cysteine which becomes the NH2 terminus of Braun's lipoprotein. On the basis of labeling, with [2-3H]glycerol, [3H]palmitate, [35S]methionine, and [35S]sulfuric acid, of an E. coli strain lysogenic for a lambda vector containing the penicillinase gene from B. licheniformis and of immunoprecipitation with rabbit antisera against purified B. licheniformis penicillinase, we conclude that B. licheniformis penicillinase synthesized in E. coli contains covalently linked glyceride and fatty acid. These results strongly suggest the operation of a modification system in E. coli, and presumably in other Gram-negative bacteria, which results in the formation of a glyceride-cysteine residue if the proper peptide sequence is present in the signal sequence of membrane proteins.

Bacillus↗
PubMed1981

Asymmetric and symmetric membrane reconstitution by detergent elimination. Studies with Semliki-Forest-virus spike glycoprotein and penicillinase from the membrane of Bacillus licheniformis.

The dissociation and reconstitution of the Semliki Forest virus membrane using the nonionic detergent octyl beta-D-glucoside was studied by sucrose density gradient centrifugation. The dissociation occurred in three stages: lysis at a free equilibrium octyl glucoside concentration of 14--18 mM, solubilization at 18--20 mM, and delipidation of the spike glycoproteins at the critical micellar concentration (22 mM) or higher. After solubilization the spike glycoproteins were present as soluble complexes with sedimentation coefficients of 19 S and 6 S. The 6-S form probably corresponded to a glycoprotein monomer complexed to detergent and the 19-S form consisted of oligomeric detergent-protein complexes. The two forms were in slow equilibrium with each other. When the soluble spike protein complexes and egg lecithin solubilized with octyl glucoside were mixed and the octyl glucoside concentration lowered either by dialysis or by dilution, reconstitution occurred. Three types of products were obtained: vesicles with 30% of the spike protein facing inwards and 70% facing outwards, vesicles with virtually all (95%) of the spike proteins pointing outwards, and small protein-rich soluble aggregates [Helenius et al. (1977) J. Cell Biol. 75, 866]. It was demonstrated that during reconstitution the symmetric vesicles were formed at 19 mM free equilibrium octyl glucoside by the association of the 6-S protein complexes with the phospholipids, and the asymmetric vesicles were formed at 10--16 mM octyl glucoside when the 19-S complexes associated with the lipids. Asymmetric membrane vesicles were also obtained when membrane penicillinase from Bacillus licheniformis was reconstituted with egg lecithin using octyl glucoside. It could be shown that the penicillinase was oligomeric at the octyl glycoside concentration where the reconstitution occurred. The results demonstrate that different mechanisms of reconstitution give rise to the symmetric and the asymmetric vesicles. The critical factor in determining the mechanism is the state of aggregation of the proteins at the octyl glucoside concentration where membranes begin to form from the solubilized lipids.

Bacillus↗
PubMed1981

Immunochemical characterization of major outer membrane components from Salmonella typhimurium.

We used crossed immunoelectrophoresis to study detergent-solubilized components of the outer membrane of Salmonella typhimurium under nondenaturing conditions. The antisera used were raised against nondenatured outer membrane preparations. Lipopolysaccharide and lipoprotein were identified easily as discrete precipitates when they were solubilized with Triton X-100. However, solubilization of the porins with Triton X-100 resulted in a complex precipitate pattern, indicating incomplete dissociation of protein-protein interactions. A clear-cut pattern was obtained when the porins were first solubilized and denatured with hot sodium dodecyl sulfate, followed by removal of the sodium dodecyl sulfate and renaturation in the presence of Triton X-100. Our findings suggested that crossed immunoelectrophoresis can be used to study the antigenicity of nondenatured porins and the antibody responses to them.

Bacterial Proteins↗
PubMed1978

Formation of protein micelles from amphiphilic membrane proteins.

The membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis, the Semliki Forest virus spike proteins, and the Sendai virus glycoproteins have each been isolated as soluble protein aggregates that are virtually free of lipid and detergent. The sedimentation coefficients of the complexes were 18 S, 29 S, and 43 S, respectively. Mixed aggregates containing both the virus glycoproteins and the penicillinase could also be formed. Such protein micelles may serve a number of useful purposes in membrane research.

Antibodies↗
PubMed1977

Teichoic acid antibody test: its use in patients with coagulase-positive staphylococcal bacteremia.

We have studied the occurrence and specificity of teichoic acid antibodies (TAAs), measured by double diffusion in agar, in 114 patients with bacteremia of whom 47 had coagulase-positive staphylococcal bacteremia. A total of 30% of the 47 patients with coagulase-positive staphylococcal bacteremia had a TAA titer of 1:8 or more, and an additional 30% had a titer of 1:2 or 1:4. High TAA titers were most often connected with coagulase-positive staphylococcal endocarditis, osteomyelitis, and deep wound infections. None of the six coagulase-negative patients with staphylococcal bacteremia nor any of the 92 controls had titers exceeding 1:1. A total of 10% of the other patients with bacteremia showed positive results on the TAA test at low titer levels. Compared to the antistaphylolysin value, the TAA test was about equally specific but more sensitive.

Adolescent↗
PubMed1976

T2 lipopolysaccharide antigen of Salmonella: genetic determination of T2 and properties of the T2, T2,S, and T2,SR Forms.

The T2 antigenic form of Salmonella bareilly was examined. The absence of O specificity in this strain was shown to be due to its nonfunctional rfb genes; when the rfb gene cluster was replaced by the rfb cluster derived from smooth donor strains, T2,S and T2,SR recombinants were produced that expressed both T2 and either 0-6,7 or 0-4,12 specificity, depending on O antigen of the donor strain. The T2, T2,S, and T2,SR forms were all unstable on culture and segregated T2-negative forms (R, S, and SR, respectively) at a high rate. In all these respects the T2 antigen closely resembled the other T-form antigen, T1. The genes responsible for the T2 antigen, rfu, were not close to rfb, but their precise location and relation to rft (which determines T1 antigen) could not be discovered because of the instability to the T2 form and low recombination frequency in the necessary interspecies crosses.

Genes↗