Biomedical subjects

M Ehrmann

Publications and source records attributed to M Ehrmann.

At least 19 recordsLinked to original sources

PubMed2001

A novel method to determine the topology of peroxisomal membrane proteins in vivo using the tobacco etch virus protease.

Most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. A prerequisite in elucidating their function is to determine their topology in the membrane. We have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast Hansenula polymorpha in vivo using the 27-kDa NIa protease subunit from the tobacco etch virus (TEVp). TEVp specifically cleaves peptides containing the consensus sequence, EXXYXQ downward arrowS (tev). We show that cytosolic TEVp and peroxisomal TEVp.SKL are selectively active on soluble cytosolic and peroxisomal tev-containing proteins in vivo, respectively, without affecting the viability of the yeast cells. The tev sequence was introduced in between the primary sequence of the peroxisomal membrane proteins Pex3p or Pex10p and the reporter protein enhanced green fluorescent protein (eGFP). Co-synthesis of these functional tev-GFP tagged proteins with either cytosolic TEVp or peroxisomal TEVp.SKL revealed that the C termini of Pex3p and Pex10p are exposed to the cytosol. Additional applications of the TEV protease to study peroxisome biogenesis are discussed.

ATP-Binding Cassette Transporters↗

PubMed2001

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY.

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.

Bacterial Proteins↗

PubMed2001

Characterization of transmembrane segments 3, 4, and 5 of MalF by mutational analysis.

MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter in Escherichia coli. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L(323)Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.

ATP-Binding Cassette Transporters↗

PubMed2000

Determinants of translocation and folding of TreF, a trehalase of Escherichia coli.

One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre(+) revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence.

Amino Acid Sequence↗

PubMed1999

A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein.

Misfolding or unfolding of polypeptides can occur as a consequence of environmental stress and spontaneous mutation. The abundance of general chaperones and proteases suggests that cells distinguish between proteins that can be refolded and "hopeless" cases fated to enter the proteolytic pathway. The mechanisms controlling this key metabolic decision are not well understood. We show here that the widely conserved heat shock protein DegP (HtrA) has both general molecular chaperone and proteolytic activities. The chaperone function dominates at low temperatures, while the proteolytic activity is present at elevated temperatures. These results show that a single cellular factor can switch between two key pathways, controlling protein stability and turnover. Implications of this finding for intracellular protein metabolism are discussed.

Amylases↗

PubMed1999

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa.

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.

Amino Acid Sequence↗

PubMed1999

Lactobacillus perolens sp. nov., a soft drink spoilage bacterium.

Lactic acid bacteria that are able to spoil soft drinks with low pH comprise a limited number of acidotolerant or acidophilic species of the genera Lactobacillus, Leuconostoc and Weissella. Various Gram-positive rods causing turbidity and off-flavour were isolated from orange lemonades. Physiological and biochemical studies including SDS-PAGE whole-cell protein analysis showed a homogeneous group of organisms. The 16S rRNA gene sequence analysis of two representatives revealed that they formed a phylogenetically distinct line within the genus Lactobacillus. All strains were facultatively heterofermentative, producing L-lactic acid. Based on the data presented a new species L. perolens is proposed. The name refers to the off-flavour caused by high amounts of diacetyl. The type strain of L. perolens is DSM 12744 (LMG 18936). A rRNA targeted oligonucleotide probe was designed that allows a fast and reliable identification of L. perolens.

Bacterial Proteins↗

PubMed1999

Membrane topology of CadA homologous P-type ATPase of Helicobacter pylori as determined by expression of phoA fusions in Escherichia coli and the positive inside rule.

The only experimental data available on the membrane topology of transition metal ATPases are from in vitro studies on two distinct P-type ATPases (CadA and CopA) of a gastric bacterium, Helicobacter pylori, both postulated to contain eight transmembrane domains (H1 to H8). In this study, H. pylori CadA ATPase was subjected to analysis of membrane topology in vivo by expression of ATPase-alkaline phosphatase (AP) hybrid proteins in Escherichia coli using a novel vector, pBADphoA. This vector contains an inducible arabinose promoter and unique restriction sites for fusion of DNA fragments to phoA. The phoA gene lacking sequences encoding its N-terminal signal peptide was linked to the C-terminal regions of the postulated five cytoplasmic and four periplasmic segments of the H. pylori pump. The results obtained by heterologous expression of ATPase-AP hybrid proteins showed consistence with a model of eight transmembrane domains. They also demonstrated that the H. pylori ATPase sequences are well assembled in the cytoplasmic membrane of E. coli, a neutralophilic bacterium. Cloning and amino acid sequence analysis of the homologous ATPase of Helicobacter felis further verified the topological model for the H. pylori pump analyzed here, although the degree of amino acid sequence identity varied between the corresponding transmembrane segments, from 25% for H1 up to 100% for H6. It was found that the topology of ATPase follows the 'positive inside rule'. With respect to the bioenergetic capacities of H. pylori, we discuss here the membrane potential as a possible factor directing insertion of ATPases in the cytoplasmic membrane of gastric bacteria.

Adenosine Triphosphatases↗

PubMed1998

The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli.

The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus. We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli. In particular, we studied the effects of prlA mutations on membrane protein topology. These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus. We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants. We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.

Bacterial Proteins↗

PubMed1998

Lactobacillus amylolyticus sp. nov., isolated from beer malt and beer wort.

Some of the strains used for the biological acidification in breweries belong to L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis or L. fermentum. However, more recent studies showed that most strains isolated are physiologically different from the above mentioned species and were tentatively allocated to Lactobacillus amylovorus. Genotypic studies of 25 strains exclusively isolated from beer malts and beer worts, showed, that there were differences to the type strain of L. amylovorus concerning DNA-DNA similarities and the sequences of their 16S and 23S rRNA genes. Therefore, we propose to combine these strains in a new species of the genus Lactobacillus, namely L. amylolyticus. Strain DSM 11664 is proposed as the type strain. An rRNA targeted oligonucleotide probe was designed that allows a fast and reliable identification of Lactobacillus amylolyticus.

Bacterial Typing Techniques↗

PubMed1998

Polypeptide binding of Escherichia coli FtsH (HflB).

The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.

ATP-Binding Cassette Transporters↗

PubMed1998

The ABC maltose transporter.

Bacterial ATP-binding cassette (ABC) transporters and their homologues in eukaryotic cells form one of the largest superfamilies known today. They function as primary pumps that couple substrate translocation across the cytoplasmic membrane to ATP hydrolysis. Although ABC transporters have been studied for more than three decades, the structure of these multi-component systems is unknown, and the mechanism of transport is not understood. This article reviews one of the most widely studied ABC systems, the maltose transporter of Escherichia coli. A first structural model of the transport channel allows discussion of possible mechanisms of transport. In addition, recent experimental evidence suggests that regulation of gene expression and transport activity is far more complex than expected.

ATP-Binding Cassette Transporters↗

PubMed1997

TnTIN and TnTAP: mini-transposons for site-specific proteolysis in vivo.

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two TnS-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.

Adenosine Triphosphatases↗

PubMed1997

Biochemical characterization and mass spectrometric disulfide bond mapping of periplasmic alpha-amylase MalS of Escherichia coli.

Periplasmic alpha-amylase of Escherichia coli, the malS gene product, hydrolyzes linear maltodextrins. The purified enzyme exhibited a Km of 49 microM and a Vmax of 0.36 micromol of p-nitrophenylhexaoside hydrolyzed per min per mg of protein. Amylase activity was optimal at pH 8 and was dependent on divalent cations such as Ca2+. MalS exhibited altered migration on SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Analytical ultracentrifugation and electrospray mass spectrometry indicated that MalS is monomeric. The four cysteine residues are involved in intramolecular disulfide bonds. To map disulfide bonds, MalS was proteolytically digested. The resulting peptides were separated by reverse phase-high performance liquid chromatography, and matrix-assisted laser desorption/ionization mass spectrometry analysis indicated the presence of two disulfide bonds, i.e. Cys40-58 and Cys104-520. The disulfide bond at Cys40-58 is located in an N-terminal extension of about 160 amino acids which has no homology to other amylases but to the proposed peptide binding domain of GroEL, the Hsp60 of E. coli. The N-terminal extension is linked to the C-terminal amylase domain via disulfide bond Cys104-520. Reduction of disulfide bonds by dithiothreitol treatment led to aggregation suggesting that the N terminus of MalS may represent an internal chaperone domain.

Amino Acid Sequence↗

PubMed1996

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.

Alkaline Phosphatase↗

PubMed1996

Genetics of lactobacilli in food fermentations.

Lactobacilli play a substantial role in food biotechnology and influence our quality of life by their fermentative and probiotic properties. Despite their obvious importance in fermentation ecology and biotechnology only recent years have brought some insight into the genetics of lactobacilli. These genetic investigations allow the elucidation of traits determinative for competitiveness and ecology and thus product safety and quality. They have concentrated only on a small selection of lactobacilli whereas others are hardly touched or remained recalcitrant to genetic analysis and manipulation. The knowledge gained on the biochemistry, physiology, ecology and especially genetics is a prerequisite for the deliberate application and improved handling of lactobacilli in traditional and novel applications. In this review, the achievements in the genetics of lactobacilli are described including detection systems, genetic elements, host vector systems, gene cloning and expression and risk assessment of genetically engineered lactobacilli.

Bioreactors↗

PubMed1996

Site-specific proteolysis of the Escherichia coli SecA protein in vivo.

A seven-amino-acid cleavage site specific for tobacco etch virus (TEV) protease was introduced into SecA at two separate positions after amino acids 195 and 252. Chromosomal wild-type secA was replaced by these secA constructs. Simultaneous expression of TEV protease led to cleavage of both SecA derivatives. In the functional SecA dimer, proteolysis directly indicated surface exposure of the TEV protease cleavage sites. Cleavage of SecA near residue 195 generated an unstable proteolysis product and a secretion defect, suggesting that this approach could be used to inactivate essential proteins in vivo.

Adenosine Triphosphatases↗

PubMed1996

Characterization of a cytoplasmic trehalase of Escherichia coli.

Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.

Amino Acid Sequence↗