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C Wandersman

Publications and source records attributed to C Wandersman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters.

Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters.

ATP-Binding Cassette Transporters↗
PubMed1996

Protein secretion in gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins, two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Using as protein substrates HasA and GST-PrtC, a chimeric protein which has a glutathione S-transferase moiety fused to a large C-terminal domain of protease C, we developed a simple system to identify proteins bound to the substrate based on substrate affinity-chromatography using heme- or glutathione-agarose. We show an ordered association between the protein substrates and the three exporter components: the substrate recognizes the ABC protein which interacts with the MFP which in turn binds the outer membrane component. Substrate binding is required for assembly of the three components.

ATP-Binding Cassette Transporters↗
PubMed1996

Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue.

The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP-binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli, TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi. By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed-milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti-TolC antibodies cross-reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens. hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug- and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.

ATP-Binding Cassette Transporters↗
PubMed1995

Protein secretion by hybrid bacterial ABC-transporters: specific functions of the membrane ATPase and the membrane fusion protein.

The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous although the secreted polypeptides share no sequence homology. Whereas protease C can use both translocators, HasA is secreted only by its specific transporter. Functional analysis of protease C and HasA secretion through hybrid transporters obtained by combining components from each system demonstrates that the ABC-protein is responsible for the substrate specificity and that inhibition of protease C secretion in the presence of HasA results from a defective interaction between HasA and the ABC-protein. We also show that the outer membrane protein, TolC, can combine with the membrane fusion protein HasE in the presence of either ABC-protein to form a functional transporter but not with the membrane fusion protein, PrtE. This indicates a specific interaction between the outer membrane component and the membrane fusion protein.

ATP-Binding Cassette Transporters↗
PubMed1995

Crystal structure of a complex between Serratia marcescens metallo-protease and an inhibitor from Erwinia chrysanthemi.

The crystal structure of the complex between the 50 kDa metallo-endoproteinase from Serratia marcescens (SMP), a member of the metzincin superfamily, and an inhibitor from Erwinia chrysanthemi (Inh) was solved by molecular replacement using the known structure of SMP, and refined at 2.30 A resolution to a crystallographic R-factor of 0.195. The E. chrysanthemi inhibitor folds into a compact eight-stranded antiparallel beta-barrel of simple up-down topology such as is found for members of the retinol binding protein family. It mainly interacts with the protease via its five N-terminal residues, which insert into the active site cleft, occupying the S' sites. The first N-terminal residue, SerI1, is partially cleaved off by the protease, while SerI2 makes a hydrogen bond with the catalytically active glutamic acid, Glu177, of the protease. Further interactions are made between one face of the inhibitor formed by the strands s3, s4 and s5 and the protease segment 218 to 228, which is located immediately after the characteristic "Met-turn" of the metzincins.

Amino Acid Sequence↗
PubMed1994

Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein.

Several pathogenic bacteria are able to use heme and hemoproteins as iron sources independent of siderophore production by mechanisms involving outer membrane heme-binding proteins and heme transport systems. Here we show that Serratia marcescens has such a property and we identify an extracellular heme-binding protein, HasA (for heme acquisition system), allowing the release of heme from hemoglobin. This protein is secreted by S. marcescens under conditions of iron depletion and is essential for heme acquisition.

Amino Acid Sequence↗
PubMed1994

C-terminal secretion signal of an Erwinia chrysanthemi protease secreted by a signal peptide-independent pathway: proton NMR and CD conformational studies in membrane-mimetic environments.

The detailed structure of a 68-residue chimeric peptide encompassing the 56 last C-terminal residues of Erwinia chrysanthemi protease G has been investigated by using circular dichroism and NMR spectroscopies. The peptide which contains the secretion signal of PrtG was solubilized either in aqueous solvent, in trifluoroethanol (TFE)/H2O mixtures, or in dodecyl beta-D-maltoside detergent. The peptide helical content increases upon TFE and detergent additions. A stable conformation is reached at 40% TFE (v:v) and at a micelle to peptide ratio higher than 1. The 1H NMR spectrum has been assigned in TFE/H2O, 2:1 (v:v), and it is shown that residues 26-29 and 50-62 form a relatively stable helix although a conformational equilibrium between a helix and probably a more random structure is observed throughout fragment 13-63. Comparison of the CterG conformation with results obtained by deletion approach could lead to the hypothesis that the C-terminal secretion signal is composed of an alpha-helix located close to the essential C-terminal tetrapeptide D65VIV.

Amino Acid Sequence↗
PubMed1994

A carboxyl-terminal four-amino acid motif is required for secretion of the metalloprotease PrtG through the Erwinia chrysanthemi protease secretion pathway.

PrtG is an extracellular metalloprotease secreted by the Gram-negative bacterium Erwinia chrysanthemi through a signal peptide-independent secretion pathway. Previous studies showed that the PrtG secretion signal is COOH-terminal and located in the last 56 residues of PrtG. We have now performed a deletion and elongation mapping of a short secretion competent COOH-terminal peptide CterG. This approach allowed us to show that: (i) the smaller COOH-terminal sequence containing the information necessary to promote the secretion of a small polypeptide is contained in the last 29 residues of PrtG; (ii) a low but significant level of secretion can be promoted by the last 15 residues of PrtG when fused to the COOH terminus of a non-secreted PrtG derivative; (iii) the extreme COOH-terminal sequence Dxxx, where xs are hydrophobic residues, is a conserved motif in all constructs that are secreted through the E. chrysanthemi transporter. (vi) This motif has to be COOH terminally exposed since addition of even one amino acid impairs the secretion of CterG. The extent of the secretion defect observed with the COOH terminally extended variants correlates with the length of the extension. These results indicate a key role for the COOH-terminal exposition of the last four amino acids in the secretion of PrtG.

Amino Acid Sequence↗
PubMed1994

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.

Amino Acid Sequence↗
PubMed1993

Involvement of lipopolysaccharide in the secretion of Escherichia coli alpha-haemolysin and Erwinia chrysanthemi proteases.

The presence of the alpha-haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram-negative envelope (owing to its large size) (M(r) 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non-haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced alpha-haemolysin secretion as well as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.

Bacterial Outer Membrane Proteins↗
PubMed1993

Identification of two components of the Serratia marcescens metalloprotease transporter: protease SM secretion in Escherichia coli is TolC dependent.

The Serratia marcescens metalloprotease (protease SM) belongs to a family of proteins secreted from gram-negative bacteria by a signal peptide-independent pathway which requires a specific transporter consisting of three proteins: two in the inner membrane and one in the outer membrane. The prtDSM and prtESM genes encoding the two S. marcescens inner membrane components were cloned and expressed in Escherichia coli. Their nucleotide sequence revealed high overall homology with the two analogous inner membrane components of the Erwinia chrysanthemi protease secretion apparatus and lower, but still significant, homology with the two analogous inner membrane components of the E. coli hemolysin transporter. When expressed in E. coli, these two proteins, PrtDSM and PrtESM, allowed the secretion of protease SM only in the presence of TolC protein, the outer membrane component of the hemolysin transporter.

ATP-Binding Cassette Transporters↗
PubMed1992

Cloning, nucleotide sequence and characterization of the gene encoding the Erwinia chrysanthemi B374 PrtA metalloprotease: a third metalloprotease secreted via a C-terminal secretion signal.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three proteases (PrtA, PrtB and PrtC) into the extracellular medium. The gene encoding the 50 kDa protease, prtA, was subcloned from a recombinant cosmid carrying a fragment of the E. chrysanthemi B374 chromosome. prtA was shown to be located immediately 3' to the structural genes for the other two extracellular proteases. The amino acid sequence of PrtA, as predicted from the prtA nucleotide sequence, showed a high level of homology with a family of metalloproteases that are all secreted via a signal peptide-independent pathway, including PrtB and PrtC of E. chrysanthemi B374, PrtC of E. chrysanthemi EC16, PrtSM of Serratia marcescens and AprA of Pseudomonas aeruginosa. PrtA secretion requires the E. chrysanthemi protease secretion factors PrtD, PrtE and PrtF. The secretion signal of PrtA is near to the carboxy-terminal end of the protein, as was previously shown to be the case for PrtB and PrtSM and for Escherichia coli alpha-hemolysin. The C-termini of these four proteins do not show extensive primary sequence homology, but PrtA, PrtB and PrtSM each have a potential amphipathic alpha-helix located close to the C-terminus.

Amino Acid Sequence↗
PubMed1992

Secretion across the bacterial outer membrane.

Many bacteria secrete extracellular proteins such as hydrolytic enzymes or toxins. In Gram-negative bacteria, secreted proteins must cross the two membranes that constitute the cell envelope. Recent studies have identified several specific secretion systems that can be classified in three distinct pathways, and related systems have been discovered in a wide range of prokaryotic and eukaryotic cells.

Adenosine Triphosphate↗
PubMed1992

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide.

Adenylyl Cyclases↗
PubMed1991

Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the gram-negative bacterium Erwinia chrysanthemi.

Erwinia chrysanthemi, a Gram-negative phythopathogenic bacterium, secretes two related extracellular metalloproteases, B and C, which do not have N-terminal signal sequences. The specific pathway by which they are secreted, which has been reconstituted in Escherichia coli, comprises three proteins -- PrtD, PrtE and PrtF. Hybrid proteins containing segments of these proteins fused to the C-terminus of protease B were purified and used to immunize rabbits. The antisera thus obtained were used to study the location and membrane topology of the three proteins. PrtD and PrtE were found to cofractionate almost exclusively with the cytoplasmic membrane, whereas PrtF was found to co-fractionate mostly with the outer membrane. Proteinase K accessibility experiments as well as sequence data lead us to propose that PrtF has one or both ends exposed to the periplasm, that PrtE has one transmembrane segment with its amino-terminus facing the cytoplasm and its C-terminal hydrophilic domain exposed to the periplasm, and that PrtD has six transmembrane segments with its N-terminus and its C-terminal hydrophilic domain in the cytoplasm.

Bacterial Outer Membrane Proteins↗
PubMed1991

The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin.

The extracellular alkaline protease produced by Pseudomonas aeruginosa is secreted by a specific pathway, independent of the pathway used by most of the other extracellular proteins of this organism. Secretion of this protease is dependent on the presence of several genes located adjacent to the apr gene. Complementation studies have shown that PrtD, E, and F, the three secretion functions for Erwinia chrysanthemi proteases B and C (Létoffé et al., 1990), can mediate the secretion of the alkaline protease by Escherichia coli. The secretion functions involved in alpha-haemolysin secretion in E. coli (hlyB, hlyD, tolC) can also be used to complement alkaline protease secretion by E. coli, although less efficiently. These data indicate that protease secretion mechanisms in Pseudomonas and Erwinia are very similar and are homologous to that of E. coli alpha-haemolysin.

Bacterial Proteins↗