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C Wandersman

Publications and source records attributed to C Wandersman.

At least 55 records · Page 3Linked to original sources
PubMed1991

Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions.

The Serratia marcescens extracellular protease SM is secreted by a signal peptide-independent pathway. When the prtSM gene was cloned and expressed in Escherichia coli, the cells did not secrete protease SM. The lack of secretion could be very efficiently complemented by the Erwinia chrysanthemi protease B secretion apparatus constituted by the PrtD, PrtE, and PrtF proteins. As with protease B and alpha-hemolysin, the secretion signal was located within the last 80 amino acids of the protease. These results indicate that the mechanism of S. marcescens protease SM secretion is analogous to the mechanisms of protease B and hemolysin secretion.

Blotting, Western↗
PubMed1990

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin.

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.

Amino Acid Sequence↗
PubMed1990

Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin.

A 5.5 kb DNA fragment carrying the functions necessary for the specific secretion of the extracellular metalloproteases B and C produced by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi has been sequenced. The fragment contains four transcribed and translated genes: inh, which codes for a protease inhibitor and is not required for protease secretion, and prtD, prtE and prtF, which share significant homology with the hlyB, hlyD and tolC genes required for alpha-haemolysin secretion in Escherichia coli. Mutations in any of the three prt genes abolish protease secretion. The prtD and prtE products (60 and 50 kd) contain at least one hydrophobic segment and the prtF gene product contains a signal sequence.

Amino Acid Sequence↗
PubMed1990

TolC, an Escherichia coli outer membrane protein required for hemolysin secretion.

Secretion of Escherichia coli alpha-hemolysin into the medium does not require the cleavage of an N-terminal signal peptide. The specific secretion apparatus was shown to consist of two proteins, HlyB and HlyD, both located in the inner membrane and encoded by genes contiguous to the hemolysin structural gene (hlyA). It was proposed that these two proteins constitute a membrane-bound translocator for hemolysin [Mackman, N., Nicaud, J. M., Gray, L. & Holland, I. B. (1986) Curr. Top. Microbiol. Immunol. 125, 159-181]. We show here that an E. coli outer membrane protein, the TolC protein, encoded by a gene not located in the hly cluster, is specifically required for hemolysin secretion. This result suggests that an outer membrane protein might be a component of the secretion apparatus allowing a specific interaction between the inner and the outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1989

Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12.

Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.

Alkaline Phosphatase↗
PubMed1989

Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide.

The gene encoding the secreted 53-kDa metalloprotease (protease B) and the 5' end of the gene encoding the secreted 55-kDa metalloprotease (protease C) of the Gram-negative bacterium Erwinia chrysanthemi have been sequenced. The predicted sequences of the two proteases do not have typical signal sequences at their NH2 termini. Both proteases are synthesized as inactive higher molecular weight precursors (zymogens proB and proC) which are secreted into the external medium where divalent cation-mediated activation occurs. The activation of proB occurs with a t1/2 of less than 5 min at 37 degrees C in Luria broth medium, whereas that of proC occurs with a t1/2 of about 150 min. The NH2 termini of purified proteases B, proB, and C were sequenced. ProB starts at the initiator methionine whereas B and C start, respectively, at residues +16 and +18 of the sequence deduced from the nucleotide sequence. A short NH2-terminal extension is therefore removed during the activation process, most likely by an autocatalytic mechanism. Protease B shows a high degree of sequence homology with the secreted 50-kDa metalloprotease of Serratia marcescens, which also lacks a signal peptide and for which an inactive higher molecular weight form has been reported.

Amino Acid Sequence↗
PubMed1989

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi.

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low-molecular-weight, heat-stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellular protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escherichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 condons. The N-terminal amino acid sequence of the purified inhibitor was also determined. It indicated the existence of an amino-terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.

Alkaline Phosphatase↗
PubMed1989

Secretion, processing and activation of bacterial extracellular proteases.

Many different bacteria secrete proteases into the culture medium. Extracellular proteases produced by Gram-positive bacteria are secreted by a signal-peptide-dependent pathway and have a propeptide located between the signal peptide and the mature protein. Many extracellular proteases synthesized by Gram-negative bacteria are also produced as precursors with a signal peptide. However, at least two species of Gram-negative bacteria secrete one or more proteases via a novel signal-peptide-independent route. Most proteases secreted by Gram-negative bacteria also have a propeptide whose length and location vary according to the protease. Specific features of protease secretion pathways and the mechanisms of protease activation are discussed with particular reference to some of the best-characterized extracellular proteases produced by Gram-positive and Gram-negative bacteria.

Bacteria↗
PubMed1987

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.

Cloning, Molecular↗
PubMed1982

Mutations that alter the transport function of the LamB protein in Escherichia coli.

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in its interaction with phages lambda and K10 but still interacts with a lambda host range mutant lambda hh*. The Dex- mutants are double lamB mutants carrying one mutation, already present in the parental strains, that reduces LamB synthesis and a second that alters LamB structure. The secondary mutations, present in different independent Dex- mutants, are clustered in the same region of the lamB gene. Dex+ revertants were isolated and analyzed: when the altered LamB protein is made in wild-type amount, due to a reversion of the first mutation, the phenotype reverts to Dex+. However, these Dex+ revertants are still very significantly impaired in maltose transport at low concentrations (below 10 microM).

Bacterial Outer Membrane Proteins↗
PubMed1982

Maltose and maltodextrin transport in Escherichia coli.

Escherichia coli K12 strains producing reduced amounts of LamB protein (one tenth the wild type amount) grow normally on dextrins but transport maltose at a concentration of 1 microM at about one tenth the normal rate. Dex--lamB missense mutants were found as derivatives of these strains. These Dex- mutants had a structurally altered LamB protein in the outer membrane, impaired in its interaction with phages lambda and K10, inefficient in maltose transport at low concentration (10 microM and below). The Dex- mutants still produced one tenth the wild type amount of the LamB protein as the parental Dex+ strains. Analysis of Dex+ revertants showed that the phenotype reverts to Dex+ when the altered LamB protein is made in wild type amounts. Even though they were Dex+, these revertants were still drastically altered in maltose transport at low concentration.

ATP-Binding Cassette Transporters↗
PubMed1980

Pleiotropic mutations rendering Escherichia coli K-12 resistant to bacteriophage TP1.

tpo mutations, located at 74 min on the genetic map, rendered Escherichia coli K-12 resistant to TP1, a phage which can use either the OmpF protein or the LamB protein as its receptor. tpo mutants synthesized decreased amounts of OmpF and LamB proteins but increased amounts of the OmpC product, another outer membrane protein. The effect of the tpo mutations in lam B gene expression was transcriptional. It is one facet of the following effect on the maltose regulon: strong decreases in the syntheses of the LamB protein and the periplasmic MalE protein occurred when the regulon was uninduced; a lesser decrease occurred in the syntheses of the LamB protein the MalE protein, and the cytoplasmic MalQ protein (amylomaltase) when the regulon was induced. The tpo mutants were found to be phenotypically identical to the perA mutant recently described by Wanner et al. (J. Bacteriol. 140:229--239, 1979) and to some of the ompB mutants described by Verhoef et al. (Mol. Gen. Genet. 169:137--146, 1979). Mapping and complementation analysis suggested that these three types of mutations belong to the same cistron. Our results bring to at least four the number of clearly distinct phenotypes which can result from mutations at, or close to, ompB, a locus which appears increasingly complex.

Bacterial Proteins↗
PubMed1979

Escherichia coli mutants impaired in maltodextrin transport.

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Bacterial Proteins↗
PubMed1978

Protein Ia and the lamB protein can replace each other in the constitution of an active receptor for the same coliphage.

Protein Ia and the lamB protein are both located in the outer membrane of Escherichia coli K-12. The lamB protein is known to be the receptor for phage lambda. Datta et al. [Datta, D. B., Arden, B. & Henning, U. (1977) J. Bacteriol. 131, 821--829] recently isolated a phage called TuIa that uses protein Ia for its adsorption. While phage TuIa fails to grow on ompB mutants, which lack protein Ia, we show here that host-range mutants of TuIa can be isolated that do grow on ompB strains. These host-range mutants fail to grow on ompB lamB double mutants, but retain the ability of the parental phage to grow on ompB+ lamB strains. They are therefore apparently able to use either protein Ia or the lamB protein for their adsorption. Genetic evidence suggests that essentially the same site on the lamB protein may be interacting with phage lambda or the host-range mutants of phage TuIa.

Bacterial Proteins↗
PubMedDate not supplied

Secretion processing and activation of Erwinia chrysanthemi proteases.

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase secretion are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the alpha-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific protease inhibitor is produced by some E chrysanthemi strains.

Bacterial Proteins↗