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W Bitter

Publications and source records attributed to W Bitter.

At least 19 recordsLinked to original sources
PubMed2001

Maturation of Pseudomonas aeruginosa elastase. Formation of the disulfide bonds.

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.

Bacterial Proteins↗
PubMed2001

Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

Amino Acid Sequence↗
PubMed2001

Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum.

The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

ATP-Binding Cassette Transporters↗
PubMed2000

Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone.

Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion. The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro. Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif. Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive. This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway. The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process. These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.

Burkholderia↗
PubMed2000

Protein secretion mechanisms in Gram-negative bacteria.

Gram-negative bacteria have developed a variety of secretion pathways to secrete toxins and enzymes into the extracellular medium. These pathways are very different with respect to their functional mechanism and complexity, and each system has its own advantages and limitations, regarding the number, size, folding state and fate of their substrates. Pseudomonas aeruginosa secretes many different proteins into the extracellular medium, using at least four secretion pathways. Most of the exoproteins are secreted via the type II system, composed of the 12 Xcp proteins. The only outer membrane protein of the system, XcpQ, belongs to a large family of proteins, designated secretins, which participate in a variety of different transport processes. Other Xcp proteins, XcpT-X, show homology to the subunits of the retractile type IV pili. Further analogies between the type II system and the assembly of retractile pili suggest a mechanism for type II secretion, in which a pilus-like structure, composed of XcpT-X, facilitates the transport of exoproteins through the channel formed by the secretin XcpQ.

Bacterial Proteins↗
PubMed2000

Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa.

One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).

Amino Acid Sequence↗
PubMed1999

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity.

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K. This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation. Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures. Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel. However, the single-channel conductances observed were not uniform. Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported.

Bacterial Proteins↗
PubMed1999

Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor.

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

Acinetobacter calcoaceticus↗
PubMed1998

Control of VSG gene expression sites in Trypanosoma brucei.

Antigenic variation in African trypanosomes continues to be one of the most elaborate and intriguing strategies ever devised by a protozoan parasite to avoid complete destruction by the immune defense of its mammalian host. Here we review some of the recent advances in our understanding of this strategy, concentrating on (unpublished) work from our laboratory.

Animals↗
PubMed1998

The role of transferrin-receptor variation in the host range of Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its surface coat, variant surface glycoprotein (VSG). Trypanosomes take up transferrin through a heterodimeric transferrin receptor, the genes for which are expressed in telomeric expression sites along with the VSG gene. There are up to 20 of these expression sites per trypanosome nucleus, but usually only one is active at a time. Different expression sites encode transferrin receptors that are similar but not identical. Here we show that these small differences between transferrin receptors can have profound effects on the binding affinity for transferrins from different mammals, and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different transferrin-receptor genes allows T. brucei to cope with the large sequence diversity in the transferrins of its hosts.

Animals↗
PubMed1998

Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to translocate proteins across both membranes of the cell envelope. Many of these proteins are transported via the type II secretion pathway and adopt their tertiary conformation in the periplasm, which implies the presence of a large transport channel in the outer membrane. The outer membrane protein, XcpQ, which is involved in transport of folded proteins across the outer membrane of P. aeruginosa, was purified as a highly stable homomultimer. Insertion and deletion mutagenesis of xcpQ revealed that the C-terminal part of XcpQ is sufficient for the formation of the multimer. However, linker insertions in the N-terminal part can disturb complex formation completely. Furthermore, complex formation is strictly correlated with lethality, caused by overexpression of xcpQ. Electron microscopic evaluation of the XcpQ multimers revealed large, ring-shaped structures with an apparent central cavity of 95 A. Purified PilQ, a homologue of XcpQ involved in the biogenesis of type IV pili, formed similar structures. However, the apparent cavity formed by PilQ was somewhat smaller, 53 A. The size of this cavity could allow for the transport of intact type IV pili.

Amino Acid Sequence↗
PubMed1998

Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa.

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions.

Bacterial Proteins↗
PubMed1997

The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex.

The YscC protein of Yersinia enterocolitica is essential for the secretion of anti-host factors, called Yops, into the extracellular environment. It belongs to a family of outer membrane proteins, collectively designated secretins, that participate in a variety of transport processes. YscC has been shown to exist as a stable oligomeric complex in the outer membrane. The production of the YscC complex is regulated by temperature and is reduced in strains carrying mutations in the yscN-U operon or in the virG gene. The VirG lipoprotein was shown to be required for efficient targeting of the complex to the outer membrane. Electron microscopy revealed that purified YscC complexes form ring-shaped structures of approximately 20 nm with an apparent central pore. Because of the architecture of the multimer, YscC appears to represent a novel type of channel-forming proteins in the bacterial outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1996

Antigenic variation in trypanosomes.

We review here antigenic variation in African trypanosomes with emphasis on genetic mechanisms and on the expression sites in which the genes for Variant Surface Glycoproteins (VSGs) are expressed. There are multiple expression sites in a trypanosome, but only one of these is active at a time. We discuss recent experiments that provide new information on expression site regulation, i.e., how inactive sites are kept inactive and how the trypanosome switches from expression of one site to expression of another one. Trypanosomes can also change the gene expressed by replacing the gene in an active expression site by another VSG gene. This replacement involves the duplicative transposition of a silent VSG gene into the expression site. We present a model for the mechanism of this transposition that incorporates new features and that explains several unusual characteristics of the transposition process. We also discuss how new knowledge of nutrient uptake, notably uptake of host transferrin by trypanosomes, might be used for vaccine development.

Animals↗
PubMed1995

Multiple outer membrane receptors for uptake of ferric pseudobactins in Pseudomonas putida WCS358.

Under iron limitation Pseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library of P. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability of P. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.

Amino Acid Sequence↗
PubMed1994

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA.

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.

Bacterial Outer Membrane Proteins↗