ABSTRACT
Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.
Subject(s)
DNA Transposable Elements/genetics , Gene Expression Regulation/genetics , Phosphoproteins , Arabinose/biosynthesis , Arabinose/pharmacology , Bacterial Proteins/genetics , Cell Division/drug effects , Dose-Response Relationship, Drug , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , GTP Phosphohydrolases/genetics , Operon/genetics , Plasmids/genetics , Promoter Regions, Genetic/genetics , TemperatureABSTRACT
Salmonella adaptation to low pH is a critical survival response and essential for virulence. Here, we show that another key virulence-associated process, flagella-mediated cell motility, is co-regulated by low pH via the PhoPQ signal transduction system. Using a proteomic approach, we found that phase 1 and phase 2 flagellin were specifically down-regulated when acid-adapted (pH 5.0) Salmonella SL1344 cells were exposed to pH 3.0. Decreased flagellin expression and cell motility was dependent on activation of the PhoPQ pathway, which directly or indirectly negatively regulated transcription of the flagellin gene fliC. In contrast, the general stress sigma factor RpoS (sigma s) positively regulated flagellar gene expression. Low external pH had no effect on the level of H-NS protein, a further regulator of flagellar gene expression. We suggest that flagellar repression at low pH conserves ATP for survival processes and helps to limit the influx of protons into the cytosol. These results highlight the power of proteomics to reveal unanticipated links between relatively well-characterised regulatory systems in bacteria.
Subject(s)
Bacterial Proteins/physiology , Proteome , Salmonella/physiology , Adaptation, Physiological , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Flagellin/genetics , Flagellin/isolation & purification , Genes, Bacterial , Hydrogen-Ion Concentration , Movement , Osmotic Pressure , Oxidative Stress , Proteome/genetics , Proteome/isolation & purification , Proteome/physiology , Salmonella/genetics , Salmonella/pathogenicity , Sigma Factor/genetics , Sigma Factor/isolation & purification , Sigma Factor/physiologyABSTRACT
Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.
Subject(s)
Bacterial Proteins , Flagellin/genetics , Gastroenteritis/etiology , Salmonella typhimurium/pathogenicity , Typhoid Fever/etiology , Animals , Bacterial Adhesion , Cattle , Disease Models, Animal , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , VirulenceABSTRACT
One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.
Subject(s)
Aspartic Acid/analogs & derivatives , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Chromatography, Affinity , Cobalt/metabolism , Escherichia coli Proteins , Escherichia coli , GTP Phosphohydrolases/isolation & purification , Histidine , Peptides/metabolism , Phosphoproteins , Repressor Proteins/isolation & purification , Repressor Proteins/metabolism , Sepharose/analogs & derivatives , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biotinylation , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Lac Operon/genetics , Lac Repressors , Mass Spectrometry , Models, Molecular , Molecular Weight , Operator Regions, Genetic/genetics , Peptide Mapping , Peptides/genetics , Protein Structure, Quaternary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/geneticsABSTRACT
Microbes present special opportunities for proteomic analysis that are not yet available for other types of organisms, due mainly to the relative abundance of information on their genomes, their low levels of functional redundancy and their experimental tractability. They are also being used to develop and validate powerful new experimental approaches that surmount some important current limitations in this field. The review surveys the different proteomic procedures that are available and considers the advantages and disadvantages of different experimental strategies. The ways in which microbiologists - and others - can exploit proteomic data are also discussed.
Subject(s)
Archaeal Proteins/analysis , Bacterial Proteins/analysis , Fungal Proteins/analysis , Proteome/analysis , AnimalsABSTRACT
For a sustained infection, enteric bacterial pathogens must evade, resist or tolerate a variety of antimicrobial host defence peptides and proteins. We report here that specific organic acids protect stationary-phase Escherichia coli and Salmonella cells from killing by a potent antimicrobial peptide derived from the human bactericidal/permeability-increasing protein (BPI). BPI-derived peptide P2 rapidly halted oxygen consumption by stationary-phase cells preincubated with glucose, pyruvate or malate and caused a 109-fold drop in cell viability within 90 min of addition. In marked contrast, O2 consumption and viability were not significantly affected in stationary-phase cells preincubated with formate or succinate. Experiments with fdhH, fdoG, fdnG, selC and sdhO mutants indicate that protection by formate and succinate requires their oxidation by the Fdh-N formate dehydrogenase and succinate dehydrogenase respectively. Protection was also dependent on the BipA GTPase but did not require the RpoS sigma factor. We conclude that the primary lesion caused by this cationic peptide is not gross permeabilization of the bacterial cytoplasmic membrane but may involve specific disruption of the respiratory chain. Because P2 shares sequence similarity with a range of other antimicrobial peptides, its cytotoxic mechanism has broader significance. Additionally, protective quantities of formate are secreted by E. coli and Salmonella during growth suggesting that such compounds are important determinants of bacterial survival in the host.
Subject(s)
Anti-Bacterial Agents/pharmacology , Blood Proteins/pharmacology , Escherichia coli Proteins , Escherichia coli/drug effects , Formates/pharmacology , Membrane Proteins , Phosphoproteins , Salmonella/drug effects , Amino Acid Sequence , Antimicrobial Cationic Peptides , Bacterial Proteins/biosynthesis , Bacterial Proteins/drug effects , Bacterial Proteins/metabolism , Cell Division/drug effects , DNA, Bacterial/biosynthesis , DNA, Bacterial/drug effects , Escherichia coli/growth & development , Escherichia coli/metabolism , Fermentation , Formate Dehydrogenases/metabolism , GTP Phosphohydrolases/drug effects , GTP Phosphohydrolases/metabolism , Glucose/pharmacology , Humans , Malates/pharmacology , Molecular Sequence Data , Oxygen/metabolism , Peptide Fragments/pharmacology , Pyruvic Acid/pharmacology , Sigma Factor/metabolismABSTRACT
Microbial proteases play diverse and important roles in bacterial virulence but their detection and characterisation is often hampered by their limited abundance or lack of expression in the absence of suitable environmental signals. We describe here a sensitive proteomic approach to detect proteases that are under the control of a virulence regulator and to characterise their recognition motifs. Using MG++-depleted growth media or a mutant strain of Salmonella in which the PhoP-PhoQ virulence regulatory system is constitutively active, truncated forms of DnaK, elongation factor G, elongation factor Tu and ribosomal protein S1 proteins were detected. Two other global regulatory mutants and cells exposed to acid or to oxidative stress failed to produce the truncated proteins, indicating specific control of the protease activity by the PhoP-PhoQ system. Our results suggest that at least two proteases are induced. To define the proteolytic cleavage sites of one of the proteases, peptides from each of the truncated proteins were identified by tryptic mass fingerprinting/nanoelectrospray mass spectrometry and mapped onto the sequence of the intact protein. Alignment of the regions around the cut site indicates that the protease recognises a dibasic amino acid motif characteristic of the omptin protease family. The induction of such proteases in bacteria depleted of Mg++ ions may contribute to the PhoPQ-mediated resistance of Salmonella to cationic antimicrobial peptides. Additionally, our results suggest it would be prudent to keep the concentration of this ion above micromolar levels during bacterial sample preparation for proteomic analyses.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Protein Kinases/metabolism , Salmonella typhimurium/enzymology , Signal Transduction , Acids , Amino Acids , Binding Sites , Enzyme Activation , HSP70 Heat-Shock Proteins/analysis , Mutation , Oxidative Stress , Peptide Elongation Factor G/analysis , Peptide Elongation Factor Tu/analysis , Peptide Mapping , Peptides/metabolism , Ribosomal Proteins/analysis , TrypsinABSTRACT
High affinity Ca(2+)-ATPases play a central role in calcium homeostasis by catalysing the active efflux of calcium from the cytoplasm. This study reports the identification of two additional type IIA (SERCA-type) Ca(2+)-ATPases from Arabidopsis (AtECA2 and AtECA3), and describes the detailed sequence analysis of these genes in comparison with AtECA1 and other plant and animal Ca(2+)-ATPases. Southern analysis suggests that each of these genes is present as a single copy and also that there may be a small family of moderately related genes that encode type IIA Ca(2+)-ATPases in Arabidopsis. Evidence is also provided from RT-PCR that these genes are expressed in Arabidopsis. Hydropathy analysis predicts that the topology of the Arabidopsis type IIA proteins is similar to the animal SERCA proteins. Sequence and phylogenetic analyses suggest that the type IIA Ca(2+)-ATPases can be further divided into sub-groups.
Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Amino Acid Sequence , Amino Acids/analysis , Animals , Blotting, Southern , Calcium-Transporting ATPases/classification , Cloning, Molecular , DNA, Complementary/analysis , Genes, Plant , Humans , Models, Genetic , Molecular Sequence Data , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidSubject(s)
Adhesins, Bacterial , Bacterial Outer Membrane Proteins/metabolism , Calcium/metabolism , Carrier Proteins , Escherichia coli Proteins , Escherichia coli/physiology , GTP Phosphohydrolases/metabolism , Phosphoproteins , Receptors, Cell Surface/metabolism , Escherichia coli/pathogenicity , GTP Phosphohydrolases/genetics , HeLa Cells , Humans , Kinetics , Phosphorylation , Receptors, Cell Surface/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-letter symbols are used for amino acid residues throughout)], displaying an increased affinity for Pi and decreased affinity for MgATP, while retaining the ability to translocate Ca2+ ions across the endoplasmic reticulum membrane. The properties of this mutant suggest that the E1-E2 equilibrium is shifted towards E2, and indicate a key role for this aromatic residue (Y295) at the end of trans-membrane helix M4. A mutant containing three amino acid residue substitutions at the end of the seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a complete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no effect on Ca2+ transport suggests that correct anchoring of the helix at the lipid-water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillosin A or t-butyl hydroquinone, suggesting that hydrogen-bonding partners for the essential -OH groups on these inhibitors lie elsewhere in the ATPase.
Subject(s)
Calcium-Transporting ATPases/genetics , Protein Folding , Adenosine Triphosphatases/metabolism , Animals , Benzoquinones/metabolism , COS Cells , Calcium-Transporting ATPases/chemistry , DNA Mutational Analysis , Enzyme Inhibitors/metabolism , Genes, Synthetic , Phosphorylation , Protein Conformation , Protein Structure, Secondary , Thapsigargin/metabolismABSTRACT
We report the functional characterization of BipA, a GTPase that undergoes tyrosine phosphorylation in an enteropathogenic Escherichia coli (EPEC) strain. BipA mutants adhere to cultured epithelial cells but fail to trigger the characteristic cytoskeletal rearrangements found in cells infected with wild-type EPEC. In contrast, increased expression of BipA enhances actin remodelling and results in the hyperformation of pseudopods. BipA appears to be the first example of a new class of virulence regulator, as it also controls flagella-mediated cell motility and resistance to the antibacterial effects of a human host defence protein. Its striking sequence similarity to ribosome-binding elongation factors suggests that it uses a novel mechanism to modulate gene expression.
Subject(s)
Bacterial Proteins/physiology , Escherichia coli/enzymology , GTP Phosphohydrolases/physiology , Amino Acid Sequence , Bacterial Adhesion/genetics , Bacterial Outer Membrane Proteins/physiology , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/pathogenicity , Flagellin/analysis , GTP Phosphohydrolases/chemistry , Gene Expression Regulation, Bacterial , HeLa Cells , Humans , Immunoblotting , Microscopy, Confocal , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation , Phosphorylation , Sequence Alignment , Tyrosine/metabolism , VirulenceABSTRACT
Salmonella typhimurium (official designation Salmonella enterica serovar Typhimurium) is an enteric pathogen and a principal cause of gastroenteritis in humans. A comprehensive description of the proteins of Salmonella and their patterns of expression under different environmental conditions would greatly increase our understanding of the virulence of this organism at the molecular level and provide insights into many other aspects of Salmonella biology. While a variety of two-dimensional studies of Salmonella have been previously carried out to address specific questions, little systematic information is available at the protein level on the numbers of Salmonella polypeptides that have homologues in other organisms, their abundance, and the frequency of post-translational modifications. To test the feasibility of determining the proteome of Salmonella, the identities of 53 randomly sequenced cell envelope proteins have been determined by N-terminal sequencing of spots from two-dimensional gels. In addition to confirming the existence of previously hypothetical proteins predicted from genomic sequencing projects, we found that approximately 20% of the proteins had no matches in sequence databases. The results suggest that proteome analysis is an efficient way to identify novel proteins from prokaryotes and that the analysis provides a useful approach to the study of Salmonella virulence.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Electrophoresis, Gel, Two-Dimensional/methods , Genome, Bacterial , Salmonella typhimurium/chemistry , Salmonella typhimurium/genetics , Amino Acid Sequence , Animals , Humans , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Molecular Sequence Data , Peptide Mapping/methods , Salmonella typhimurium/classification , Sequence Homology, Amino AcidABSTRACT
Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.
ABSTRACT
MxaJ is a protein of unknown function encoded by mxaJ in the mxaFJGI operon. We have constructed a mxaJ mutant of M. extorquens with a deletion which does not affect transcription of downstream genes. It contained cytochrome cL (MxaG), but neither subunit of methanol dehydrogenase (MxaF and MxaI). MxaJ is probably involved in processing this enzyme. We have sequenced the region between mxaFJGI and five other methanol oxidation genes, mxaACKLD; it includes one open reading frame (mxaR) and a possible second open reading frame (mxaS), demonstrating the presence in M. extorquens of the following gene cluster: mxaFJGIR(S) ACKLD.
Subject(s)
Genes, Bacterial , Gram-Negative Aerobic Bacteria/genetics , Gram-Negative Aerobic Bacteria/metabolism , Methanol/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , Base Sequence , DNA, Bacterial/genetics , Molecular Sequence Data , Multigene Family , Oxidation-Reduction , Restriction Mapping , Sequence DeletionABSTRACT
Forty-nine cell envelope proteins of Salmonella typhimurium SL1344 have been identified by microsequencing and assigned to a two-dimensional reference map. Ten of the sequenced proteins appear to be novel. Several others closely match currently hypothetical proteins or proteins found in other bacteria but not previously reported in salmonellae.
Subject(s)
Bacterial Proteins/analysis , Membrane Proteins/analysis , Salmonella typhimurium/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Escherichia coli/chemistry , Escherichia coli/metabolism , Information Systems , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , Molecular Sequence Data , Peptide Fragments/chemistry , Salmonella typhimurium/chemistry , Species SpecificityABSTRACT
Bactericidal/permeability-increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene. Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell-surface component.
Subject(s)
Blood Bactericidal Activity , Blood Proteins/pharmacology , Membrane Proteins , Neutrophils/metabolism , Salmonella typhimurium/drug effects , Amino Acid Sequence , Antimicrobial Cationic Peptides , Bacterial Proteins/metabolism , Base Sequence , Cell Membrane Permeability/drug effects , DNA Primers/genetics , DNA, Bacterial/genetics , Genes, Bacterial/drug effects , Humans , In Vitro Techniques , Molecular Sequence Data , Polymyxin B/pharmacology , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
The interaction of propionyl-CoA and acetyl-CoA with E. coli citrate synthase has been studied in order to gain insight into the structural requirements for substrate binding by this enzyme. In contrast to the enzyme from pig heart, the E. coli enzyme was unable to catalyse significant exchange of the methylene protons of propionyl-CoA while overall activity was very low with this enzyme. Carboxymethyl-CoA is a presumptive transition state analogue of acetyl-CoA using pig heart citrate synthase. The effect of carboxymethyl-CoA on both the native enzyme from E. coli and a catalytically active aspartate mutant (D362E) was investigated. Whereas the native enzyme was inhibited by carboxymethyl-CoA, the mutant enzyme (D362E) shows either no inhibition or minimal inhibition depending on the assay conditions. The binding of acetyl-CoA is not inhibited as a result of the mutation. The results with propionyl-CoA and carboxymethyl-CoA suggest that the active site of the E. coli enzyme is more restricted as compared with the enzyme from pig heart and, in the case of propionyl-CoA, this restriction prevents the formation of a catalytically productive enzyme-substrate complex.
Subject(s)
Acyl Coenzyme A/metabolism , Citrate (si)-Synthase/metabolism , Escherichia coli/metabolism , Acyl Coenzyme A/pharmacology , Animals , Binding Sites , Citrate (si)-Synthase/antagonists & inhibitors , Escherichia coli/enzymology , Hydrogen-Ion Concentration , Myocardium/enzymology , SwineABSTRACT
The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.
Subject(s)
Citrate (si)-Synthase/chemistry , Anilino Naphthalenesulfonates/chemistry , Arginine , Aspartic Acid , Base Sequence , Binding Sites , Catalysis , Citrate (si)-Synthase/isolation & purification , Citrate (si)-Synthase/metabolism , Dithionitrobenzoic Acid/chemistry , Escherichia coli/enzymology , Histidine , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , NAD/metabolism , Protein Conformation , Structure-Activity RelationshipABSTRACT
Bactericidal/permeability-increasing protein (BPI) is a potent antimicrobial agent produced by polymorphonuclear leucocytes that specifically interacts with and kills Gram-negative bacteria. An 825 bp gene determining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli. The recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined. Folding was critically dependent on the urea and salt concentrations as well as the pH. BPI' bound with high affinity to Salmonella typhimurium cells (apparent Kd = 36 nM), permeabilized their outer membranes to actinomycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity. In contrast with the native protein, however, it could not be efficiently released from the cell surface by the addition of high concentrations of Mg2+ ions. Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxicity. However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able to function even when presented to cells in an unfolded form. A BPI' derivative, containing a 13-residue foreign antigenic determinant genetically inserted between Ala115 and Asp116, was also produced. The derivative was functional in the above assays and bound with high affinity to S. typhimurium (apparent Kd = 74 nM). These results imply that the region defined by these residues is not involved in the lipopolysaccharide-binding or bactericidal activities of BPI. The availability of functional, nonglycosylated recombinant derivatives of BPI should greatly aid detailed studies on its structure, interactions with lipopolysaccharide and mechanism of action.