Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli.

Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo. HlyA activated in E. coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity. Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site. The intact repeat domain of HlyA was not required for the activation. The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.     

Active and inactive forms of hemolysin (HlyA) from Escherichia coli

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.     

In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3′-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3′-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.     

Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.     

Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.     

The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes

Summary The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca²⁺ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca²⁺ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca²⁺ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca²⁺. These data indicate that the repeat domain of haemolysin is responsible for Ca²⁺-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca²⁺ in haemolysis is presented.     

Oxidation of fatty aldehydes to fatty acids by Escherichia coli cells expressing the Vibrio harveyi fatty aldehyde dehydrogenase (FALDH)

Fatty acids represent an important renewable feedstock for the chemical industry. To enable biotechnological one carbon truncations of fatty acids, the enzymes α-dioxygenase and fatty aldehyde dehydrogenase (FALDH) have to be combined in a two-step process. We expressed an FALDH from V. harveyi in E. coli and characterized its substrate spectrum with a focus on the number and position of double bonds in the fatty aldehyde molecules. Synthesis of the expected fatty acid products was proven by analysis of whole cell biotransformation products. Coexpression of a H₂O-forming NADPH oxidase (NOX) from Lactobacillus sanfranciscensis led to the implementation of a cofactor regeneration cycle in in vitro oxidation experiments. The presence of NOX in whole cell biotransformations improved reaction velocity but did not result in higher product yields. We could further demonstrate that at least part of the endogenous NAD(P)⁺ regeneration capacity in the resting cells results from the respiratory chain. The whole cell catalyst with the high broad range FALDH activity described here is an important biotechnological module for lipid biotransformation processes, especially the shortening of fatty acids.     

Generation of Novel Plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn²⁺ (48 mM), Cu²⁺ (12 mM), Ni²⁺ (12 mM), chloramphenicol (50 μg/ml), and tetracycline (25 μg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 mM Zn²⁺, 12 mM Cu²⁺, and 12 mM Ni²⁺, but remained chloramphenicol and tetracycline resistant—the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell. Received: 5 April 2002 / Accepted: 5 July 2002     

Generation of novel plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn(2+) (48 m M), Cu(2+) (12 m M), Ni(2+) (12 m M), chloramphenicol (50 microg/ml), and tetracycline (25 microg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 m M Zn(2+), 12 m M Cu(2+), and 12 m M Ni(2+), but remained chloramphenicol and tetracycline resistant-the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell.     

YfcX enables medium-chain-length poly(3-hydroxyalkanoate) formation from fatty acids in recombinant Escherichia coli fadB strains

Expression of Escherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional beta-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHA(MCL)s when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB(-) YfcX(-) PhaC(+) strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.     

Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation

Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170-400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic alpha-helix spanning residues 272-298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.     

An integrated computational and experimental study for overproducing fatty acids in Escherichia coli

Increasing demands for petroleum have stimulated sustainable ways to produce chemicals and biofuels. Specifically, fatty acids of varying chain lengths (C₆–C₁₆) naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids from plants and other microbial production hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways. In addition, precursors for fatty acids are used along other central metabolic pathways for the production of amino acids and biomass, which further complicates the engineering of microbial hosts for higher yields. Here, we demonstrate an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis techniques to meet this challenge. The OptForce procedure was used for suggesting and prioritizing genetic manipulations that overproduce fatty acids of different chain lengths from C₆ to C₁₆ starting with wild-type E. coli. We identified some common but mostly chain-specific genetic interventions alluding to the possibility of fine-tuning overproduction for specific fatty acid chain lengths. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose (～39% maximum theoretical yield) of C_14–16 fatty acids in minimal M9 medium. These results highlight the benefit of using computational strain design and flux analysis tools in the design of recombinant strains of E. coli to produce free fatty acids.     

Alkylthioacetic acids (3-thia fatty acids) are metabolized and excreted as shortened dicarboxylic acids in vivo

The metabolism of 1-14C-labeled long-chain alkylthioacetic acids (3-thia fatty acids) which are blocked for normal beta-oxidation by a sulfur atom in the beta-position has been investigated in vivo. Most of the injected radioactivity (greater than 50%) was excreted in the urine within the first 48 h. The recovered and identified metabolites were all short sulfoxydicarboxylic acids. The main metabolite from dodecylthioacetic acid was carboxypropylsulfoxy acetic acid. Some bis(carboxymethyl)sulfoxide (dithioglycolic acid sulfoxide) was also found. The main metabolite from nonylthioacetic acid was carboxyethylsulfoxyacetic acid. No sulfones were found. Less than 1% of the 1-14C from the dodecylthioacetic acid was recovered in respiratory CO2 and about 3% of the 1-14C from nonylthioacetic acid. [1-14C]Dodecyl-sulfonylacetic acid was recovered almost quantitatively as carboxypropylsulfonylacetic acid in the urine after 3 h. A significant fraction (10% of the dodecylthioacetic acid was recovered in the phospholipids and triacylglycerols from liver and epidymal fat pad 4 h after injection. These experiments show that the alkylthioacetic acids undergo an initial omega-oxidation followed by beta-oxidation to short dicarboxylic acids.     

Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.     

Expression and purification of uniformly (15)N-labeled amyloid beta peptide 1-40 in Escherichia coli

For biophysical studies using heteronuclear NMR analysis of amyloid beta peptide, construction of an efficient and high yield expression system of uniformly isotopic labeled amyloid beta peptide is desirable. Here we succeeded in obtaining (15)N-labeled amyloid beta 1-40 expressed by attachment to hen egg white lysozyme as a fusion protein.     

Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC.

Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of residue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level. Complete inactivation of HlyC was caused by a double mutation, replacement of Gly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dimensional gel electrophoresis revealed the production and simultaneous secretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites.     

Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.