Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo

We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL⁺ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb⁺ background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

Suppression of the ssb-1 and ssb-113 mutations of Escherichia coli by a wild-type rep gene, NaCl, and glucose

The ssb-1 mutation confers severe temperature sensitivity and UV sensitivity on many strains of Escherichia coli K-12 and C, including strain C1412. However, ssb-1 confers only slight temperature sensitivity and slight UV sensitivity on strain C1a, suggesting that strain C1a contains extragenic suppressors of ssb-1. We found that introduction of the wild-type rep gene from C1a into strain C1412 ssb-1 gave strong suppression of temperature sensitivity and moderate suppression of UV sensitivity. Also, the C1a rep+ gene mildly suppressed the temperature sensitivity conferred by the ssb-113 mutation, formerly called lexC113. Suppression of the C1412 ssb-1 growth defect by C1a rep+ rendered the cells Gro- for phi X174. In contrast to the positive suppression of ssb-1 and ssb-113 by a wild-type rep gene, mutant rep alleles enhanced the severity of the ssb-1 defect, with several C1a ssb-1 double mutants being either more temperature sensitive or more UV sensitive than C1a ssb-1, depending on which mutant rep allele was used. As a control, the same rep alleles in combination with a dnaB mutation gave an allele-independent increase in temperature sensitivity. Our results on suppression of ssb-1 by rep and on the role of the genetic background in this suppression suggested that the rep and ssb proteins interact to form a subcomplex of the total DNA replication complex and that this subcomplex has some function in repair. The effects of NaCl and glucose on suppression of both the temperature sensitivity and the UV sensitivity conferred by ssb-1 and ssb-113 are described. The degree of suppression of temperature sensitivity by salt or glucose was dependent on the source of the wild-type rep allele, as well as on the genetic background.     

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.     

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.     

Construction of a chimeric aminopeptidase by a combination of gene shuffling and mutagenesis

Three chimeric genes were constructed by gene shuffling of aminopeptidases from Aeromonas caviae and Vibrio proteolyticus. Although expressed chimeric enzymes formed inclusion bodies in Escherichia coli, the introduction of two amino acid mutations into the chimeric genes by site-saturated mutagenesis and a random mutation on error-prone PCR resulted in solubilization of the chimeric enzyme. In addition, active chimeric enzyme showed a different thermostability and thermoactivity to parental enzymes.     

Study on thermostability of phospholipase D from Streptomyces sp

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.     

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.     

Chimeric genes as a source of rapid evolution in Drosophila melanogaster

Chimeric genes form through the combination of portions of existing coding sequences to create a new open reading frame. These new genes can create novel protein structures that are likely to serve as a strong source of novelty upon which selection can act. We have identified 14 chimeric genes that formed through DNA-level mutations in Drosophila melanogaster, and we investigate expression profiles, domain structures, and population genetics for each of these genes to examine their potential to effect adaptive evolution. We find that chimeric gene formation commonly produces mid-domain breaks and unites portions of wholly unrelated peptides, creating novel protein structures that are entirely distinct from other constructs in the genome. These new genes are often involved in selective sweeps. We further find a disparity between chimeric genes that have recently formed and swept to fixation versus chimeric genes that have been preserved over long periods of time, suggesting that preservation and adaptation are distinct processes. Finally, we demonstrate that chimeric gene formation can produce qualitative expression changes that are difficult to mimic through duplicate gene formation, and that extremely young chimeric genes (d(S) < 0.03) are more likely to be associated with selective sweeps than duplicate genes of the same age. Hence, chimeric genes can serve as an exceptional source of genetic novelty that can have a profound influence on adaptive evolution in D. melanogaster.     

Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.     

Deletion Formation between the Two Salmonella Typhimurium Flagellin Genes Encoded on the Mini F Plasmid: Escherichia Coli Ssb Alleles Enhance Deletion Rates and Change Hot-Spot Preference for Deletion Endpoints

Deletion formation between the 5'-mostly homologous sequences and between the 3'-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5' sequences, but not between the 3' sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the ΔrecA allele. Rates of the deletion between the 3' sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA(+) background and 88% in ΔrecA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background >50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3' sequences. The deletion formation between the homeologous sequences is RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors.     

A chimeric Plasmodium falciparum Pfnbp2b/Pfnbp2a gene originated during asexual growth

The Plasmodium falciparum line 3D7-A has an unusual invasion phenotype, such that it can invade enzyme-treated and mutant red blood cells that are resistant to invasion by other parasite lines. 3D7-A has a chimeric Pfnbp2b gene that contains part of the repeat region of the paralogous gene Pfnbp2a. This chimeric gene originated by spontaneous gene conversion during normal maintenance in culture, indicating that ectopic recombination and gene conversion during asexual growth are potentially important mechanisms participating in the evolution of paralogous genes in Plasmodium. However, the presence of the chimeric Pfnbp2b gene in 3D7-A was not associated with its peculiar invasion phenotype.     

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46.

Previous studies from our laboratory have shown that an allele of the heat shock protein GroEL (groEL411) is able to specifically suppress some of the physiological defects of the single-stranded DNA-binding protein mutation ssb-1. A search for additional alleles of the groE genes which may act as suppressors for ssb mutations has led to the identification of groEL46 as a specific suppressor of ssb-113. It has very little or no effect on ssb-1 or ssb-3. All of the physiological defects of ssb-113, including temperature-sensitive growth, temperature-sensitive DNA synthesis, sensitivity to UV irradiation, methyl methanesulfonate, and bleomycin, and reduced recombinational capacity, are restored to wild-type levels. The ssb-113 allele, however, is unable to restore sensitivity of groEL46 cells to phage lambda. The mechanism of suppression of ssb-113 by groEL46 appears to differ from that of ssb-1 by groEL411. The data suggest that GroEL may interact with single-stranded DNA-binding protein in more than one domain.     

Immobilized phospholipase D]

Conditions of phospholipase D adsorption on silica gels have been studied. The immobilized phospholipase D is shown to differ from the soluble form in thermostability, pH optima and activation conditions. A question is discussed as to the connection of the use of activators and the adsorption immobilization. It is assumed that phospholipase D belongs to enzymes, functioning only in the immobilized state.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.     

Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains. CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins. To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes. CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined. Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B. stearothermophilus NO2. Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence. Base substitutions were found at the third letter of five codons among the three genes. Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence. The molecular weight of the mature enzyme was estimated to be 75,374. The CGTase gene (cgtM) of B. macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence. The molecular weight of the mature enzyme was estimated to be 74,008. The sequence determined in this work was quite different from that reported previously by other workers. From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B. stearothermophilus NO2 and cgtM from B. macerans IFO3490. We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability. It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.