Gene expression of Bacillus subtilis subtilisin E in Thermus thermophilus

The Bacillus subtilis subtilisin E gene was cloned into an expression vector of the extreme thermophile, Thermus thermophilus. Active subtilisin E was produced in E. coli, indicating that the Thermus promoter functions in E. coli. When the plasmid was further introduced into T. thermophilus, the subtilisin E gene was expressed and the gene product accumulated as an inactive pro-form, because the autoprocessing of the wild-type enzyme to the active-form did not occur at 50°C or above. Received 17 March 1999/ Accepted in revised form 28 June 1999     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for β-isopropylmalate dehydrogenase (leuB).     

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes

To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.     

Expression study with the Escherichia coli lep gene for leader peptidase in phototrophic purple bacteria

Synthesis and assembly of leader peptidase of Escherichia coli (signal peptidase I), was studied by heterologous expression of its lep gene in three species of phototrophic purple bacteria. Cell extracts of the recipient species showed neither cross reaction with antibodies against E. coli leader peptidase nor cleavage of the model substrate M13-procoat in vitro. The lep gene was transferred via conjugation using the plasmid expression vector for phototrophic bacteria pJAJ9. Plasmidborne leader peptidase enzyme was identified by immunochemical means. However, extracts of transconjugant cells showed no cleavage function. Trypsin digestion studies revealed that the enzyme was not properly integrated across the host membranes. The data suggest that cleaving enzymes for protein export and/or their assembly pathway in purple bacteria differ from the E. coli type.Abbreviations DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway.     

Screening of stable proteins in an extreme thermophile, Thermus thermophilus

The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.     

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

Summary The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.Abbreviations GDH glutamate dehydrogenase - PMS phenazine methosulphate - MTT 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium-bromide - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - DTT dithiothreitol - bp base pairs - kbp kilo base pairs - dNTP deoxynucleoside triphosphate     

Overproduction of Biologically-active Human Nerve Growth Factor in Escherichia coli

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the β-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the β-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions

Branch migration of Holliday junctions, which are central DNA intermediates in homologous recombination, is promoted by the RuvA-RuvB protein complex, and the junctions are resolved by the action of the RuvC protein in Escherichia coli. We report here the cloning of the ruvB gene from a thermophilic eubacterium, Thermus thermophilus HB8 (Tth), and the biochemical characterization of the gene product expressed in E. coli. The Tth ruvB gene could not complement the UV sensitivity of an E. coli ruvB deletion mutant and made the wild-type strain more sensitive to UV. In contrast to E. coli RuvB, whose ATPase activity is strongly enhanced by supercoiled DNA but only weakly enhanced by linear duplex DNA, the ATPase activity of Tth RuvB was efficiently and equally enhanced by supercoiled and linear duplex DNA. Tth RuvB hydrolyzed a broader range of nucleoside triphosphates than E. coli RuvB. In addition, Tth RuvB, in the absence of RuvA protein, promoted branch migration of a synthetic Holliday junction at 60° C in an ATP-dependent manner. The protein, as judged by its ATPase activity, required ATP for thermostability. Since a RuvA protein has not yet been identified in T. thermophilus, we used E. coli RuvA to examine the effects of RuvA on the activities of Tth RuvB. E. coli RuvA greatly enhanced the ability of Tth RuvB to hydrolyze ATP in the presence of DNA and to promote branch migration of a synthetic Holliday junction at 37° C. These results indicate the conservation of the RuvA-RuvB interaction in different bacterial species, and suggest the existence of a ruvA homolog in T. thermophilus. Although GTP and dGTP were efficiently hydrolyzed by Tth RuvB, these nucleoside triphosphates could not be utilized for branch migration in vitro, implying that the conformational change in RuvB brought about by ATP hydrolysis, which is necessary for driving the Holliday junction branch migration, cannot be accomplished by the hydrolysis of these nucleoside triphosphates. Received: 26 November 1998 / Accepted: 19 April 1999     

Introduction of genes for leucine biosynthesis from Clostridium pasteurianum into C. acetobutylicum by cointegrate conjugal transfer

Summary A Clostridium pasteurianum gene bank was constructed in Escherichia coli, using plasmid pAT153, and several chromosomal fragments found which complemented both leuB and leuC mutations in auxotrophic E. coli K12 strains. No fragments capable of complementing leuA or leuD mutations were identified. Conjugal transfer of the LeuB/leuC genes from Bacillus subtilis into two different Leu^- C. acetobutylicum auxotrophic strains was elicited by their incorporation into a large plasmid cointegrate composed of the conjugal plasmid pAM1 and a specially constructed gram-positive, replication-deficient plasmid, pMTL21 EC. Inheritance of the cointegrate plasmid restored one of the auxotrophic C. acetobutylicum strains to prototrophy. The second strain remained Leu^-.     

Cloning of the β-Isopropylmalate Dehydrogenase Gene from Acetobacter aceti and Its Use for Construction of a New Host-vector System for Acetobacter

The β-isopropylmalate dehydrogenase (β-IPMDH) gene of Acetobacter aceti No. 1023, which complemented the leuB mutation of Escherichia coli, was cloned and expressed in E. coli. Plasmids pCAL1 and pCAL4, carrying a 5.44 kilobase pairs (kb) HindIII-fragment in the opposite orientation, conferred the same β-IPMDH activity as that of the wild type strain of E. coli. Restriction mapping and deletion analysis indicated that the β-IPMDH gene was located on a 1.65 kb AatII-HindIII fragment. Plasmids pMVL1 and pMVL2, composing the cloned β-IPMDH gene and plasmid pMV102, a plasmid indigenous to Acetobacter, were constructed as plasmid cloning vectors which allow selection of leu⁺ transformants in an A. aceti leu^- host. The A. aceti leu^- host was obtained through the insertional inactivation occurring as a result of homologous recombination between the chromosome of A. aceti and the cloned β-IPMDH gene, which was disrupted by insertion of the kanamycin resistance gene from pACYC177 into the BamHI site in the AatII-HindIII fragment. This system constitutes a relatively simple technique for gene disruption or replacement in Acetobacter that requires a single transformation.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.