Expression of bacterial chitinase protein in tobacco leaves using two photosynthetic gene promoters

Summary A bacterial chitinase gene from Serratia marcescens (chiA) was fused to (i) a promoter of the ribulose bisphosphate carboxylase small subunit (rbcS) gene and (ii) two different chlorophyll a/b binding protein (cab) gene promoters from petunia. The resulting constructions were introduced into Agrobacterium Ti plasmid-based plant cell transformation vectors and used to generate multiple independent transgenic tobacco plants. ChiA mRNA and protein levels were measured in these plants. On average, the rbcS/chiA fusion gave rise to threefold more chiA mRNA than either cab/chiA fusion. We investigated the influence of sequences around the translational initiation ATG codon on the level of ChiA protein. The rbcS/chiA and cab/chiA fusions in which the sequence in the vicinity of the translational initiation codon is ACC ATGGC gave rise to transformants with higher levels of ChiA protein than those carrying a cab/chiA fusion with the sequence CAT ATGCG in the same region. This difference in translational efficiency is consistent with previous findings on preferred sequences in this region of the mRNA. In those transformants showing the highest level of ChiA expression, ChiA protein accumulated to about 0.25% of total soluble leaf protein. These plants contained significantly higher chitinase enzymatic activity than control plants.     

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Construction of a Promoter-probe Vector for <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>: the Identification of <Emphasis Type="Italic">cis</Emphasis>-acting Elements of the <Emphasis Type="Italic">chiA</Emphasis> Locus

The expression and application of Bacillus thuringiensis (Bt) chitinase genes have been extensively investigated. However, little information is available regarding the regulation of chitinase gene expression in Bt. In this study, a shuttle promoter-probe vector was constructed incorporating the thermostable β-galactosidase gene bgaB of B. stearothermophilus as the reporter for the study of Bt promoters. Using this plasmid, the activity of the chiA gene promoter in Bt was investigated. Deletion analysis of the putative chiA promoter region revealed that the sequence located ~75 bp DNA from positions −116 to −42, with respect to the translation start site, is the core promoter of chiA gene. Furthermore, a site for chitin induction was identified near position −36. This site for negative regulation was indicated downstream of the RNA polymerase binding sites of the promoter of chiA. The expression of chiA started in cell grown for about 6 h and reached the maximum after 60 h of incubation. Induction of chiA expression by chitin was demonstrated by an increase in β-galactosidase activity of ~2.5-fold.     

Expression of human antithrombin III in the cellular slime mould Dictyostelium discoideum

Summary In order to test the biotechnological potential of the cellular slime mould Dictyostelium discoideum the cDNA coding for human antithrombin III was expressed in this microorganism. The 1392-bp antithrombin III cDNA was fused to the N-terminal coding part of the D. discoideum actin 6 gene. In constructs carrying this artificial N-terminal coding region only low amounts of antithrombin III were detected. However, constructs from which all actin coding nucleotides were removed produced significant amounts of antithrombin III, most of which was secreted into the culture broth. Stationary cultures (1.5 × 10⁷ cells/ml) of certain stable transformants accumulated up to 1.0 g antithrombin III/ml culture medium within 24 h. The recombinant protein has a slightly smaller molecular weight in sodium dodecyl sulphate-polyacrylamide gels than authentic plasma antithrombin III and it is glycosylated, as determined by concanavalin A labelling. Offprint requests to: T. Dingermann     

Effect of local secondary structure of mRNA on expression of recombinant human lymphotoxin inEscherichia coli

We substituted a few alternative codons close to the N-terminal coding region of human lymphotoxin (LT) gene for natural ones to avoid stable local secondary structures of mRNA, predicted in the 5-terminal 130 nucleotides of mRNA. With this modification, we have overexpressed a recombinant lymphotoxin (rLT) inEscherichia coli under the control of trc promoter. Most of the produced rLT was a soluble and active form.     

Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.     

Bacterial chitinase is modified and secreted in transgenic tobacco

The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway.     

Cloning, sequencing and expression of the amylase isozyme gene from Pseudomonas sp. KFCC 10818

Summary A gene coding for amylase was cloned and sequenced from an alkalophilic Pseudomonas sp. KFCC 10818. The coding region for the amylase precursor contained 1,692 nucleotides. The presumed Shine-Dalgarno sequence, AAGG, was located at 8 nucleotides upstream from the ATG initiation codon. The precursor protein had a putative signal peptide of 25 amino acid residues at its amino terminus. The Pseudomonas amylase had four highly conserved regions as other -amylases. The amylase expressed from E. coli harboring the Pseudomonas gene produced maltose and maltotriose from soluble starch.The nucleotide sequence reported in this paper has been submitted in the GenBank/EMBL database with accession number(s) U40056.     

Cloning of Penicillium canescens Endo-1,4-β-xylanase Gene and Construction of Multicopy Strains

The complete gene xylA that encodes endo-1,4--xylanase secreted byPenicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven to eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30–50% of the total secreted protein.     

Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene

We have determined the nucleotide sequences of two structural genes of the Escherichia coli gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway: gabD, coding for succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16) and gabP, coding for the 4-aminobutyrate (GABA) transport carrier (GABA permease). We have previously reported the nucleotide sequence of the third structural gene of the cluster, gabT, coding for glutamate: succinic semialdehyde transaminase (EC 2.6.1.19). All three gab genes are transribed unidirectionally and their orientation within the cluster is 5-gabD-gabT-gabP-3. gabT and gabP are separated by an intergenic region of 234-bp, which contains three repetetive extragenic palindromic (REP) sequences. The gabD gene consists of 1,449 nucleotides specifying a protein of 482 amino acids with a molecular mass of 51.7 kDa. The protein shows significant homologies to the NAD⁺-dependent aldehyde dehydrogenase (EC 1.2.1.3) from Aspergillus nidulans and several mammals, and to the tumor associated NADP⁺-dependent aldehyde dehydrogenase (EC 1.2.1.4) from rat. The permease gene gabP comprises 1,401 nucleotides coding a highly hydrophobic protein of 466 amino acids with a molecular mass of 51.1 kDa. The GABA permease shows features typical for an integral membrane protein and is highly homologous to the aromatic acid carrier from E. coli, the proline, arginine and histidine permeases from Saccharomyces cerevisiae and the proline transport protein from A. nidulans. Uptake of GABA was increased ca. 5-fold in transformants of E. coli containing gabP plasmids. Strong overexpression of the gabP gene under control of the isopropyl-2-d-thiogalactoside (IPTG) inducible tac promoter, however, resulted in a severe growth inhibition of the transformed strains. The GABA carrier was characterized using moderately overexpressing transformants. The K _m of GABA uptake was found to be 11.8 M and the V_max 0.33 nmol/min · mg cells. Uptake of GABA was stimulated by ammonium sulfate and abolished by 2,4-dinitrophenol. Aspartate competed with GABA for uptake.     

Endochitinase Is Transported to the Extracellular Milieu by the eps-Encoded General Secretory Pathway of Vibrio cholerae

The chiA gene of Vibrio cholerae encodes a polypeptide which degrades chitin, a homopolymer of N-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans. chiA has a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of the chiA open reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity by V. cholerae required chiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for β,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes of V. cholerae and by the closely related bacterium Aeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by the eps system. The eps system is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential for eps-dependent secretion.Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is a major component of the cell walls of fungi and the integuments of crustaceans and insects (38). The molecule is one of the most abundant biopolymers in nature and is used by many microorganisms as a source of carbon. Utilization of chitin as a nutrient usually requires degradation of the molecule to GlcNAc monomers. Complete degradation of chitin in both prokaryotes and eukaryotes is a two-step process which involves successive hydrolysis of the β,1-4 glycosidic bonds linking the GlcNAc subunits. In the first stage, endochitinase binds and degrades tetrameric and longer polymeric forms of GlcNAc to produce the disaccharide chitobiose. In the second step, chitobiase hydrolyzes chitobiose to GlcNAc monomers. The enzymes for chitin degradation are usually coordinately regulated and in several organisms are induced by chitosan, chitobiose, GlcNAc, or glucosamine (2, 7, 44).Members of the family Vibrionaceae thrive in marine environments where chitin is abundant. It is not surprising that many Vibrionaceae evolved systems for utilizing chitin as a nutrient source. Chitinases have been identified in Vibrio vulnificus (56, 61), V. harveyi (57), and V. parahemolyticus (29, 30). Nucleotide sequence analysis indicated that the chitinase of V. harveyi is homologous with human hexosamindase, indicating that the two enzymes, as well as other chitinases, are members of a phylogenically related group (56).V. cholerae is a human intestinal pathogen that resides in brackish and marine waters. In vitro experiments established that V. cholerae has the potential to use chitin as a sole source of carbon for growth (15). It is likely, therefore, that production of chitinase (29, 30, 42) by V. cholerae provides the bacterium with a readily available nutrient source in aquatic environments. Hydrolysis of chitin by V. cholerae is an extracellular process that requires expression of epsE, one of a cluster of genes in the eps locus (43, 46–48). Several proteins of V. cholerae are dependent on the eps system for extracellular transport, including cholera toxin (CT), an undefined protease, and a chitinase activity (43, 48). Although expression of chitinase activity has been reported for V. cholerae, the enzyme responsible for the activity has not been identified. To further characterize the extracellular chitinase of V. cholerae, we cloned a gene encoding chitinase activity. Here we report the nucleotide sequence of a cloned endochitinase gene and establish that the protein encoded by that gene is secreted to the extracellular environment by an eps-dependent mechanism.     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11

Here, three different suicide vectors were evaluated for the possibility of performing gene mutagenesis in strain OH11 using the chiA gene (accession number: DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied for disruption and in-frame deletions in the chiA gene in strain OH11, which was confirmed by PCR amplification and Southern hybridization. The chiA-deletion mutant OH11-3 did not have the ability to produce chitinase on chitine selection medium. Interestingly, the chiA-deletion mutants displayed wild-type antimicrobial activity against Saccharomyces cerevisiae, Magnaporthe grisea, Phytophthora capsici, Rhizoctonia solani, Sclerotinia sclerotiorum and Pythium ultimum. Our data suggest that chitinase might not be a unique lytic enzyme in controlling S. cerevisiae, M. grisea, P. capsici, and P. ultimum. R. solani, S. sclerotiorum. Also, suicide vector pEX18GM might be explored as a potential tool for gene deletions in L. enzymogenes, which will facilitate the molecular study of mechanisms of biological control in L. enzymogenes.     

Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct

Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5 end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectinesterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit.This paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.     

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties

A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10–17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34269, calculated pI 4.7), and a 235 nucleotide 3-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-termininal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).