Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase.

In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of cellulose, the major cell wall polymer of plants. We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are homologs of the bacterial celA genes that encode the catalytic subunit of cellulose synthase. Three regions in the deduced amino acid sequences of the plant celA gene products are conserved with respect to the proteins encoded by bacterial celA genes. Within these conserved regions, there are four highly conserved subdomains previously suggested to be critical for catalysis and/or binding of the substrate UDP-glucose (UDP-Glc). An overexpressed DNA segment of the cotton celA1 gene encodes a polypeptide fragment that spans these domains and binds UDP-Glc, while a similar fragment having one of these domains deleted does not. The plant celA genes show little homology at the N- and C-terminal regions and also contain two internal insertions of sequence, one conserved and one hypervariable, that are not found in the bacterial gene sequences. Cotton celA1 and celA2 genes are expressed at high levels during active secondary wall cellulose synthesis in developing cotton fibers. Genomic Southern blot analyses in cotton demonstrate that celA forms a small gene family.

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers.

In animals, the small GTP-binding proteins, Rac and Rho, of the ras superfamily participate in the signal transduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.

Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco.

The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.

Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase.

Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.

Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch.

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.

Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants.

Cyclodextrins (CDs) are cyclic oligosaccharides containing six (alpha), seven (beta), or eight (gamma) glucose molecules, respectively. The cyclodextrin glycosyltransferases (CGT), which produce CDs from starch, are found only in bacteria and are used in batch fermentors with hydrolyzed starch to produce CDs commercially. Using a CGT gene from Klebsiella, we attempted to engineer the tubers of developing potatoes to produce these novel, high-value carbohydrates. A chimeric gene, consisting of (1) the patatin promoter for tuber-specific expression, (2) the small subunit of ribulose bisphosphate carboxylase (SSU) transit peptide for plastid targeting, (3) the CGT structural gene from Klebsiella and (4) the nopaline synthase 3' region, was introduced into potatoes. Both alpha and beta CDs were produced in tubers of transgenic potatoes at levels corresponding to 0.001-0.01% of the starch being converted to CDs.

Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide.

Ribulose bisphosphate carboxylase small subunit protein is synthesized in the cytoplasm as a precursor and transported into the chloroplast where the amino-terminal portion, the transit peptide, is removed proteolytically. To obtain chloroplast delivery of the 43-kDa 5-enolpyruvyl 3-phosphoshikimate (EPSP) synthase of Salmonella typhimurium, we constructed fusion proteins between the bacterial EPSP synthase and the ribulose bisphosphate carboxylase small subunit. A fusion protein consisting of the transit peptide fused to the EPSP synthase was not transported in vitro or in vivo into chloroplasts. A second fusion protein consisting of the transit peptide and 24 amino acids of the mature small subunit fused to the EPSP synthase was transported both in vitro and in vivo into chloroplasts. It was processed into two polypeptides of 46 and 47 kDa, respectively. This heterogeneity in processing was not caused by the presence of the aroA start codon, since its removal resulted in the same pattern. Substituting 24 different amino acids for the 24 amino acids of the mature small subunit resulted in a fusion protein that was not transported into the chloroplast. It was concluded that a portion of the mature small subunit was needed for efficient chloroplast delivery.

Herbicide resistance in transgenic plants expressing a bacterial detoxification gene.

The herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) is a photosynthetic (photosystem II) inhibitor in plants. A gene, bxn, encoding a specific nitrilase that converts bromoxynil to its primary metabolite 3,5-dibromo-4-hydroxybenzoic acid, was cloned from the natural soil bacterium Klebsiella ozaenae. For expression in plants, the bxn gene was placed under control of a light-regulated tissue-specific promoter, the ribulose bisphosphate carboxylase small subunit. Transfer of this chimeric gene and expression of a bromoxynil-specific nitrilase in leaves of transgenic tobacco plants conferred resistance to high levels of a commercial formulation of bromoxynil. The results presented indicate a successful approach to obtain herbicide resistance by introducing a novel catabolic detoxification gene in plants.

Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene.

A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.

Cloning and expression in Escherichia coli of a Klebsiella ozaenae plasmid-borne gene encoding a nitrilase specific for the herbicide bromoxynil.

An enzyme (nitrilase) that converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to its metabolite 3,5-dibromo-4-hydroxybenzoic acid was shown to be plasmid encoded in the natural soil isolate Klebsiella ozaenae. The bromoxynil-specific nitrilase was expressed in Escherichia coli by direct transfer and stable maintenance in E. coli of a naturally occurring 82-kilobase K. ozaenae plasmid. Irreversible loss of the ability to metabolize bromoxynil both in E. coli and K. ozaenae was associated with the conversion of the 82-kilobase plasmid to a 68-kilobase species. In E. coli this conversion was the result of a host recA+-dependent recombinational event. A gene, designated bxn, encoding the bromoxynil-specific nitrilase was constitutively expressed in K. ozaenae and E. coli and subcloned on a 2.6-kilobase PstI DNA segment. The polarity and the location of the gene were determined by assaying hybrid constructs of the bromoxynil-specific nitrilase gene fused with the heterologous lac promoter.

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae.

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K.

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.

Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication.

Plasmid pRK419, a derivative of the naturally occurring antibiotic resistance plasmid R6K, contains the pir gene that codes for the pi initiation protein and the beta and gamma replication origins of R6K. A mutation in plasmid pRK419, designated cos405, results in an elevated plasmid copy number in Escherichia coli growing at 42 degrees C and an even greater increase in copy number when the cells are shifted to 30 degrees C. This mutation was assigned to the structural gene for the pi protein on the basis of suppression of the mutant phenotype in E. coli when the wild-type pi protein is supplied in trans. Nucleotide sequence analysis of the cos405 mutant confirmed the pir gene location of the mutation and showed that this mutation results in a single amino acid substitution (glycine to aspartic acid) at the 81st position of the 305-amino acid pi protein. The properties of this mutant suggest that the pi protein plays a role in the negative control of the frequency of R6K initiation in addition to its requirement for the initiation of plasmid replication.

An Altered aroA Gene Product Confers Resistance to the Herbicide Glyphosate.

The hypothesis that the herbicide glyphosate (N-phosphonomethylglycine) acts on plants and microorganisms by inhibiting synthesis of 5-enolpyruvyl-3-phosphoshikimate, a precursor to aromatic amino acids, was tested. Salmonella typhimurium was treated with ethyl methanesulfonate, and mutants mapping at the aroA locus, which encodes 5-enolpyruvyl-3-phosphoshikimate synthetase, were isolated by selection for glyphosate resistance. One of the mutants results in the synthesis of a 5-enolpyruvyl-3-phosphoshikimate synthetase that is resistant to inhibition by glyphosate. The mutant aroA gene and the corresponding wild-type allele were cloned. The mutation confers high resistance to glyphosate when introduced in Escherichia coli in the presence or absence of the wild-type aroA allele.

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2.

A DNA sequence consisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G + C content is present. Deletion evidence indicates that the A-T rich and possibly the G + C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.

Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K.

A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and a structural gene for a protein, designated pi, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the pi structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the pi gene. A possible role of the tandem repeats in the regulation of expression of the pi protein and the control of initiation of replication of the plasmid R6K is discussed.