DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δrtx::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.     

Rhizobitoxine modulates plant-microbe interactions by ethylene inhibition

Bradyrhizobium elkanii produces rhizobitoxine, an enol-ether amino acid, which has been regarded as a phytotoxin because it causes chlorosis in soybeans. However, recent studies have revealed that rhizobitoxine plays a positive role in establishing symbiosis between B. elkanii and host legumes: rhizobitoxine enhances the nodulation process by inhibiting ACC (1-aminocyclopropane-1-carboxylate) synthase in the ethylene biosynthesis of host roots. B. elkanii rtxA and rtxC genes are required for rhizobitoxine production. In particular, rtxC gene is involved in the desaturation of dihydrorhizobitoxine into rhizobitoxine. A legume with a mutated ethylene receptor gene produced markedly higher numbers of rhizobial infection threads and nodule primordia. Thus, endogenous ethylene in legume roots negatively regulates the formation of nodule primordia, which is overcome by rhiozbitoxine. Although a plant pathogen Burkholderia andropogonis has been known to produce rhizobitoxine, the genome sequence of Xanthomonas oryzae showed the existence of a putative rhizobitoxine transposon in the genome. The cumulative evidence suggests that rhizobitoxine-producing bacteria modulate plant-microbe interactions via ethylene in the rhizosphere and phyllosphere environments. In addition, rhizobitoxine-producing capability might be utilized as tools in agriculture and biotechnology.     

Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum.

To explore the role of rhizobitoxine in Bradyrhizobium-legume symbiosis, 11 rhizobitoxine mutants of B. japonicum USDA61 were isolated on the basis of their inability to synthesize the toxin in culture. Each mutant is prototrophic and symbiotically effective on soybean, cowpea, siratro, and Glycine soja. The rhizobitoxine mutants differ in their chlorosis phenotypes and rhizobitoxine production in planta. As expected, one group of mutant fail to make toxin in planta, resulting in the absence of chlorosis. Another group of mutants causes severe chlorosis on all cultivars of soybean tested. Surprisingly, this group of mutants makes more rhizobitoxine in soybean nodules than the wild-type strain does. This phenotype is only observed on soybean and not on other hosts such as cowpea, siratro, or G. soja. The remaining mutants all produce rhizobitoxine in planta but vary in the amount of toxin they produce and the severity of chlorosis they induce in soybean plants. Biochemical analysis of mutants demonstrates that one mutant is unable to synthesize serinol, a molecule hypothesized to be an intermediate in rhizobitoxine biosynthesis. By using these mutants, it was found that rhizobitoxine plays no apparent role in the nodulation of rj1 soybeans. Recently, it was found that inhibition of ethylene biosynthesis allows Rhizobium meliloti to overcome nitrate inhibition of nodule formation on alfalfa. Because rhizobitoxine also inhibits ethylene biosynthesis, we tested the ability of mutants which accumulate high levels of toxin in planta to overcome nitrate inhibition of nodule formation on soybean plants and found that the nodule formation induced by the wild type and that induced by mutant strains were equally suppressed in the presence of nitrate.     

Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and competitiveness on Macroptilium atropurpureum

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.     

Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. strain SCRC-2738.

The 38 kb eicosapentaenoic acid (EPA) biosynthesis gene cluster of Shewanella sp. strain SCRC-2738 was cloned into the cosmid vector (pEPA). A 27 kb nucleotide sequence of the XhoI to SpeI region of pEPA showed EPA production (6.3%) in E. coli JM109. Among the nine open reading frames (ORFs) in this sequence, only five (ORFs 2 and 5-8) were essential for EPA production. High levels of production (16%-22%) were found in E. coli JM109 transformed with a multicopy pNEB vector carrying only the five essential ORFs and in that transformed with a pNEB vector that integrated ORFs 3, 5, 6, 7 and 8, and vector pSTV28 that integrated the ORF2 encoding phosphopantetheinyl transferase (PPTase). Thus, production of EPA appears to be regulated by the presence of all the biosynthesis gene products and by the ratio of PPTase to the other gene products. The temperature -EPA production relationship in E. coli strain DH5alpha varied between constructs, suggesting that it is controlled not only by EPA biosynthesis enzymes but also by other factors in vivo. There was a strict upper temperature limit for EPA biosynthesis: no EPA was synthesized at 30 degrees C in E. coli transformants carrying any gene construct for EPA biosynthesis.     

Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of amphicarpaea.

Reciprocal inoculations with Bradyrhizobium sp. isolates from the North American legume Amphicarpaea bracteata (L.) Fern. (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var. japonica were performed to analyze relative symbiotic compatibility. Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A. bracteata, but all A. bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A. edgeworthii. However, one group of A. bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine. The beta-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia. No North American bradyrhizobial isolate from A. bracteata induced chlorosis on any plants, and the beta-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates. The role of rhizobitoxine in A. edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine. Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant. Thus, efficient nodule development in A. edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains.     

Biotechnological conversion of glycerol to 2-amino-1,3-propanediol (serinol) in recombinant Escherichia coli

Microbial conversion is an important technology for the refinement of renewable resources. Here, we describe the biotechnological conversion of glycerol to 2-amino-1,3-propanediol (serinol), a relevant intermediate in several chemical syntheses processes. Either the dihydroxyacetone phosphate aminotransferase/dihydrorhizobitoxine synthase (RtxA) of Bradyrhizobium elkanii USD94 or only the N-terminal domain (RtxA513) comprising the first reaction, respectively, was expressed in recombinant Escherichia coli. Serinol contents of up to 3.3 g/l were achieved in batch cultures. We could further clarify that glutamic acid is the preferred cosubstrate for the transamination of dihydroxyacetone phosphate to serinolphosphate, which is the essential step in serinol synthesis. An in vivo detoxification of serinol employing wax ester synthase/acyl-CoA:diacyl-glycerol acyl transferase from Acinetobacter baylyi ADP1 was not accomplished. This study paves the way for biotechnological production of serinol from glycerol derived from the biodiesel industry.     

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.     

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.     

Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. Strain SE19 by in vitro transposition

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.     

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosomes deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1

The general property of the Magnetospirillum spp is capable of forming magnetosomes in their cells, which are nanometer-sized, membrane-bound and chain- linked particles of magnetite (Fe3O4)[1,2]. The magne-tosomes may be useful for some aspects; for example, as carriers of antibodies for highly sensitive immuno-assay[3,4] and as carriers of drugs for targeting therapy of tumors[5]. The findings of magnetic particles in the presence of bees[6] and human brain[7], aroused interest in studying …     

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosomes deleted mutant NM4 of <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> MSR-1

A magnetosome deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 transposon mutagenesis, and a 5045-bp fragment flanking mini-Tn5 in NM4 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved six putative open reading frames (ORFs); the mini-Tn5 was inserted into ORF4. Functional complementary test indicated that the 5045-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The protein encoded by ORF4 had 25% of identity with the chemotaxis protein CheYIII of Caulobacter crescentus CB15, and the protein encoded by ORF4 contained a conserved signal receiver domain that can receive the signal from the sensor partner of the bacterial two-component systems. It was suggested that the protein encoded by ORF4 may take part in the signal transduction relating to biosynthesis of magnetosomes.     

Rhizobitoxine Production by Bradyrhizobium elkanii Enhances Nodulation and Competitiveness on Macroptilium atropurpureum

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.     

Acyl-CoA Z9- and Z10-desaturase genes from a New Zealand leafroller moth species,Planotortrix octo

Two cDNAs encoding acyl-CoA Z9-desaturase from the fat body and Z10-desaturase from the pheromone gland of the greenhead leafroller moth, Planotortrix octo, were obtained by RACE PCR. The Z9-desaturase (Pocto-Z9) cDNA spans 2291 nt with an ORF encoding a 352 amino-acid protein, which has 65% identity to Trichoplusia ni Delta 9 desaturase (Tni-Z9). The Z10-desaturase (Pocto-Z10) cDNA spans 2777 nt with an ORF encoding a protein with 356 amino acids. Pocto-Z10 shows lower identity to Pocto-Z9 and Tni-Z9 (48 and 46%, respectively) and relatively higher identity to the Delta 11 desaturases of T. ni and Helicoverpa zea (57 and 56%, respectively). The ORFs of these two P. octo cDNAs were constructed into an expression vector, YEpOLEX, that complemented the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae. Expression of Pocto-Z9 produced a 5:2 ratio of Z9-16 and Z9-18 acids, with minor amounts (<4%) of Z9-14, Z9-15, and Z9-17 acids. Pocto-Z10 was successfully expressed in the YEpOLEX system when complemented with Z11-18:Me, and the major desaturase product proved to be Z10-16:Acid. The results confirm the regio- and stereo-selectivity of this unusual Delta 10 desaturase.     

The molecular basis of the high linoleic acid content in Petunia seed oil: analysis of a seed-specific linoleic acid mutant

From a random transposon mutagenesis experiment, using Petunia line W138, a seed-specific linoleic acid mutant was isolated. The tagged gene was cloned and identified as a microsomal Delta(12) desaturase. Expression of the gene, however, was constitutive and not, as might have been expected, seed-specific. Moreover, self-fertilized homozygous mutants still contain 40% 18:2 in the seed lipid fraction. This suggests that at least two (seed-specific) Delta(12) desaturase genes are responsible for the high linoleic acid content in Petunia seed oil. Five members of the microsomal Delta(12) desaturase gene family have been identified and isolated. Data are presented on the molecular characterization and tissue-specific expression of these genes, which suggest that, in Petunia, the flux through the prokaryotic and eukaryotic pathways of lipid synthesis might be different from the situation found in Arabidopsis.     

rRNA and nifD phylogeny of Bradyrhizobium from sites across the Pacific Basin

Many undomesticated legumes harbor nodule bacteria related to the soybean symbiont Bradyrhizobium elkanii, but little is known about their phylogenetic relationships or geographic distribution. Sequences of ribosomal genes (16S rRNA and partial 23S rRNA) and the nitrogenase alpha-subunit gene (nifD) were analyzed in 22 isolates of this group sampled from diverse legumes in Korea, Japan, the USA, Mexico, Costa Rica and Panama. Some strains from Asia and North America shared identical sequences for both ribosomal genes. However, pairs of strains with closely related nifD sequences were almost never found in different regions. The major exceptions involved North American isolates B. elkanii USDA 76 and USDA 94, which had nifD sequences highly similar to certain Korean strains. However, 16S rRNA sequences of USDA 76 and USDA 94 were closely related to Central American rather than Asian bradyrhizobia, implying that these strains are genetic mosaics combining sequences from distinct ancestral areas. Several other conflicts between rRNA and nifD tree topologies indicated that the genealogical histories of these loci have been influenced by recurrent lateral gene transfer events.     

Trypanosoma brucei oleate desaturase may use a cytochrome b5-like domain in another desaturase as an electron donor.

An open reading frame with fatty acid desaturase similarity was identified in the genome of Trypanosoma brucei. The 1224 bp sequence specifies a protein of 408 amino acids with 59% and 58% similarity to Mortierella alpina and Arabidopsis thaliana Delta12 desaturase, respectively, and 51% with A. thaliana omega3 desaturases. The histidine tracks that compose the iron-binding active centers of the enzyme were more similar to those of the omega3 desaturases. Expression of the trypanosome gene in Saccharomyces cerevisiae resulted in the production of fatty acids that are normally not synthesized in yeast, namely linoleic acid (18:2Delta9,12) and hexadecadienoic acid (16:2Delta9,12), the levels of which were dependent on the culture temperature. At low temperature, the production of bi-unsaturated fatty acids and the 16:2/18:2 ratio were higher. Transformed yeast cultures supplemented with 19:1Delta10 fatty acid yielded 19:2Delta10,13, indicating that the enzyme is able to introduce a double bond at three carbon atoms from a pre-existent olefinic bond. The expression of the gene in a S. cerevisiae mutant defective in cytochrome b5 showed a significant reduction in bi-unsaturated fatty acid production, although it was not totally abolished. Based on the regioselectivity and substrate preferences, we characterized the trypanosome enzyme as a cytochrome b5-dependent oleate desaturase. Expression of the ORF in a double mutant (ole1Delta,cytb5Delta) abolished all oleate desaturase activity completely. OLE1 codes for the endogenous stearoyl-CoA desaturase. Thus, Ole1p has, like Cytb5p, an additional cytochrome b5 function (actually an electron donor function), which is responsible for the activity detected when using the cytb5Delta single mutant.