Enhanced Nodulation and Nitrogen Fixation by a Revertant of a Nodulation-Defective Bradyrhizobium japonicum Tryptophan Auxotroph.

In greenhouse studies, the symbiotic properties of a prototrophic revertant (TA11 NOD) of a nodulation defective tryptophan auxotroph of Bradyrhizobium japonicum were compared with those of the normally nodulating wild-type strain, B. japonicum I-110 ARS. Strain I-110 ARS was the parent of auxotrophic mutant TA11. Plants inoculated with TA11 NOD contained significantly more nitrogen per plant than did plants inoculated with wild-type bacteria (275.9 +/- 35 versus 184 +/- 18 mg). Also, plants that received the revertant were larger, averaging 8.4 +/- 0.9 g (dry weight) versus 6.4 +/- 0.6 g for those that received the wild-type bacterial strain. Additionally, plants that received the NOD strain had 56% more nodules and 41% more nodule mass than did control plants. With both inocula, average nodule size and amount of nitrogen fixed per gram of nodule were about the same. These data indicated that the improvement in nitrogen fixation observed with the TA11 NOD resulted from an increase in the overall nodule number. The physiological basis for this increase in nodulation is not known, but enhanced tryptophan catabolism does not appear to be involved.

Determination of dimethylselenide and dimethyldiselenide by gas chromatography-photoionization detection.

A simple method for the determination of volatile selenium compounds employing a gas chromatograph equipped with a photoionization detector is described. The method involves the direct injection of dimethylselenide (DMS) or dimethyldiselenide (DMDS) into the gas chromatograph; no derivatization of the sample was required. The photoionization detector was capable of detecting 60 pg (0.55 pmol) of DMS and 150pg (0.80pmol) DMDS. Sensitivity was 10-50 times greater with DMS and 4-20 times greater with DMDS when the photoionization detector was employed than when the flame ionization detector was employed.

Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts.

As of February 2003, bacteria that form nitrogen-fixing symbiotic associations with legumes have been confirmed in 44 species of 12 genera. Phylogenies of these taxa containing legume symbionts based on the comparative analysis of 16S rDNA sequences show that they are not clustered in one lineage but are distributed in the classes Alphaproteobacteria and Betaproteobacteria, and dispersed over the following nine monophyletic groups, being intermingled with other taxa that do not contain legume symbionts (shown in parentheses below): Group 1, which comprises Rhizobium and Allorhizobium species containing legume symbionts (intermingled with Agrobacterium and Blastobacter species, which are nonsymbionts); Group 2, Sinorhizobium and Ensifer species (with unclassified nonsymbionts); Group 3, Mesorhizobium species (with nonsymbiotic Aminobacter and Pseudaminobacter species); Group 4, Bradyrhizobium species and Blastobacter denitrificans (with nonsymbiotic Agromonas, Nitrobacter, Afipia, and Rhodopseudomonas species); Group 5, 'Methylobacterium nodulans" (with nonsymbiotic Methylobacterium species); Group 6, Azorhizobium species (with nonsymbiotic Xanthobacter and Aquabacter species); Group 7, 'Devosia neptuniae" (with nonsymbiotic Devosia species and unclassified nonsymbionts); Group 8, symbiotic Burkholderia strains (with nonsymbiotic Burkholderia species); and Group 9, Ralstonia taiwanensis (with nonsymbiotic Ralstonia species). For Groups 5, 8, and 9, the present classification, in which 'each monophyletic group comprises one genus wherein legume symbionts and nonsymbionts are intermingled with each other, " is considered to be retained as is because they are clearly separated from other genera at high bootstrap values and have already been sufficiently characterized based on polyphasic taxonomy. As for the remaining six monophyletic groups, on the other hand, there are currently three options for emending their current classification (definitions and circumscriptions) at the generic level: A) the current classification shall be retained as is; B) all the genera within each monophyletic group shall be amalgamated into one single genus in conformity with the results of phylogenetic analysis; or C) each subordinate lineage in each monophyletic group shall be proposed as a genus. It is considered that research and discussions will be continuously conducted for emending the classification of these monophyletic groups based chiefly on Options B and C as preferable candidates.

Conservation of gene order and content in the circular chromosomes of 'Candidatus Liberibacter asiaticus' and other Rhizobiales.

'Ca. Liberibacter asiaticus,' an insect-vectored, obligate intracellular bacterium associated with citrus-greening disease, also called "HLB," is a member of the Rhizobiales along with nitrogen-fixing microsymbionts Sinorhizobium meliloti and Bradyrhizobium japonicum, plant pathogen Agrobacterium tumefaciens and facultative intracellular mammalian pathogen Bartonella henselae. Comparative analyses of their circular chromosomes identified 514 orthologous genes shared among all five species. Shared among all five species are 50 identical blocks of microsyntenous orthologous genes (MOGs), containing a total of 283 genes. While retaining highly conserved genomic blocks of microsynteny, divergent evolution, horizontal gene transfer and niche specialization have disrupted macrosynteny among the five circular chromosomes compared. Highly conserved microsyntenous gene clusters help define the Rhizobiales, an order previously defined by 16S RNA gene similarity and herein represented by the three families: Bartonellaceae, Bradyrhizobiaceae and Rhizobiaceae. Genes without orthologs in the other four species help define individual species. The circular chromosomes of each of the five Rhizobiales species examined had genes lacking orthologs in the other four species. For example, 63 proteins are encoded by genes of 'Ca. Liberibacter asiaticus' not shared with other members of the Rhizobiales. Of these 63 proteins, 17 have predicted functions related to DNA replication or RNA transcription, and some of these may have roles related to low genomic GC content. An additional 17 proteins have predicted functions relevant to cellular processes, particularly modifications of the cell surface. Seventeen unshared proteins have specific metabolic functions including a pathway to synthesize cholesterol encoded by a seven-gene operon. The remaining 12 proteins encoded by 'Ca. Liberibacter asiaticus' genes not shared with other Rhizobiales are of bacteriophage origin. 'Ca. Liberibacter asiaticus' shares 11 genes with only Sinorhizobium meliloti and 12 genes are shared with only Bartonella henselae.

'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction.

Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored 'Ca. Liberibacter asiaticus,' is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. Only two 'Ca. Liberibacter asiaticus' proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na(+)/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and 'Ca. Liberibacter asiaticus' orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤ E-10) with 'Ca. Liberibacter asiaticus' proteins, often present as multiple orthologs of single 'Ca. Liberibacter asiaticus' proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational analysis and is consistent with the hypothesis that these proteins may be of particular importance in host/microbe interaction and their duplication likely facilitates their ongoing evolution.

Genes Encoding Callose Synthase and Phytochrome A Are Adjacent to a MAP3Kα-Like Gene in Beta vulgaris US H20.

MAP3Kα, a gene that encodes a key conserved protein kinase, is responsible for initiating a rapid cascade of cellular events leading to localized cell death. Hypersensitive response, as it is termed, enables genetically resistant plants to limit microbial invasion under the right environmental conditions. Since knowledge of close physically linked genes is important for genome analysis and possibly for improving disease resistance, systematic DNA sequence analysis, gene annotation, and protein BLASTs were performed to identify and characterize genes in close physical proximity to a MAP3Kα-like gene in Beta vulgaris L. US H20. On the same 125 Kb BAC, callose synthase (BvCS) and phytochrome A (PhyA) genes were within 50 Kb of MAP3Kα. The close physical linkage of these genes may result from selection for coordinated responses to disease pressure. Bert, a new chromodomain-carrying gypsy-like LTR retrotransposon, resides within an intron of the BvCS gene, where it is transcribed from the opposing strand.

Comparison of the 'Ca. Liberibacter asiaticus' genome adapted for an intracellular lifestyle with other members of the Rhizobiales.

An intracellular plant pathogen 'Candidatus Liberibacter asiaticus,' a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, nitrogen fixing endosymbionts, Agrobacterium tumefaciens, a plant pathogen, and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 50 clusters of conserved orthologous genes found on the chromosomes of all five metabolically diverse species. The intracellular pathogens 'Ca. Liberibacter asiaticus' and Bartonella henselae have genomes drastically reduced in gene content and size as well as a relatively low content of guanine and cytosine. Codon and amino acid preferences that emphasize low guanosine and cytosine usage are globally employed in these genomes, including within regions of microsynteny and within signature sequences of orthologous proteins. The length of orthologous proteins is generally conserved, but not their isoelectric points, consistent with extensive amino acid substitutions to accommodate selection for low GC content. The 'Ca. Liberibacter asiaticus' genome apparently has all of the genes required for DNA replication present in Sinorhizobium meliloti except it has only two, rather than three RNaseH genes. The gene set required for DNA repair has only one rather than ten DNA ligases found in Sinorhizobium meliloti, and the DNA PolI of 'Ca. Liberibacter asiaticus' lacks domains needed for excision repair. Thus the ability of 'Ca. Liberibacter asiaticus' to repair mutations in its genome may be impaired. Both 'Ca. Liberibacter asiaticus and Bartonella henselae lack enzymes needed for the metabolism of purines and pyrimidines, which must therefore be obtained from the host. The 'Ca. Liberibacter asiaticus' genome also has a greatly reduced set of sigma factors used to control transcription, and lacks sigma factors 24, 28 and 38. The 'Ca. Liberibacter asiaticus' genome has all of the hallmarks of a reduced genome of a pathogen adapted to an intracellular lifestyle.

Carbohydrate and ethane release with Erwinia carotovora subspecies betavasculorum--induced necrosis.

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.

Reduction of selenite to elemental red selenium by Rhizobium sp. strain B1.

A bacterium that reduces the soluble and toxic selenite anion to insoluble elemental red selenium (Se(0)) was isolated from a laboratory bioreactor. Biochemical, morphological, and 16S rRNA gene sequence alignment identified the isolate as a Rhizobium sp. that is related to but is genetically divergent from R. radiobacter (syn. Agrobacterium tumefaciens) or R. rubi (syn. A. rubi). The isolate was capable of denitrification and reduced selenite to Se(0) under aerobic and denitrifying conditions. It did not reduce selenate and did not use selenite or selenate as terminal e(-) donors. Native gel electrophoresis revealed two bands, corresponding to molecular weights of approximately 100 and approximately 45 kDa, that reduced selenite. Tungsten inhibited in vivo selenite reduction, suggesting that a molybdenum-containing protein is involved in selenite reduction. This organism, or its enzymes or DNA, might be useful in bioreactors designed to remove selenite from water.

Identification and characterization of an Aeromonas salmonicida (syn Haemophilus piscium) strain that reduces selenite to elemental red selenium.

A bacterium that reduces toxic and mobile selenite to insoluble elemental selenium (Se0) was isolated from a laboratory scale permeable reactive biobarrier. Biochemical tests and 16S rRNA gene sequence alignment identified the isolate as Aeromonas salmonicida. Two colony types were isolated, one more resistant to selenite than the other. Both grew on agar plates containing 16 mM: selenite, although the colony diameter was reduced to 8% of controls with the small colony type and to 18% with the large colony type. Further study was done with the large colony type. In anaerobic culture, this bacterium was able to use nitrate as a term electron acceptor but not selenate or selenite. In aerobic culture, when no nitrate was present, early log phase cells removed selenite at a rate of 2.6 +/- 0.42 micromol SeO3 (-2)/mg protein/day. Reduction was retarded by 25 mM: nitrate. Mutants with a diminished ability to reduce selenite to Se0 also had a reduced ability to reduce nitrate to nitrous oxide. This bacterium, or perhaps its enzymes or DNA, might be used to remove selenite from contaminated groundwaters.

Removing selenite from groundwater with an in situ biobarrier: laboratory studies.

Laboratory biobarriers were evaluated for their ability to remove selenite from flowing groundwater. Microbial activity in aquifers is usually limited by substrate availability, and biobarriers stimulate microbial activity by providing a substrate; for these studies soybean oil was used. Water containing 10 mg L(-1) selenite-Se was pumped through the biobarriers for 74 days and the amount present in the effluent monitored. The amounts remained high for the first 2 weeks of the study but then declined. From day 28 until the end of the study the amount of selenite-Se in the column effluents averaged 0.20 +/- 0.04 mg L(-1), a decrease of approximately 98%. At the end of the study about half of the selenite-Se applied to the columns was recovered as immobilized selenium trapped by the biobarrier. This study suggests that biobarriers containing vegetable oil might be used as a process for removing selenite from contaminated groundwater.

Expression in sugar beet of the introduced cercosporin toxin export (CFP) gene from Cercospora kikuchii, the causative organism of purple seed stain in soybean.

The Cercospora kikuchii cercosporin export gene, CFP, introduced into Beta vulgaris L. by conjugation with Rhizobium radiobacter, was stably maintained during vegetative propagation as verified by PCR using primers specific for the CFP gene. Transcriptional expression of the CFP gene in leaves was determined by RT-PCR using CFP-specific primers. CFP protein was detected using Western analysis with an affinity-purified polypeptide-specifc antibody. Analysis of the relative susceptibility of CFP-transgenic and non-transgenic sugar beet plants is planned but will probably take several years to complete.

Rhizobium radiobacter conjugation and callus-independent shoot regeneration used to introduce the cercosporin export gene cfp from Cercospora into sugar beet (Beta vulgaris L.).

Leaf spot disease caused by Cercospora is responsible for crop and profitability losses in sugar beet crops in the US and worldwide. The cfp gene that encodes a protein that exports phytotoxic cercosporins from Cercospora was conjugally transferred to sugar beet using Rhizobium radiobacter (Agrobacterium tumefaciens), to improve Cercospora-induced leafspot resistance. Conditions for shoot regeneration were optimized to increase regeneration/transformation efficiencies. Low-light and room-temperature conditions were favorable to sugar beet regeneration without callus when cytokinin had been added to the tissue culture medium. Using this procedure adventitious shoots from leaf pieces were obtained in a simple, one-step regeneration procedure. T7, a cfp-transgenic clone verified by PCR with gene-specific primers, is being propagated for leaf spot disease resistance evaluation.