Division of Rhizobitoxine-Producing and Hydrogen-Uptake Positive Strains of Bradyrhizobium japonicum by nifDKE Sequence Divergence

Genomic digests from 25 strains of Bradyrhizobium japonicum,for which the phenotypes have been determined with respect toproduction of rhizobitoxine, hydrogen uptake (Hup) and compositionof extracellular polysaccharide (EPS), were hybridized withprobe DNAs of the nifDK and nifE genes of B. japonicum USDA110. The degree of the estimated base substitution in and aroundnifDKE clearly divided the strains of B. japonicum into twomarkedly divergent groups, which were designated as genotypeI and II. Moreover, a strict correlation was observed betweenthese genotypes, production of rhizobitoxine and EPS composition.The genotype I strains produced no rhizobitoxine and an EPScomposed of glucose, mannose, galactose, 4-O-methyl galactoseand galacturonic acid, whereas the genotype II strains producedrhizobitoxine and an EPS composed of rhamnose and 4-O-methylglucuronic acid. Hup⁺ strains were confined exclusively to thegenotype I. Hind III digests of genomic DNAs from the 25 strainswere hybridized with probe DNA of structural genes for the uptakehydrogenase from B. japonicum. In 23 wild-type strains, Hup⁺strains generated a 5.9-kb band that hybridized to the probeunder high-stringency conditions, while Hup^– strains didnot generate the band. These results suggest that the genotypesI and II are two highly divergent evolutionary lines that definea marked division of various phenotypes, such as productionof rhizobitoxine, EPS composition and hydrogen uptake. (Received July 10, 1989; Accepted November 4, 1989)     

Comparison of Extracellular Polysaccharide Composition, Rhizobitoxine Production, and Hydrogenase Phenotype among Various Strains of Bradyrhizobium japonicum

A survey of 51 strains of Bradyrhizobium japonicum was performedwith respect to the composition of extracellular polysaccharide(EPS), the production of rhizobitoxine and the hydrogenase phenotype.A good correlation was found among these three different characteristics.Thirty-six strains producing an EPS composed of glucose, mannose,galactose, 4-O-methyl galactose and galacturonic acid did notsynthesize rhizobitoxine, whereas 14 strains producing an EPScomposed of rhamnose and 4-O-methyl glucuronic acid were allfound to synthesize rhizobitoxine. Hydrogen-uptake positive(Hup⁺) strains were confined exclusively to the former groupof strains which produced an EPS composed of glucose, mannose,galactose, 4-O-methyl galactose and galacturonic acid. Theseresults suggest that the phenotype with respect to rhizobitoxineproduction and hydrogen uptake is involved in the phylogenyof Bradyrhizobium japonicum as well as in the productivity ofnodulated soybeans. (Received March 24, 1989; Accepted June 16, 1989)     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)     

Indolepyruvate Pathway for Indole-3-Acetic Acid Biosynthesis in Bradyrhizobium elkanii

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatantof Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp)was incorporated into IAAId and indole-3-acetic acid (IAA),suggesting that B. elkanii produces IAA via IAAId from Trp.In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA)was converted to IAAId, and exogenously added IAAId was rapidlyconverted to IAA. Furthermore, the activity of indolepyruvatedecarboxylase (IPDC), which catalyzes the decarboxylation ofIPyA to produce IAAId and is a key enzyme for IPyA pathway,was detected in B. elkanii cell-free extract. The IPDC activitydepended on Mg²⁺ and thiamine pyrophosphate, cofactors of decarboxylation.This mounting evidence strongly suggests that IAA synthesisoccurs via IPyA pathway (Trp IPyA p IAAId IAA) in B. elkanii. (Received December 11, 1995; Accepted March 4, 1996)     

Increased Production of IAA by Rhizoctonia solani is Induced by Culture Filtrate from Rice Suspension Cultures

To investigate the role of the plant hormones produced by fungi,we tried to construct a system to examine the interaction betweenRhizoctonia solani Kühn MAFF305219 and rice cells in suspensionculture (Oc). R. solani was previously found to produce IAA,with the main biosynthetic pathway via the indole-3-pyruvatepathway. The amount of IAA in the medium produced by R. solaniwas increased by cocultivation with rice cells (Oc) and by culturefiltrate (CF) of Oc. Further analysis revealed that the factor(s)that induced the enhanced accumulation of IAA was sensitiveto heat, to freezing and thawing and lyophilization, and themolecular weight was estimated to more than 10,000. These resultssuggest that the active agent(s) in the medium was (a) proteinor a proteinous substance. Among suspension cultures of variousplants, Oc and another line of rice cells (Ok) had the abilityto induce the accumulation of IAA in the fungal medium 4 h afterinoculation but other cultures of plant cells were ineffective.The promotive effect of rice CF on the accumulation of IAA wasalso observed with some strains of R. solani that belong toa different anastmosis group from MAFF305219. Thus, the accumulationof IAA was not related to the host specificity. (Received July 28, 1997; Accepted October 27, 1997)     

RESTRICTION ENZYME ANALYSIS OF DNA FROM SPECIES OF BULINUS (BASOMMATOPHORA: PLANORBIDAE) USING A CLONED RIBOSOMAL RNA GENE PROBE

A ribosomal RNA gene probe (pSM889) has been used to study restrictionenzyme digests of various species of Bulinus. In order to minimiseproblems of DNA shearing associated with snail tissues a methodof extracting nucleic acids from material embedded in agaroseblocks has been used. Restriction enzyme digests with Bgl IIand Bam HI hybridised to pSM889 showed clear differences betweenB. truncatus, B. wrighti, B. africanus and B. forskalii, representingthe four species groups of Bulinus. No differences were observedbetween samples of B. tropicus and B. truncatus digested withBam HI, Bgl II and Pst I. Intra-specific variation was observedbetween samples of B. forskalii from Säo Tomé andAngola digested with Bgl II and Hind III although restrictionprofiles for Bam HI, Pst I and Bst EII digests were similar.Intra-specific variation was also observed between two differentpopulation samples of B. wrighti from South Yemen using BamHI and Bgl II digested genomic DNA hybridised to pSM889. (Received 5 December 1989; accepted 19 April 1990)     

REMARKABLE CONSERVATION OF INTERNALLY TRANSCRIBED SPACER SEQUENCES OF ARTHROSPIRA (“SPIRULINA” ) (CYANOPHYCEAE,CYANOBACTERIA) STRAINS FROM FOUR CONTINENTS AND OF RECENT AND 30‐YEAR‐OLD DRIED SAMPLES FROM AFRICA1

The internally transcribed spacer (ITS) sequences of 21 Arthrospira clonal strains from four continents and assigned to four different species (A. platensis, A. maxima, A. fusiformis, A. indica) in the culture collections were determined. Two main clusters, I and II, were differentiated by 49 positions out of 475 nt or 477 nt, respectively. Each cluster was further subdivided into two subclusters. Subclusters I.A and I.B were separated by two substitutions, whereas subclusters II.A and II.B were distinguished by four substitutions. After direct sequencing of the PCR products, three dried samples from Chad aged between 3 months and 35 years yielded a sequence belonging to subcluster I.A, as did a recent commercial product. The strains grown in production plants belonged to the same (sub)clusters as strains from culture collections, mainly I.A and II. PCR primers specific for each cluster and subcluster were designed and tested with crude cell lysates of Arthrospira strains. One dried sample (“dihé” 1) and a herbarium sample from Lake Sonachi (Kenya) only contained I.A sequences, whereas the commercial product was a mixture of the four genotypes and the other two dried samples contained minor polymorphisms characteristic of different clusters. Five clonal Arthrospira strains, thought to be duplicates, showed the simultaneous presence of the two forms of the four diagnostic positions that distinguish subclusters genotype II.A and genotype II.B. This is likely to be caused by multiple copies of the rDNA operon, in a intermediate stage of homogenization between subcluster II.A and subcluster II.B. The high conservation of ITS sequences is in contrast with the assignment to four different species, the great morphological variability of the strains, and their wide geographic distribution.     

The Production of Pyruvic and {alpha}-Oxoglutaric Acids by Mucor Species

Six out of the seven species of Mucor studied (M. plumbeus,M. racemosus, M. rouxii, M. hiemalis, M. ramannianus, and M.mucedo) are shown to produce pyruvic acid when grown on a mediumcontaining glucose, glutamate, and mineral salts. Mucor pusillusproduces no detectable pyruvate on this medium. The proportionof the carbohydrate metabolized which is excreted as pyruvateis greatest in species having a high thiamine requirement forgrowth. Addition of thiamine to the culture medium reduces pyruvateexcretion. However, in the species which need added thiaminefor growth, the growth requirement is almost completely satisfiedby 20 µg thiamine per litre whereas suppression of pyruvateexcretion requires about 200 µg thiamine per litre. Experimentswith M. plumbeus show that the yield of pyruvate is reducedwhen xylose is substituted for glucose and also when alanineis substituted for glutamate in the medium. -Oxoglutaric acidis produced by all seven species on the glucose/glutamate medium,the yield varying with the species. This acid is partly derivedfrom the glutamate in the medium.     

Rhizobitoxine: A phytotoxin of unknown function which is commonly produced by bradyrhizobia

Summary Fifty-six percent of 93 strains ofBradyrhizobium japonicum andBradyrhizobium sp. (various hosts) from diverse geographical areas were found to produce a chlorosis-inducing toxin. Toxin production was common among bradyrhizobia originating from the USA, Africa, Central America, and South America. Toxin produced by West African strains was compared with rhizobitoxine by cation exchange chromatography, paper chromatography, and soybean (Glycine max (L.) Merr.) bioassay. The comparison suggested that the chlorosis-inducing toxin produced by West African bradyrhizobia is rhizobitoxine. Purified toxin from a West AfricanBradyrhizobium sp. (Vigna) strain inhibited the growth ofBacillus subtilis on minimal medium. The growth inhibition was reduced by addition of yeast-extract or casamino acids but not by any of 21 individual amino acids, including methionine. The same toxin did not inhibit the growth of 14 Bradyrhizobium strains, including eight strains that did not produce toxin. Mixed inoculum experiments revealed that a toxin-producing West African strain could not assist toxin non-producingB. japonicum strains in nodulating non-nodulating (rj₁ rj₁) soybeans.     

Isolation of Rhizobium leguminosarum biovar viciae Variants with Indole-3-acetamide Hydrolase Activity

The IAA biosynthetic pathway from tryptophan to IAA via IAM(IAM pathway) was investigated in Rhizobium spp. (fast-growingrhizobium). Southern hybridization with the bam gene, a structuralgene for IAM hydrolase (the enzyme that converts IAM to IAA)cloned from Bradyrhizobium japonicum J1063, indicated that homologoussequences exist among wild-type Rhizobium spp. However the IAMpathway has not been detected biochemically in free-living bacteria.When 5-methyltryptophan-resistant strains were screened forRhizobium leguminosarum biovar viciae K5 which has DNA sequenceswith high homology to the bam gene, spontaneous mutants showingIAM hydrolase activity were isolated. The results suggest thepossibility that the activity of IAM hydrolase is suppressedin free-living state in Rhizobium leguminosarum biovar viciaeK5. In addition we detected the peak at the same t_R of IAM byHPLC analysis using two columns when a large amount of L-tryptophanwas added to the suspension of 5-methyltryptophan-resistantvariants. Whether or not tryptophan-2-monooxygenase activity,however, actually works in Rhizobium cells remained to be solved. (Received September 20, 1989; Accepted March 6, 1990)     

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 new strains of nonphotosynthetic mutants of Chlamydomonas reinhardtii I. Fluorescence, photochemical activities, chlorophyll-protein complexes

Thirty-six new strains of nonphotosynthetic mutants of Chlamydomonasreinhardtii, which had been induced by UV irradiation then screenedphotographically for strong chlorophyll fluorescence of theircolonies, were characterized with respect to pigment contents,photochemical activities of Hill and Mehler reactions and chlorophyllfluorescence induction, and by SDS-polyacrylamide gel electrophoresisof the chlorophyllprotein complexes. Eight strains did not show any Photosystem II activity and fiveshowed only very weak activity. Analysis of the chloroplastmembranes of ten of these strains showed that all containedboth complexes CP I and CP II. In the case of one of these mutants,Fl 50, which was totally unable to perform the Hill reactionfrom H₂O, addition of DPC restored about 25% of the DCIP photoreductionactivity. This could be interpreted tentatively in terms ofan impaired accessibility of the Photosystem II centers to electrondonors and acceptors. Ten other mutants showed the following anomalies: no PhotosystemI activity, lack of complex CP I, and higher chlorophyll fluorescenceyield and lower Chl a/Chl b ratio than the wild type. Thesefeatures appeared to be related. (Received February 8, 1979; )     

Production of Indole-3-Acetic Acid in the Plant-Beneficial Strain Pseudomonas chlororaphis O6 Is Negatively Regulated by the Global Sensor Kinase GacS

Certain plant growth–promoting bacteria, such as Pseudomonas fluorescens 89B61 and Bacillus pumilus SE34, secreted high levels of indole-3-acetic acid (IAA) in tryptophan-amended medium in stationary phase as determined by chromogenic analysis and high-performance liquid chromatography. Two other growth-promoting strains, P. chlororaphis O6 and Serratia marcescens 90-166, did not produce these high levels of IAA. However, when the gacS mutant of P. chlororaphis O6 was grown in tryptophan-supplemented medium, IAA was detected in culture filtrates. IAA production by the gacS mutant in P. chlororaphis O6 was repressed in the tryptophan medium by complementation with the wild-type gacS gene. Thus, the global regulatory Gac system in P. chlororaphis O6 acts as a negative regulator of IAA production from trypophan.     

Production of volatile compounds in liquid cultures by six strains of coryneform bacteria

Summary The aromatic profiles of four strains of Brevibacterium linens, one strain of Brevibacterium sp. and one strain of Microbacterium sp. were determined with some pure cultures of these microorganisms in standard trypcase soy liquid medium, which enabled four of these six strains to produce flavour compounds of ripened cheese. Thirty-two flavour compounds were identified by gas chromatography and mass spectrometry. The identified flavour compounds included the following: fatty acids, alcohols, methylketones, sulphur compounds, aromatic compounds and a pyrazine. Some important differences were found among the six strains studied. The four strains of B. linens had similar flavour profiles. Their typical flavour was probably due to dimethyltrisulphide. The two other strains did not appear to produce this compound. Three strains produced significant amounts of the floral aromas phenylethanol and phenylpropanone.Offprint requests to: J.-M. Belin     

The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19^th century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins.     

Formation of Novel Polysaccharides by Bradyrhizobium japonicum Bacteroids in Soybean Nodules

Certain strains of Bradyrhizobium japonicum form a previously unknown polysaccharide in the root nodules of soybean plants (Glycine max (L.) Merr.). The polysaccharide accumulates inside of the symbiosome membrane—the plant-derived membrane enclosing the bacteroids. In older nodules (60 days after planting), the polysaccharide occupies most of the symbiosome volume and symbiosomes become enlarged so that there is little host cytoplasm in infected cells. The two different groups of B. japonicum which produce different types of polysaccharide in culture produce polysaccharides of similar composition in nodules. Polysaccharide formed by group I strains (e.g., USDA 5 and USDA 123) is composed of rhamnose, galactose, and 2-O-methylglucuronic acid, while polysaccharide formed by group II strains (e.g., USDA 31 and USDA 39) is composed of rhamnose and 4-O-methylglucuronic acid. That the polysaccharide is a bacterial product is indicated by its composition plus the fact that polysaccharide formation is independent of host genotype but is dependent on the bacterial genotype. Polysaccharide formation in nodules is common among strains in serogroups 123, 127, 129, and 31, with 27 of 39 strains (69%) testing positive. Polysaccharide formation in nodules is uncommon among other B. japonicum serogroups, with only 1 strain in 18 (6%) testing positive.     

Epidermal Cells Do Not Contribute to Auxin-Induced Ethylene Production in Mung Bean Stem Sections

Auxin-induced and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependentethylene production in mung bean (Vigna radiata [L] Wilczek)hypocotyl sections, from which epidermis had been removed, wasinvestigated. Ethylene production in hypocotyl sections withoutepidermis was induced by treatment with IAA, and also occurredfrom exogenously supplied ACC in the presence of 0.2 M mannitol.Isolated epidermal strips alone failed to produce substantialamounts of ethylene in response to IAA or from exogenous ACC.3,4-[¹⁴C]-Methionone was incorporated into both ACC and ethylenein peeled sections treated with IAA, but not in the isolatedepidermal strips. Radioactive ACC, however, was detected inthe epidermal strips separated from the unpeeled sections previouslyfed with 3,4-[¹⁴C]-methionine in the presence of IAA. We concludethat the Site of auxin-induced ethylene production is not inthe epidermis, but in other hypocotyl cells, and that epidermalcells lack the activity which converts ACC to ethylene. (Received January 28, 1985; Accepted May 4, 1985)     

Stability of Related Human and Chicken Campylobacter jejuni Genotypes after Passage through Chick Intestine Studied by Pulsed-Field Gel Electrophoresis

The genomic stability of 12 Campylobacter jejuni strains consisting of two groups of human and chicken isolates was studied by analysis of their PFGE (pulsed-field gel electrophoresis) patterns after passage through newly hatched chicks’ intestines. The patterns of SmaI, SalI, and SacII digests remained stable after intestinal passage, except for those of two strains. One originally human strain, FB 6371, changed its genotype from II/A (SmaI/SacII) to I/B. Another strain, BTI, originally isolated from a chicken, changed its genotype from I/B to a new genotype. The genomic instability of the strains was further confirmed by SalI digestion and ribotyping of the HaeIII digests. In addition, heat-stable serotype 57 of strain FB 6371 changed to serotype 27 in all isolates with new genotypes but remained unchanged in an isolate with the original genotype. Serotype 27 of strain BTI remained stable. Our study suggests that during intestinal colonization, genomic rearrangement, as demonstrated by changed PFGE and ribopatterns, may occur.     

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.