Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Ubiquinone system,GC contents and cellular fatty acid composition of species of the form-genusMalbranchea andCoccidioides immitis for chemotaxonomic study

Ubiquinone system, GC contents and cellular fatty acid (CFA) composition were analyzed as criteria for chemotaxonomy ofMalbranchea species andCoccidioides immitis, which are suggested to be phylogenetically related. Based on the major ubiquinone,Malbranchea spp. were divided into two groups, of which one group possessed the same major ubiquinone asC. immitis. Similar GC contents and CFA profiles were obtained for the species ofMalbranchea andC. immitis. On the basis of these criteria the relationships between the fungi are discussed.     

Lipopolysaccharide structures from Agrobacterium and Rhizobiaceae species

This review reports and discusses the structural and the biological data available for the lipopolysaccharides from the Gram-negative bacterium Agrobacterium together with those of other related Rhizobiaceae species.     

Suitability of lipid materials for culture ofMalassezia as evaluated from its cellular fatty acid composition

Malassezia is a facultative or obligatory lipophilic yeast. We devised new lipid-supplemented media suitable for the culture ofMalassezia. Malassezia furfur andM. pachydermatis grew well on both solid and liquid media supplemented with creaming powder preparations which are commercially available at moderate prices. Striking differences were found between the cellular fatty acid compositions ofM. furfur grown on media supplemented with creaming powder and that grown on media with conventional olive oil.Malassezia furfur grown on media with olive oil had nearly the same fatty acid composition as olive oil, with C18:1 amounting to 80%, while that grown on media supplemented with creaming powder had C16, C18:1 and C18:2 as the principal components. The use of these supplementary lipids appeared not to inhibit the normal synthesis of fatty acid inM. furfur. For the culture ofM. pachydermatis, media supplemented with creaming powder were also found more suitable than lipid-free media. The media devised are considered excellent, because they appear to provide a more natural growth environment forMalassezia.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Grouping of isolates in AG 2 ofRhizoctonia solani by total cellular fatty acid analysis

Total-cellular fatty acid compositions of 34 isolates ofRhizoctonia solani belonging to intraspecific groups (ISGs) of anastomosis group (AG) 2, i.e., AG 2-1, AG 2-2 IIIB (mat rush), AG 2-2 IV (sugar beet), AG 2-2 LP (turfgrass), and AG 2–3 (soybean), were compared. The major fatty acids identified were palmitic, stearic, and oleic acids. Principal component analysis based on the percentage composition of total cellular fatty acids revealed consistently low variability among isolates of a single ISG of AG 2. Average linkage cluster analysis showed that isolates obtained from turfgrass representing a newly proposed group, AG 2-2 LP, were differentiated from other AG 2 ISGs. Isolates of another newly proposed group AG 2–3, from diseased soybean were also closely related to AG 2-1 and AG 2-2 IIIB but distinguishable from the AG 2-1 and AG 2-2 LP isolates by the average linkage cluster analysis. These results suggested that the percentage composition of total-cellular fatty acids is a distinct characteristic for the five ISGs belonging to AG 2, and fatty acid analysis is useful for the differentiation and characterization of these ISGs of AG 2 inR. solani.     

Chemotaxonomy of the Rubiaceae family based on leaf fatty acid composition

With 10,700 species distributed in 637 genera, the Rubiaceae family is one of the largest of the angiosperms. Since it was previously evidenced that the fatty acid composition of photosynthetic tissues can be a tool for chemotaxonomic studies, the fatty acid composition of leaves from 107 Rubiaceae species highly representative of the diversity of the family was determined. Principal component analysis allowed a clear-cut separation of Coffeae, Psychotrieae and Rubieae. The occurrence of C16:3 fatty acid, a marker of the prokaryotic plastidial lipid biosynthetic pathway, concerned at least two branches: Theligoneae/Rubieae and Anthospermeae-Anthosperminae which appeared to be in close relationship. Additional experiments were carried out to ensure the correlation between the presence of C16:3 fatty acid and the prokaryotic biosynthetic pathway.     

Effect of salinity of medium on cellular fatty acid composition of marine algaPorphyridium cruentum (Rhodophyceae)

Porphyridium cruentum Näg. (clone 161) was found to grow best in medium containing between 0.45 M and 0.8 M NaCl. From studies done on growing cultures, the palmitic acid content of the cells decreased with increasing NaCl concentration of the medium. Conversely, when the culture was transferred from a 0.8 M NaCl medium to 0.2 M NaCl, the amount of palmitic acid in thePorphyridium cells increased with time of incubation and it contributed up to 64.5% of the total fatty acid content. There appears to be a negative correlation between the cellular content of palmitic acid and the growth lag. The oleic acid content varied only marginally with increasing NaCl concentration. The poly-unsaturated acid content (linolenic and arachidonic acids) decreased initially and then increased with NaCl concentration up to and beyond ca. 0.8 M NaCl respectively. At 1.5 M NaCl, the poly-unsaturated fatty acids amounted to 78.2% of the total fatty acids in the cell. For stationary phaseP. cruentum cultures, a similar relationship existed between fatty acids and NaCl concentration. However, palmitic acid was accumulated up to three-fold more when compared to the exponential culture grown in low salinity. In addition stearic acid was also found in significant quantities.     

Lipid content and fatty acid composition in hypogeous organs ofHelleborus species (Ranunculaceae)

Root and rhizome of fourHelleborus species:H. viridis, H. odorus, H. niger, H. foetidus were examined throughout a two-year period in order to determine the variation of lipid content and fatty acid composition during the ontogenetic cycle. In the deciduous geophytesH. viridis andH. odorus subsp.laxus the lipid content reaches its maximum during the quiescent phase, whereas in the evergreen geophyteH. niger the lipid content is highest during the main growth period. In the chamaephyteH. foetidus lipid content is always very low and it does not show variation throughout the year. In all species the fatty acids detected in the neutral fraction are myristic, palmitic, stearic, oleic and linoleic acid. Palmitic and linoleic acid are always present in larger quantities. The pattern of the relative content of saturated/unsaturated fatty acids seems to be linked to the life form of these species.     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Identification of catalase-producingCampylobacter species based on biochemical characteristics and on cellular fatty acid composition

A total of 50 catalase-positive campylobacters from human and animal sources were studied. The nomenclatural type strains ofCampylobacter coli, C. jejuni, C. fetus, andC. fetus subsp.venerealis, a typical strain of the nalidixic acid-resistant thermophilic group, and various clinical isolates were characterized by bacteriological tests and by gas-liquid chromatographic analysis of their cellular fatty acids. The tests most useful in the differentiation of the various catalase-positive species were growth at 25 and 42°C, H₂S production, tolerance to nalidixic acid and to 2,3,5-triphenyltetrazolium chloride, and hippurate hydrolysis. The latter test was the only reliable means to differentiate betweenC. coli andC. jejuni. Differences between.C. fetus andC. jejuni/coli were confirmed by cellular fatty acid compositions. The bacteriological results indicated thatC. fetus andC. jejuni were distinct species, although within the thermophilic campylobacters there were several related taxa that included bothC. coli andC. jejuni strains with typicalC. coli and some thermophilic strains ofC. fetus subsp.fetus at the extremes.     

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border

Summary The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the T_c-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the T_c-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the T_L-DNA and ends approximately 3 bp from an IS66 homolog in the T_c-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with T_c-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).     

Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici

Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features. The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules. Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici. This work shows that R. gallicum and R. tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia. Moreover, some of these host plants can be nodulated by both species. The results confirm the great promiscuity of R. tropici and also support the hypothesis that the species R. gallicum may be native from America or cosmopolitan and worldwide spread.     

Comparative effects of Glomus mosseae and P fertilizer on foliar mineral composition of Acacia senegal seedlings inoculated with Rhizobium

Summary A factorial experiment with two controlled factors was conducted in the greenhouse with Acacia Senegal seedlings. The substrate was a degraded sandy soil (Dior soil) poor in available P (11 ppm — Olsen). The first controlled factor was soil sterilization, with two levels: (A) sterilized soil; (B) non-sterilized soil. The second factor was fertilization, with six levels: (1) uninoculated control; (2) inoculation with Rhizobium (ORS 1007); (3) inoculation with Glomus mosseae; (4) double inoculation with ORS 1007 and G. mosseae; (5) inoculation with ORS 1007 and 30 ppm phosphorus per plant; (6) inoculation with ORS 1007 and 60 ppm phosphours. The combination of the two factors and their levels led to 12 different plant treatments (A1–A6 and B1–B6). Compared to the control B1, the B5 and B6 treatments containing phosphorus increased: nodule dry weight about 7 times ; leaf dry weight about 4 times ; total N, P and Mg 4–5 times; total K and Ca 3–4 times. The mycorrhizal inoculation had the same positive effect on plant growth and mineral composition but with lower values. Plants inoculated with Rhizobium alone gave the lowest results. The A1 treatment gave lower values than B1. Foliar mineral contents varied within a narrow range (20–30%).     

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     

Diet-induced variations in fatty acid content and composition of two on-grown stages of Artemia Salina

Three species of microalgae, the freshwater Euglena gracilis and themarine Dunaliella salina and Tetraselmis suecica, were fed tothe brine shrimp Artemia salina in order to compare their suitabilityin terms of fatty acid enrichment, and their effect on the biometric parametersof the zooplankter. The fatty acid content and composition were analyzed for the post-larval and pre-adult stages of Artemia fed the algae and theresults compared to the initial content of unfed 24-hour post-hatch nauplii.Differences in the total fatty acid content occurred between the three stages,the fatty acid profile being determined by the composition of the diet. A decreasing trend for almost all the individual fatty acids occurred throughdevelopment from post-larva to pre-adult with each of the three algal diets.Biometrical differences between Artemia fed the marine algae and that fed Euglena were not consistent in the post-larval stage, but became considerable in the pre-adult stage. Artemia fed with Euglena achieved twice the weight of animals fed the marine algae and showed thehighest length. The implications for the use of on-grown Artemia as afeed in larviculture of marine and freshwater fish and crustaceans are considered.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Polar lipids and fatty acid composition ofThermus strains from New Zealand

The polar lipids and fatty acid composition ofThermus aquaticus YT-1 and YS 041,T. filiformis Wai33 A1 and eighteen isolates from New Zealand, several of which are attributed toT. filiformis, were compared to complement the taxonomy of these organisms. The polar lipid patterns were essentially similar in all strains and consisted of one major phospholipid and one major glycolipid. The fatty acid analysis produced three basic groups corresponding toT. filiformis Wai33 A1,T. aquaticus and the third to the other New Zealand strains. The presence of hydroxy fatty acids is reported inThermus spp. for the first time.     

Rhizobium nepotum sp. nov. isolated from tumors on different plant species

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7(T) from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these isolates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeeping genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA-DNA hybridization of strain 39/7(T) with the type strain of R. radiobacter LMG 140(T) revealed 45% DNA-DNA hybridization. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7(T)=LMG 26435(T)=CFBP 7436(T)) is proposed.