Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils

Generation times, acid production, carbon utilization, immunological properties, plasmid content, protein profile and symbiotic properties of 15 isolates of rhizobia nodulating Lotus subbiflorus were studied. Based on specific growth rates, carbon source utilization and acid production, 13 out of the 15 isolates could be assigned to the slow-growing group of rhizobia (bradyrhizobia). Using antisera against whole cells of three isolates, we separated the 15 isolates into three serogroups. Only the slow-growing isolate Ls4 and the fast-growers Ls5 and Ls552 lacked cross-reactivity with any of the sera tested. Electrophoretic mobilities of whole cell protein from seven out of the eight isolates included in the serogroup represented by strain Ls31 were identical. Similarly, isolates Ls1B3 and Ls1B4, both in serogroup Ls1B3, had the same pattern of cell proteins. In contrast, isolates Ls3 and Ls7, belonging to serogroup Ls7, differed in protein profile. Plant growth experiments carried out under bacteriologically controlled conditions revealed that all of the isolates effectively nodulated L. subbiflorus and L. pedunculatus, but were unable to form effective nodules on L. tenuis and L. corniculatus. All isolates showed similar effectiveness in symbiosis with L. subbiflorus, except isolate Ls7, which gave significantly higher plant dry weight.Abbreviations ELISA enzyme linked immuno-sorbent assay - kDa kiloDalton - MM mineral medium - PBS phosphate-buffered saline - RE relative efficiency - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - YEM deyeast extract mannitol     

Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485

A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid as the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5?g/l pyruvic acid was obtained from 50?g/l glucose after the culture for 32–40?h at pH?6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermentor culture was discussed.     

Lipoic acid and diabetes II: Mode of action of lipoic acid

Intraperitoneal administration of lipoic acid (10 mg/100 g) does not effect changes in serum insulin levels in normal and alloxan diabetic rats, while normalising increased serum pyruvate, and impaired liver pyruvic dehydrogenase characteristic of the diabetic state. Dihydrolipoic acid has been shown to participate in activation of fatty acids with equal facility as coenzyme A. Fatty acyl dihydrolipoic acid however is sparsely thiolyzed to yield acetyl dihydrolipoic acid. Also acetyl dihydrolipoic acid does not activate pyruvate carboxylase unlike acetyl coenzyme A. The reduced thiolysis of Β-keto fatty acyl dihydrolipoic acid esters and the lack of activation of pyruvic carboxylase by acetyl dihydrolipoic acid could account for the antiketotic and antigluconeogenic effects of lipoic acid     

Relationships Amongst the Fast-growing Rhizobia of Lablab purpureus, Leucaena leucocephala, Mimosa spp., Acacia farnesiana and Sesbania grandiflora and their Affinities with Other Rhizobial Groups

Colony characteristics, growth in litmus milk, precipitation in calcium glycerophosphate medium and utilization of carbon sources of the root-nodule bacteria isolated from the tropical legumes Leucaena, Mimosa, Acacia, Sesbania and Lablab were similar to fast-growing rhizobia of temperate legumes, particularly Rhizobium meliloti. In agglutination tests, isolates from each host shared antigens with one or more of five Rhizobium strains from Leucaena. Infective characteristics of the fast-growing rhizobia were studied in modified Leonard jars and in agar culture. Cross-infections by rhizobia between these plants were common and the association often effective. Lablab was effectively nodulated by its own fast-growing isolate but only formed root swellings, possibly ineffective pseudonodules, with the other isolates. Slow-growing rhizobia which were able to nodulate Macroptilium atropurpureus were unable to form nodules on these legumes except Lablab which was considered more akin to the cowpea group. All fast-growing isolates nodulated, often effectively, Vigna unguiculata and V. unguiculata ssp. sesquipedalis. The isolate from Lablab also effectively nodulated a number of other tropical legumes which have previously only been reported to nodulate with slow-growing nodule bacteria and it also produced ineffective nodulation on Medicago sativa. This is the first record of an effective fast-growing isolate from Lablab.     

Diverse Exopolysaccharide Producing Bacteria Isolated from Milled Sugarcane: Implications for Cane Spoilage and Sucrose Yield

Bacterial deterioration of sugarcane during harvesting and processing is correlated with significant loss of sucrose yield and the accumulation of bacterial polysaccharides. Dextran, a homoglucan produced by Leuconostoc mesenteroides, has been cited as the primary polysaccharide associated with sugarcane deterioration. A culture-based approach was used to isolate extracellular polysaccharide (EPS) producing bacterial strains from milled sugarcane stalks. Ribosomal RNA sequencing analysis grouped 25 isolates into 4 genera. This study identified 2 bacterial genera not previously associated with EPS production or sucrose degradation. All isolates produced polysaccharide when grown in the presence of sucrose. Monosaccharide analysis of purified polymers by Gas Chromatography revealed 17 EPSs consisting solely of glucose (homoglucans), while the remainder contained traces of mannose or fructose. Dextranase treatment of polysaccharides yielded full digestion profiles for only 11 extracts. Incomplete hydrolysis profiles of the remaining polysaccharides suggest the release of longer oligosaccharides which may interfere with sucrose crystal formation.     

Structure of Cyl-2, a Novel Cyclotetrapeptide from Cylindrocladium scoparium

Fermentative production of pyruvic acid by yeasts was studied using extracts from citrus natsudaidai peel as a carbon source. Many yeasts showed good growth. Of these yeasts, Debaryomyces coudertii IFO 1381 produced pyruvic acid at high yield. Pretreatment of the peel extract with Amberlite IR-120B (Na⁺) led to increased production of pyruvic acid. Under optimum conditions, the accumulation of pyruvic acid reached a maximum of 970 mg/100ml at 48 hr-fermentation. The pyruvic acid from the fermentation broth was identified with lactic acid dehydrogenase and by comparisons of properties of its 2,4-dinitrophenylhydrazone with those of authentic pyruvic acid in paper chromatography, IR spectrometry and elemental analysis.     

The origin of chloroplastic acetyl coenzyme A

Pyruvic dehydrogenase activity has been examined in a number of highly purified leaf organelles. In spinach leaf cell, the major activity is in the mitochrondrion with low activity in isolated chloroplasts. The major source of CO₂ derived from pyruvic acid metabolism in the isolated chloroplast is via the acetolactic synthase reaction localized in the chloroplast. Evidence is presented that the leaf mitochondrion contains both the pyruvic acid dehydrogenase and an acetyl coenzyme A hydrolase. It is suggested that free acetic acid is generated in the mitochrondrion and then moves to the chloroplast where acetyl coenzyme synthetase converts it from the metabolically inert acid to the very metabolically active acetyl coenzyme A.     

Improved Detection of Invasive Pulmonary Aspergillosis Arising during Leukemia Treatment Using a Panel of Host Response Proteins and Fungal Antigens

Invasive pulmonary aspergillosis (IPA) is an opportunistic fungal infection in patients undergoing chemotherapy for hematological malignancy, hematopoietic stem cell transplant, or other forms of immunosuppression. In this group, Aspergillus infections account for the majority of deaths due to mold pathogens. Although early detection is associated with improved outcomes, current diagnostic regimens lack sensitivity and specificity. Patients undergoing chemotherapy, stem cell transplantation and lung transplantation were enrolled in a multi-site prospective observational trial. Proven and probable IPA cases and matched controls were subjected to discovery proteomics analyses using a biofluid analysis platform, fractionating plasma into reproducible protein and peptide pools. From 556 spots identified by 2D gel electrophoresis, 66 differentially expressed post-translationally modified plasma proteins were identified in the leukemic subgroup only. This protein group was rich in complement components, acute-phase reactants and coagulation factors. Low molecular weight peptides corresponding to abundant plasma proteins were identified. A candidate marker panel of host response (9 plasma proteins, 4 peptides), fungal polysaccharides (galactomannan), and cell wall components (β-D glucan) were selected by statistical filtering for patients with leukemia as a primary underlying diagnosis. Quantitative measurements were developed to qualify the differential expression of the candidate host response proteins using selective reaction monitoring mass spectrometry assays, and then applied to a separate cohort of 57 patients with leukemia. In this verification cohort, a machine learning ensemble-based algorithm, generalized pathseeker (GPS) produced a greater case classification accuracy than galactomannan (GM) or host proteins alone. In conclusion, Integration of host response proteins with GM improves the diagnostic detection of probable IPA in patients undergoing treatment for hematologic malignancy. Upon further validation, early detection of probable IPA in leukemia treatment will provide opportunities for earlier interventions and interventional clinical trials.     

Proteome analysis of Halobacterium salinarum and characterization of proteins related to the degradation of isopropyl alcohol

We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M.     

Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.     

The extracellular polysaccharides of Rhizobium japonicum: Compositional studies

The extracellular polysaccharides of seven strains of Rhizobium japonicum were investigated by using a gas-chromatographic scheme developed for determination of the various sugars present. These polysaccharides were more heterogeneous in their composition than those of any other species of Rhizobium yet examined. Five strains (1809, 110, 123, 127, and 709) produced polysaccharides containing the same constituents, although in varying relative amounts: glucose (36–44%), galactose (7–25%), mannose (18–20%), 4-O-methylgalactose (5–13%), galacturonic acid (12–16%), and acetyl groups (4–8%). The sugars of the polysaccharide of strain 1809 were all of the d series. These are the first bacterial polysaccharides reported to contain 4-O-methylgalactose and the first Rhizobium polysaccharides in which galacturonic acid has been found. In contrast to this, the polysaccharide of strain 129 consisted of glucose (7%), galactose (51%), mannose (5%), xylose (5%), glucuronic acid (5%), and pyruvic acid (2%). The polysaccharide of strain 711 contained glucose (34%), galactose (13%), mannose (27%), and pyruvic acid (6%).     

Analysis of bla(CTX-M)-carrying plasmids from Escherichia coli isolates collected in the BfT-GermVet study

In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored bla(CTX-M-1), and the canine isolate 913 harbored bla(CTX-M-15), as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The bla(CTX-M-1) upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the bla(CTX-M-15) gene on plasmid pCTX913 differed distinctly from that of both bla(CTX-M-1) genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the bla(CTX-M) gene regions.     

Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase-rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae.     

Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485

A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid aas the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5 g/l pyruvic acid was obtained from 50 g/l glucose after the culture for 32–40 h at pH6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermentor culture was discussed.     

Characterisation and differentiation of indigenous rhizobia isolated from Canarian shrub legumes of agricultural and ecological interest

In the course of a study on rhizobia nodulating six indigenous legume shrubs from the Canary Islands, one Rhizobium and 27 Bradyrhizobium Canarian isolates were characterised. It was found that those ascribed to Bradyrhizobium were promiscuous and formed effective nodules not only in their original host but on Chamecytisus proliferus subsp. proliferus (Tagasaste) as well. However, Rhizobium isolate RES-1 was more specific and only nodulated on its host (Teline canariensis). The serotyping of these isolates required a broad antisera panel due to the great antigenic diversity of these rhizobia, that appeared to be due to differences in their lipopolysaccharides, the main antigenic determinants, that showed great structural diversity. The 28 isolates studied produced 22 easily distinguishable electrophoretic profiles of lipopolysaccharides. Protein or plasmid electrophoretic profiles were equally or less discriminating than the lipopolysaccharides profiles and were more difficult to compare. The comparison of the lipopolysaccharide electrophoretic patterns is a more reliable and discriminating method than serotyping or electrophoretic protein and plasmid profile analysis for the identification of Bradyrhizobium strains. No correlation between the lipopolysaccharide profiles of the isolates and the plant from which they were obtained or their geographical origin was observed.     

Genetic diversity of rhizobia from Leucaena leucocephala nodules in Mexican soils

Leucaena species are leguminous plants native to Mexico. Using two L. leucocephala cultivars grown in different soils, we obtained 150 isolates from the nodules. Twelve rDNA types were identified which clustered into groups corresponding to Mesorhizobium, Rhizobium , and Sinorhizobium by restriction fragment length polymorphism (RFLP) of amplified 16S rRNA genes. Types 2, 4, 5, 6, 10, 11, and 12 were distinct from all the defined species. Others had patterns indistinguishable from some recognized species. Most of the isolates corresponded to Sinorhizobium . Forty-one electrophoretic types (ETs) were identified among the isolates based on the different combinations of electrophoretic patterns of 13 metabolic enzymes. ETs were clustered into groups in general agreement with the rDNA types. Diverse plasmid patterns were obtained among the isolates, but common plasmids were observed among most isolates within rDNA types 5, 10, and 11. The symbiotic plasmids were identified among most of the isolates, except for the Mesorhizobium isolates. The affinities of host cultivars for different rhizobial groups and the impact of soil cultivation on the soil populations of rhizobia were analysed from the estimation of isolation frequencies and diversity. The results showed differences in rhizobial populations in cultivated and uncultivated soils and also differences in rhizobia trapped by L. leucocephala cv. Cunningham or Peruvian.     

Extracellular polysaccharides of Erwinia futululu,a bacterium associated with a fungal canker disease of Eucalyptus spp

Extracellular polysaccharides (EPSs) produced by an Erwinia spp. associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii. The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6-O-[(R)-1-carboxyethylidene)-Galp. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures:The molecular weights of the polysaccharides range from 1.3-2.1x10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Differential soluble protein expression between Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes

A comparative analysis of proteomic maps of long-term grown and fresh clinical Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes, respectively, was performed using two-dimensional gel electrophoresis and mass spectrometry. Of 29 protein spots differentially expressed between the isolates, 19 were over-expressed in the isolate exhibiting high virulence phenotype: proteins associated with cytoskeletal dynamics, such as coronin and several isoforms of actin, as well as proteins involved in signal transduction, protein turnover, proteolysis, and energetic and polyamine metabolisms were identified. Some malate dehydrogenase, fructose-1,6-bisphosphate aldolase and ornithine cyclodeamidase isoforms were exclusively expressed by the highly virulent isolate. During interaction assays with VEC, parasites exhibiting high virulence phenotype rapidly adhered and switched to amoeboid forms. In contrast, low adhesion and no morphological transformation were observed in parasites displaying low virulence phenotype. Our findings demonstrate that expression of specific proteins by high and low virulence parasites could be associated with the ability of each isolate to undergo morphological transformation and interact with host cells. Such data represent an important step towards understanding of the complex interaction network of proteins that participate in the mechanism of pathogenesis of this protozoan.     

Proteomic investigation of the impact of oxygen on the protein profiles of hyaluronic acid‐producing Streptococcus zooepidemicus

Hyaluronic acid (HA) is a linear and negatively charged polysaccharide regularly used in medicine and cosmetics. Recently Streptococcus zooepidemicus has been exploited in the fermentation industry to produce HA. Many studies showed that higher amounts of HA were produced under aerobic condition compared to anaerobic conditions. To explore the effect of oxygen on the HA synthesis in S. zooepidemicus, 2‐DE was used to compare the proteomes of aerobically and anaerobically fermented bacteria to identify proteins, which might be associated with the influence of oxygen on the HA synthesis. Totally nine pairs of 2‐DE gels collected from three batches were compared and nine overexpressed proteins were observed in aerobically fermented bacteria. These proteins were identified by LC/tandem MS as dihydrolipoamide dehydrogenase, UDP‐acetyl‐glucosamine pyrophosphoylase, dihydrolipoamide‐S‐acetyltransferase and acetoin dehydrogenase α and β chains, respectively. These upregulated proteins were involved in acetoin dissimilation, the central carbon metabolism and the HA anabolic pathway, implicating that oxygen might augment the expression of genes that are involved in central energy metabolism, acetoin reutilization and HA biosynthesis to enhance the amount of acetyl‐CoA as such that more acetyl‐CoA can be diverged from the central carbon metabolism to replenish acetyl‐CoA for the HA synthesis.     

Numerical Taxonomy of Sarothamnus scoparius Rhizobia

Twenty nodule isolates from Sarothamnus scoparius (broom) growing in Poland and nine strains from plants growing in Japan were studied for phenotypic properties, plasmid presence, phage sensitivity, and host plant specificity. By numerical analysis of phenotypic properties, it was found that the studied nodule bacteria, originating from geographically different countries, constitute two separate groups affiliated to the bradyrhizobium cluster. The membership of S. scoparius rhizobia in the Bradyrhizobium genus was also supported by their long generation time, alkaline reaction in YEM medium with mannitol, lack of plasmids, and wide host plant range. Received: 12 June 2000 / Accepted: 17 July 2000