The role of cell surface bacterial lectins in the aggregation of Azospirilla

The mutant strain Azospirillum brasilense Sp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilense Sp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilense Sp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense 75, A. brasilense Sp7, and A. lipoferum 59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilense Sp7 cells with Bacillus polymyxa 1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab(Sp59b) and Ab(Cd) were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.     

Chemical and serological studies of liposaccharides of bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis>

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS_Cd, LPS_Sp59b, LPS_Sp7, LPS_S17, and LPS_KBC1 preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab_Sp59b and Ab_Cd were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.     

Lipopolysaccharides in four strains of the unicellular cyanobacterium Synechocystis

The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir⁺) which react with FA from Sp7 the type strain. None of the nir^- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir^- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.Abbreviations Fa fluorescent antibody - FITC fluorescein isothiocyanate - Rh ITC gelatin-rhodamine isothiocyanate - nir⁺ nitrite reductase positive - nir^- nitrite reductase negative     

Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245

Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC.     

Catabolism of carbohydrates and organic acids and expression of nitrogenase by azospirilla

Fructose, galactose, L-arabinose, gluconate, and several organic acids support rapid growth and N2 fixation of Azospirillum brasiliense ATCC 29145 (strain Sp7) as a sole source of carbon and energy. Growth of Azospirillum lipoferum ATCC 29707 (strain Sp59b) is also supported by glucose, mannose, mannitol, and alpha-ketoglutarate. Oxidation of fructose and gluconate by A. brasiliense Sp7 and of glucose, gluconate, and fructose by A. lipoferum Sp59b was achieved through inducible enzymatic mechanisms. Both strains exhibited all of the enzymes of the Embden-Meyerhof-Parnas pathway, and strain Sp59b also possesses all the enzymes of the Entner-Doudoroff pathway. Fluoride inhibited growth on fructose (strains Sp7 and Sp59b) or on glucose (strain Sp59b) but not on malate. There was no activity via the oxidative hexose monophosphate pathway in either strain. There was greater activity with 1-phosphofructokinase than with 6-phosphofructokinase in both strains. Strain Sp59b formed fructose-6-phosphate via hexokinase, an enzyme that is lacking in strain Sp7. A. brasiliense and A. lipoferum exhibited the enzymes both of the tricarboxylic acid cycle and of the glyoxylate shunt; iodoacetate, fluoropyruvate, and malonate were inhibitory. A. brasiliense Sp7 could not transport [14C]glucose and alpha-[14C]ketoglutarate into its cells.     

Serological relationships of azospirilla revealed by their motility patterns in the presence of antibodies to lipopolysaccharides

Motility of the serologically different Azospirillum brasilense strains Sp245 (serogroup I) and Sp7 (serogroup II) was studied in the presence of antibodies to their lipopolysaccharides (LPS). A procedure was proposed in order to determine the motility patterns indicating the specificity of the interaction between the anti-LPS antibodies and bacteria. Analysis of the effect of such antibodies on motility of 25 strains (A. brasilense, A. lipoferum, A. irakense, and Azospirillum sp.) revealed bacteria exhibiting antigenic cross reactions with A. brasilense Sp7 or Sp245. The effect of anti-LPS antibodies on motility of azospirilla was in agreement with the results of immune agglutination analysis of bacterial cells and of immunodiffusion analysis of the LPS preparations. According to our results, strains Azospirillum sp. SR81 and A. brasilense SR14 should be included into serogroups I and II, respectively.     

Plasmid-encoded gamma-hexachlorocyclohexane degradation genes and insertion sequences in Sphingobium francense (ex-Sphingomonas paucimobilis Sp+)

The lin genes encode the gamma-hexachlorocyclohexane (gamma-HCH or lindane) catabolic pathway in lindane-degrading strains. The location and stability of these genes have been explored in the lindane-degrading Sphingobium francense strain Sp+, and in two non-lindane-degrading mutants (Sp1- and Sp2-). The lin genes, linA, linB, linE and linX were localized by hybridization on three of the six plasmids of the S. francense strain Sp+ showing dispersal within the genome. The linC gene was detected by PCR, but was not detected by hybridization on any of the plasmids. The hybridization of the linA and linX genes was negative with the two non-lindane-degrading mutants S. francense strains, Sp1- and Sp2-. The dynamic of this genome associated with gene loss and acquisition, and plasmid rearrangement was explored by a search for associated insertion sequences. A new insertion sequence, ISSppa4, belonging to the IS21 family was detected and compared with IS6100 and ISsp1. Insertion sequence localization was explored on different hybridization patterns (plasmid, total genome) with the lindane-degrading Sp+ strain and the two non-degrading derivatives (Sp1-, Sp2-). Insertion sequence movement and plasmid rearrangement could explain the emergence of the non-lindane-degrading mutants.     

Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1–specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-endophytic strains of Azospirillum brasilense.

The plant-associated nitrogen-fixing rhizobacterium Azospirillum brasilense attracts world-wide attention owing to its plant growth-promoting activities. Among hundreds of its strains known up to date, wild-type strain Sp245 has been proved to be capable of colonising both the plant-root interior and exterior (i.e. a facultative endophyte), whereas others are non-endophytes colonising the root surface only. Thus, the different ecological niches occupied by these strains in the rhizosphere suggest that their responses to environmental conditions might differ as well. In this study, responses of A. brasilense strains Sp245 and Sp7 to several heavy metals (Co2+, Cu2+, Zn2+), present in the medium at tolerable concentrations (up to 0.2 mmol/l) and taken up by the bacteria, were compared. Fourier transform infrared (FTIR) spectroscopy was used for controlling the compositional features of whole cells. The results obtained show that in strain Sp7 (non-endophyte) the heavy metals induced an enhanced accumulation of polyester compounds (poly-3-hydroxybutyrate; PHB). In contrast, the response of the endophytic strain Sp245 to heavy metal uptake was found to be much less pronounced. These dissimilarities in their behaviour may be caused by different adaptation abilities of these strains to stress conditions owing to their different ecological status. It was also found that adding 0.2 mmol/l Cu2+ or Cd2+ in the culture medium resulted in noticeably reducing the levels of indole-3-acetic acid (IAA, auxin) produced by both the strains of the bacterium. This can directly affect the efficiency of associative plant-bacterial symbioses involving A. brasilense in heavy-metal-contaminated soil.     

Haloalkane dehalogenase LinB is responsible for beta- and delta-hexachlorocyclohexane transformation in Sphingobium indicum B90A

Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform beta- and delta-hexachlorocyclohexane (beta- and delta-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with beta- and delta-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including beta-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade beta- and delta-HCH.     

Diversity of infectious pancreatic necrosis virus strains isolated from fish, shellfish, and other reservoirs in Northwestern Spain

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.     

Diversity of Infectious Pancreatic Necrosis Virus Strains Isolated from Fish, Shellfish, and Other Reservoirs in Northwestern Spain

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.     

In‐planta sporulation phenotype: a major life history trait to understand the evolution of Alnus‐infective Frankia strains

Two major types of Frankia strains are usually recognized, based on the ability to sporulate in‐planta: spore‐positive (Sp+) and spore‐negative (Sp?). We carried out a study of Sp+ and Sp? Frankia strains based on nodules collected on Alnus glutinosa, Alnus incana and Alnus viridis. The nodules were phenotyped using improved histology methods, and endophytic Frankia strain genotype was determined using a multilocus sequence analysis approach. An additional sampling was done to assess the relation between Sp+ phenotype frequency and genetic diversity of Frankia strains at the alder stand scale. Our results revealed that (i) Sp+ and Sp? Alnus‐infective Frankia strains are genetically different even when sampled from the same alder stand and the same host–plant species; (ii) there are at least two distinct phylogenetic lineages of Sp+ Frankia that cluster according to the host–plant species and without regard of geographic distance and (iii) genetic diversity of Sp+ strains is very low at the alder stand scale compared with Sp? strains. Difference in evolutionary history and genetic diversity between Sp+ and Sp? Frankia allows us to discuss the possible ecological role of in‐planta sporulation.     

Lipopolysaccharides of two strains of the phototrophic bacterium Rhodopseudomonas capsulata

The lipopolysaccharides of Rhodopseudomonas capsulata strains St. Louis (ATCC 23782) and Sp 11 both contain L-acofriose, rhamnose, glucose and glucosamine as the main sugar constituents. 2-Keto-3-deoxyoctonate and neuraminic acid were tentatively identified. The fatty acid spectrum found with both strains comprises 3-OH-C10 and C12:1 (ester-linked) and 3-oxo-C14 (amide-linked). Isolated lipid A from strain Sp 11 contains glucosamine, glucosamine-phosphate and the total of the fatty acids of the lipopolysaccharide. Methylation analysis of the degraded polysaccharide of this lipopolysaccharide shows L-acofriose in both terminal and 1 leads to 2 chain-linked positions in a 1:4 molar ratio. Rhamnose is exclusively chain-linked (1 leads to 2), glucose is both terminally and chain-linked (1 leads to 6) in a 1:1 molar ratio. The serological activity of the lipopolysaccharide of both the R. capsulata strains is low in antisera against living or heat-killed cells when tested by passive hemagglutination, Ouchterlony immunoprecipitation or gel-immunoelectrophoresis. No crossreaction was observed among the lipopolysaccharides of R. capsulata strains St. Louis, Sp 11 and 37b4 in immunoprecipitation. Lipopolysaccharide of strain Sp 11 was found to lack lethal toxicity in galactosamine-sensitized mice.     

Determination of the structure of the repeated unit of the Azospirillum brasilense SR75 O-specific polysaccharide and homology of the lps loci in the plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the OPSs of previously studied A. brasilense strain Sp245, isolated from surface-sterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains was localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects.