Effect of genomic rearrangement on heavy metal tolerance in the plant-growth-promoting rhizobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245

A derivative of Azospirillum brasilense Sp245, Sp245.5, which spontaneously lost 85 and 120 MDa replicons upon the formation of a new megaplasmid, has been shown to produce a novel lipopolysaccharide and to lose Calcofluor-binding polysaccharides. As compared to Sp245, the derivative displays notably increased heavy metal tolerance. The phenotypes of Sp245 and Sp245.5 are characterized by the following minimal inhibitory concentrations (MICs) of heavy metals: 0.5 and 0.9 μmol l⁻¹ of Ag⁺, 0.4 and 0.7 mmol l⁻¹ of Co²⁺, 0.9 and 4.7 mmol l⁻¹ of Cu²⁺, and 3.1 and 11.5 mmol l⁻¹ of Zn²⁺, respectively. In Sp245, in the presence of a nonlethal concentration (0.625 μmol l⁻¹) of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), the MIC of cobalt, copper, and zinc drop 1.3- to 1.6-fold, but the low tolerance to silver is unaffected. In Sp245.5, CCCP does not affect cobalt tolerance, suppresses tolerance to copper and silver to the wild-type levels, and causes a 1.4-fold decrease in resistance to zinc. Therefore, significant elevation of heavy metal tolerance in Sp245.5 seems caused by the induction/overexpression of the proton-dependent efflux of certain metal ions. The novel cell surface and other unknown factors could also be responsible for the increased tolerance of A. brasilense Sp245.5 to heavy metals.     

O-polysaccharide structure in serogroup I azospirilla

Lipopolysaccharides (LPS) and O-specific polysaccharides (OPS) were obtained from the outer membrane of four Azospirillum strains previously assigned to serogroup I based on the serological affinity revealed by the antibodies (AB) to the LPS of A. brasilense Sp245. Investigation, including determination of monosaccharide composition, methylation analysis, and one- and two-dimensional NMR spectroscopy, was carried out to determine the OPS structure. The OPSs of A. brasilense Sp107 and S27 and of A. lipoferum RG20a were found to have an identical structure of repeating units represented by a linear penta-D-rhamnan, as was previously described for the OPSs of A. brasilense Sp245 and SR75. The OPS of A. brasilense SR15 was found to consist of tetrasaccharide repeating units of the following structure: → 2)-α-D-Rhap-(1 → 2)-β-D-Rhap-(1 → 3)-α-D-Rhap-(1 → 2)-α-D-Rhap-(1 →. An opine compound, N^δ-(1-carboxyethyl)-ornithine, closely associated with the LPS of A. brasilense SR15, was identified in azospirilla for the first time. The presence of a 6-deoxisugar (D-rhamnose) in the OPS structure was shown to be the chemical basis of the serological similarity and the reason for classification of these strains within the serogroup I.     

Hemagglutinating activity and motility of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in the presence of various nitrogen sources

Hemagglutinating activity of the Azospirillum brasilense strain Sp245 grown in liquid media and the swarming motility of those bacteria grown in semisolid media vary significantly depending on the nitrogen source. In media with nitrate or nitrite, an increase in the hemagglutinating activity and a decrease in the swarming circles’ diameter of Sp245 were observed, compared to bacteria grown in the presence of ammonium or N₂. A ∼67-kDa hemagglutinin exhibiting affinity to the O-specific polysaccharide, an acidic D-rhamnan (OPS-I), was isolated from the surface of Sp245 cells. Introduction of the hemagglutinin into the media resulted in a decrease in the Sp245 cell motility while not affecting its mutants lacking the acidic D-rhamnan or the Sp245.5 mutant with a different OPS structure. Cells of strain Sp245.5 demonstrated hemagglutinating activity two times higher than that of the parent Sp245 strain and formed “diffuse” colonies, rather than distinct swarming circles Sp245 formed when grown in a semisolid medium. The data obtained demonstrate that intercellular contacts mediated by the interaction between the surface hemagglutinin and OPS-I, which is sensitive to environmental factors, affect the collective motility of cells.     

Analysis of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> plasmid loci coding for (Lipo)polysaccharides synthesis enzymes

Lipopolysaccharides LpsI and LpsII containing the same O-specific polysaccharide (OPS), yet different in antigenic structure and charge, have been revealed in the rhizobacterium Azospirillum brasilense Sp245. In the present work, four putative glycosyltransferase genes were identified in a 14-kbp fragment of a 120-MDa plasmid of Sp245, p120-lpsKM348X. Insertional mutagenesis of one of them, encoding the predicted ADP-heptose:LPS-heptosyltransferase, resulted in LpsI loss. By means of DNA hybridizations and PCR with primers specific towards several sites of p120-lpsKM348X, it was demonstrated that homologous segments of 120-MDa plasmids of A. brasilense strains Sp245 and Sp107, which are characterized by identical structures of the OPS repeating units, are practically identically organized. In an 85-MDa plasmid of Sp245, a locus was identified with high homology to the plasmid genes of glycosyltransferases and conserved membrane-bound proteins from a wide range of soil bacteria.     

The Use of Fragments of the 85- and 120-MDa Plasmids of Azospirillum brasilense Sp245 to Study the Plasmid Rearrangement in This Bacterium and to Search for Homologous Sequences in Plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of theAzospirillum brasilenseSp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilenseSp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

Structure of the O-specific polysaccharide of the marine bacterium Arenibacter palladensis KMM 3961T containing 2-acetamido-2-deoxy-L-galacturonic acid

The O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Arenibacter palladensis type strain KMM 3961^T and studied by chemical methods and ¹H and ¹³C NMR spectroscopy including 2D COSY, TOCSY, ¹H,¹³C HSQC, and HMBC experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units containing two mannose residues (Man), one 2-acetamido-2-deoxy-D-galactose residue (D-GalNAc), and one 2-acetamido-2-deoxy-L-galacturonic acid residue (L-GalNAcA) and having the following structure: ? 2) - a- D - Manp - (1 ? 6) - a- D - Manp - (1 ? 4) - a- L - GalpNAcA - (1 ? 3) - b- D - GalpNAc - (1 ?\to 2) - \alpha - D - Manp - (1 \to 6) - \alpha - D - Manp - (1 \to 4) - \alpha - L - GalpNAcA - (1 \to 3) - \beta - D - GalpNAc - (1 \to.     

Structural properties of capsular and O-specific polysaccharides of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 under varying cultivation conditions

Effect of the carbon source in the culture medium and of the growth phase on the composition and structure of the capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) of the bacterium Azospirillum brasilense Sp245 was studied. Growth with fructose resulted in an increased carbohydrate content in the CPSs, while long-term cultivation resulted in an increased content of phosphorus in both CPSs and LPSs. The LPSs produced on the medium with fructose (regardless of the cultivation duration) and the LPSs of the bacteria grown with sodium malate until the stationary phase were characterized by higher levels of unsaturated fatty acids than the LPSs of the bacteria grown with sodium malate to the late exponential phase. The structures of the polysaccharides from the isolated glycopolymers were established using monosaccharide analysis, including determination of the absolute configurations and 1D and 2D NMR spectroscopy. This study is the first to report that the CPS of A. brasilense Sp245 grown with sodium malate to the end of the exponential phase is structurally identical to the O-polysaccharide from the LPS of this bacterium and that the LPS and CPS of A. brasilense Sp245 grown with fructose contain an additional homoglucan of the following structure: [→3)-α-D-Glcp-(1→] _n .     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

D-Aminoacylase from a novel producer: Stenotrophomonas maltophilia ITV-0595

A novel bacterial strain producing D-aminoacylase was isolated from organic waste and identified as Stenotrophomonas maltophilia ITV-0595. The isolation was performed using N-acetyl-D-phenylglycine (NAcDPG) as the sole source of C and N. The optimum pH for enzyme expression was 8 at 37°C. Using N-Ac-DPG concentrations from 0.5 up to 3% w/v, it was observed that at the 1% level, the microorganism showed acceptable responses in both enzyme activities and cell growth. From the different tested compounds N-acetyl-D-methionine (1%) was the best enzyme inducer (Sp. act. = 4.14 U mg⁻¹ protein, Vol. act. = 0.17 U ml⁻¹) and the only one that increased cell growth. Received 13 June 1997/ Accepted in revised form 29 October 1998     

Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense

Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified. The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis. Mn²⁺, Mg²⁺, and Co²⁺ were found to be effective in supporting biosynthetic activity of the A. brasilense GS. Some kinetic properties of Mn²⁺-activated and Mg²⁺-activated unadenylylated GS were characterized. Circular dichroism analysis of the enzyme showed that the A. brasilense GS is a highly structured protein: 59% of its residues form -helices and 13% -strands. Removal of the metal ions from the A. brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure.     

Energy transduction efficiencies in nitrogenous oxide respirations of Azospirillum brasilense Sp7

For Azospirillum brasilense Sp7, the energy transformation efficiencies were measured in anaerobic respirations with either nitrate, nitrite or nitrous oxide as respiratory electron acceptors by determining the maximal molar growth yields and the H⁺-translocations using the oxidant pulse method. In continuous cultures grown with malate limiting, the maximal molar growth yields (Y _s ^max -values) were essentially the same with O₂ or N₂O but were 1/3 and 2/3 lower with NO ₂ ^- or NO ₃ ^- , respectively, as respiratory electron acceptors. Both the maximal molar growth yields and the maintenance energy coefficients were surprisingly high when Azospirillum was grown with nitrite as the sole electron acceptor and source for N-assimilation. Growth under N₂-fixing conditions drastically reduced the Y _s ^max -values in the N₂O and O₂-respiring cells. In the H⁺-translocation measurements, the /oxidant ratios were 5.6 for O₂→H₂O, 2.5–2.8 for NO ₃ ^- →NO ₂ ^- , 2.2 for NO ₂ ^- →N₂O and 3.1 for N₂O→N₂ respirations when the cells were preincubated with valinomycin and K⁺. All the values were enhanced when the experiments were performed with valinomycin plus methyltriphenylphosphonium (=TPMP⁺) cation. The uncoupler carbonyl cyanide-m-chlorophenyl-hydrazone diminished the H⁺-excretion indicating that this translocation was due to vectorial flow across the membrane. In the absence of any ionophore, nitrate and nitrite respirations were accompanied by a H⁺-uptake . Any significant H⁺-translocation could not be detected in N₂O- and O₂-respirations under these conditions. It is concluded that nitrate reduction proceeds inside the cytoplasmic membrane, whereas nitrite is reduced extramembraneously. The data are not conclusive for the location of nitrous oxide reductase. The maximal molar growth yield determinations and the absence of any H⁺-uptake in untreated cells indicate a cytoplasmic orientation of the enzyme similar to the terminal cytochrome oxidase of respiration. The low H⁺-extrusion values for N₂O-respiration compared to O₂-respiration in cells treated with valinomycin plus TPMP⁺ are, however, not in accord with such an interpretation.     

Oxygen transport in the green sea turtle

Summary Green sea turtles (Chelonia mydas) are well known as endurance swimmers and divers. Physiological correlates of these traits were studied in 9 adult sea turtles (mean body mass=87 kg) at a body temperature of 25°C. The respiratory properties of the blood were similar to those of other turtles except for a higher oxygen affinity (P ₅₀=18.2 Torr, pH 7.6), which may be an allometric function. Resting, systemic blood flow, calculated from the Fick principle was 21.5 ml·kg⁻¹. min⁻¹, similar to values reported for other turtles. Pulmonary blood flow, measured by mass spectrometry of acetylene uptake in the lungs was 24.0 ml·kg⁻¹·min⁻¹, not significantly different from the calculated systemic flow. Other evidence of a small (net) intracardiac shunt is the high arterial saturation (ca. 90%) of arterial blood. This distinctive feature of O₂ transport inC. mydas provides an content difference of 4.1 ml· dl⁻¹. This results in a relatively low blood convection requirement at rest =24.4 mlbtps·mlstpd ⁻¹), similar to that for many mammals. This would favor a high maximum O₂ uptake, as measured by others in this species. The relatively high O₂ affinity of blood in this species could be adaptive to “loading” O₂ during intermittent breathing while swimming and to utilizing the lung O₂ store during the progressive hypoxia of diving.     

Kinetics of anaerobic biodegradation of glycerol by sulfate-reducing bacteria

A kinetic model has been developed and kinetic parameters of anaerobic degradation of glycerol, an abundant by-product of biofuel manufacturing, by a consortium of sulfate reducing bacteria (SRB) in a closed system have been determined. The following main species of SRB has been identified in the consortium: Desulfovibrio baarsii, Desulfomicrobium sp., and Desufatomaculum sp. The proposed model included processes of glycerol degradation, sulfate reduction, and inhibition by metabolic products, as well as effects of pH and temperature. The suggested equation for the anaerobic glycerol degradation was based on Edward and Andrew’s equation. The following kinetic parameters of the anaerobic glycerol degradation were obtained for the initial glycerol concentration from 0.15 to 4 ml/l and sulfate concentration of 2760 mg/l at 22°C: maximum specific growth rate of SRB μ_max = 0.56 day⁻¹, economic coefficient of ashless biomass from glycerol of 0.08 mol SRB/mol COC, and yield of ashless biomass from sulfate of 0.020 mol SRB/mol SO₄. It was shown that the optimum molar ratio of $ {{C_{Gl} } \mathord{\left/ {\vphantom {{C_{Gl} } {C_{SO_4 } }}} \right. \kern-\nulldelimiterspace} {C_{SO_4 } }} $ {{C_{Gl} } \mathord{\left/ {\vphantom {{C_{Gl} } {C_{SO_4 } }}} \right. \kern-\nulldelimiterspace} {C_{SO_4 } }} for SRB growth was 0.8. Initial boundary concentration of inhibition by undissociated hydrogen sulfide was 70 mg/l. Dependence of the specific growth rate of bacteria on the temperature was approximated by the Arrhenius equation in the temperature range of 20–30°C with the goodness of fit R² = 0.99.     

Detection of putative polysaccharide biosynthesis genes in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains from serogroups I and II

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.     

Phytoplankton dynamics,primary productivity and community metabolism in a north-central Texas pond

Phytoplankton diversity, primary productivity and community metabolism were measured for 1 year in a 0.94 ha pond located in north-central Texas. Gross primary production ranged from 4.5 to 46.8 kcal m⁻² day⁻¹ ( =22.0 kcal m⁻² day⁻¹) and community metabolism ranged from 7.3 to 32.4 kcal m⁻² day⁻¹ ( =14.8 kcal m⁻² day⁻¹). Average production/respiration ratio (1.5) showed that the pond was principally autotrophic. Photosynthetic efficiency (gross primary production/0.5 total solar radiation) ranged from 0.32 to 2.8 with a mean of 1.2. Phytoplankton diversity based on numbers and biomass fluctuated greatly. Highest gross primary productivity occurred during Cyanophyta blooms in late summer-early fall when species diversity was minimal. Water temperature and turbidity, which governed light penetration, were the principal determinants of primary production.     

Changes in cell surface properties and biofilm formation efficiency in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 mutants in the putative genes of lipid metabolism <Emphasis Type="Italic">mmsB1</Emphasis> and <Emphasis Type="Italic">fabG1</Emphasis>

The previously obtained insertion mutants of Azospirillum brasilense Sp245 in the genes mmsB1 and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense strains Sp245, SK039, and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide preparations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.     

Sialic Acids and Related Substances

N-Acetyl-D-galactosamine, N-acetyl-D-mannosamine and N-acetyl-D-glucosamine were allowed to react with oxalacetic acid under alkaline conditions, and the condensation products purified by ion-exchange chromatography. Properties of these products on the whole are similar to each other, though there is a minor but significant diference in the condensation product with N-acetyl-D-galactosaminc. Paper chromatograms of the condensation products suggest that N-acetyl-D-galactosamine as well as N-acetyl-D-glucosamine are epimerized partly before they condense with oxalacetic acid to givc each two sialic acids with different configurations at C-5 from each other.     

RBE of quasi-monoenergetic 60 MeV neutron radiation for induction of dicentric chromosomes in human lymphocytes

The production of dicentric chromosomes in human lymphocytes by high-energy neutron radiation was studied using a quasi-monoenergetic 60 MeV neutron beam. The average yield coefficient of the linear dose–response relationship for dicentric chromosomes was measured to be (0.146±0.016) Gy⁻¹. This confirms our earlier observations that above 400 keV, the yield of dicentric chromosomes decreases with increasing neutron energy. Using the linear-quadratic dose–response relationship for dicentric chromosomes established in blood of the same donor for ⁶⁰Co γ-rays as a reference radiation, an average maximum low-dose RBE (RBE_M) of 14±4 for 60 MeV quasi-monoenergetic neutrons with a dose-weighted average energy of 41.0 MeV is obtained. A correction procedure was applied, to account for the low-energy continuum of the quasi-monoenergetic spectral neutron distribution, and the yield coefficient α for 60 MeV neutrons was determined from the measured average yield coefficient . For α, a value of (0.115±0.026) Gy⁻¹ was obtained corresponding to an RBE_M of 11±4. The present experiments extend earlier investigations with monoenergetic neutrons to higher energies.