Optimization, purification, characterization and antioxidant activity of an extracellular polysaccharide produced by Paenibacillus polymyxa SQR-21

The optimization, purification and characterization of an extracellular polysaccharide (EPS) from a bacterium Paenibacillus polymyxa SQR-21 (SQR-21) were investigated. The results showed that SQR-21 produced one kind of EPS having molecular weight of 8.96 × 10⁵ Da. The EPS was comprised of mannose, galactose and glucose in a ratio of 1.23:1.14:1. The ratio of monosaccharides and glucuronic acid was 7.5:1. The preferable culture conditions for EPS production were pH 6.5, temperature 30 °C for 96 h with yeast extract and galactose as best N and C sources, respectively. The maximum EPS production (3.44 g L⁻¹) was achieved with galactose 48.5 g L⁻¹, Fe³⁺ 242 μM and Ca²⁺ 441 μM. In addition, the EPS showed good superoxide scavenging, flocculating and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities were determined. These results showed the great potential of EPS produced by SQR-21 to be used in industry in place of synthetic compounds.     

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.     

Siderophore Cross-Utilization Amongst Rhizospheric Bacteria and the Role of Their Differential Affinities for Fe3+ on Growth Stimulation Under Iron-Limited Conditions

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.     

溶藻弧菌铁载体合成及外膜蛋白表达的研究

初步研究了海洋动物病原菌溶藻弧菌的铁摄取机制。溶藻弧菌能够在高浓度铁螯合剂2-2二联吡啶的培养基中存活。在限铁环境中,溶藻弧菌生长受到抑制,补加铁可以消除这种抑制作用。通过铁载体定量检测,发现分离于发病鱼体的溶藻弧菌MVP01产铁载体量大于分离于海水的菌株No·1·1587。互补实验证明溶藻弧菌的铁载体粗提物能够被铁载体合成缺陷的大肠杆菌突变株AN93利用。在铁限制培养环境中,溶藻弧菌合成了约80kD铁调控外膜蛋白。铁摄取系统在溶藻弧菌的生存和致病性方面,都有重要的作用。     

Differential Siderophore Utilization and Iron Uptake by Soil and Rhizosphere Bacteria

The differential availabilities of the hydroxamate siderophores ferrioxamine B (FOB) and ferrichrome (FC) and the pseudobactin siderophores St3, 7NSK₂, and WCS 358 as sources of Fe for soil and rhizosphere bacteria were studied. About 20% of the total bacterial CFU from the rhizospheres of four plant species were able to use FOB as the sole Fe source in an Fe-deficient medium, while about 12, 10, 2, and > 1% were able to use FC and pseudobactins 7NSK₂, St3, and WCS 358, respectively. Of the 165 colonies isolated from plates containing pseudobactins, 64 were able to use the pseudobactin on which they were isolated as the sole Fe source in pure culture. Cross-feeding tests showed that almost all of these 64 strains were also able to use at least one of the other siderophores studied (pseudobactin, FOB, or FC). Pseudomonas putida StS2, Pseudomonas maltophilia 7NM1, and Vibrio fluvialis WS1, which were originally isolated on pseudobactins St3, 7NSK₂, and WCS 358, respectively, were selected for their ability to grow with pseudobactin St3 as the sole Fe source. They incorporated ⁵⁵Fe³⁺ mediated by pseudobactin St3 at various rates (71.5, 4, and 23 pmol/min/mg [dry weight] of cells, respectively). Similarly, P. putida St3 was shown to incorporate ⁵⁵Fe³⁺ mediated by FOB and FC. We suggest that the ability of bacteria to utilize a large variety of siderophores confers an ecological advantage.     

Production,characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

The production, characterization and antioxidant activities in vitro of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3 were investigated. For EPS production, the preferable culture conditions were 24 °C and pH 8 for 60 h with sucrose and yeast extract as the carbon and nitrogen sources, respectively. Notably, sucrose concentration was the prominent factor, and the maximum yield of EPS (22.82 g/L) was obtained at a sucrose concentration of 160 g/L. The crude EPS was purified by chromatography of DEAE-52 and Sephadex G-100, affording EPS-1 and EPS-2 with molecular weights of 1.22 × 10⁶ and 8.69 × 10⁵ Da, respectively. They were composed of mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. In addition, both crude and purified EPS showed strong scavenging activities on superoxide and hydroxyl radicals, and their antioxidant activities decreased in the order of crude EPS > EPS-2 > EPS-1.     

Siderophore Production and Iron Reduction by Pseudomonas mendocina in Response to Iron Deprivation

In aerobic environments microorganisms are faced with a discrepancy of ~10 orders of magnitude between the available Fe (~10^-17M) and their metabolic requirement for it (~10^-7M). In contrast to facultative anaerobic environments, where dissimilatory iron-reducing bacteria (DIRB) are often abundant, few studies have detailed microbial interactions with Fe(III) (hydr)oxides in aerobic environments. To better understand acquisition of Fe from Fe(III) (hydr)oxides, we investigated the production of siderophore and Fe(III) reduction by a strict aerobe in the presence of synthetic hematite as a source of Fe. Pseudomonas mendocina grew best when Fewas supplied as FeEDTA (~1.8x10⁸ colony-forming units [CFU] ml^-1), grew abundantly when Fe was supplied as hematite (~1.2x10⁸ CFU ml^-1), and grew poorly when Fe was withheld from the medium (~5.5x10⁷ CFU ml^-1). As expected, negligible siderophore was produced per cell when Fe was supplied as FeEDTA and more siderophore was produced in the hematite flasks than in the controls. Thus, growth of P. mendocina and the production of siderophore in the presence of hematite present compelling evidence that siderophore was produced as a mechanism to acquire Fe from hematite. For the Fe reduction experiments, Fe reduction by components of the supernatant fluid was induced weakly when Fe was supplied as hematite or as FeEDTA, but much more when the cells were cultured under extreme Fe deprivation. In fact, 16 times as much Fe reduction occurred in the controls as in the presence of either of the FeEDTA or hematite amendments. Our results, which contravene the long-held assumptions that Fe acquisition was facilitated solely by siderophores, provides a new perspective regarding microbial interactions with Fe bearing minerals.     

Siderophore Production by Pseudomonas stutzeri under Aerobic and Anaerobic Conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.     

N2-Fixation and Seedling Growth Promotion of Lodgepole Pine by Endophytic Paenibacillus polymyxa

We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus polymyxa P2b-2R, a diazotrophic bacterium previously isolated from internal stem tissue of a naturally regenerating pine seedling to evaluate biological nitrogen fixation and seedling growth promotion by this microorganism. Seedlings generated from pine seed inoculated with strain P2b-2R were grown for up to 13 months in a N-limited soil mix containing 0.7 mM available N labeled as Ca(¹⁵NO₃)₂ to facilitate detection of N₂-fixation. Strain P2b-2R developed a persistent endophytic population comprising 10²–10⁶?cfu?g^?1 plant tissue inside pine roots, stems, and needles during the experiment. At the end of the growth period, P2b-2R had reduced seedling mortality by 14 % and ¹⁵N foliar N abundance 79 % and doubled foliar N concentration and seedling biomass compared to controls. Our results suggest that N₂-fixation by P. polymyxa enhanced growth of pine seedlings and support the hypothesis that plant-associated diazotrophs capable of endophytic colonization can satisfy a significant proportion of the N required by tree seedlings growing under N-limited conditions.     

Nitrate Reductase and Nitrous Oxide Production by Fusarium oxysporum 11dn1 Under Aerobic and Anaerobic Conditions

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively. Received: 2 February 2000 / Accepted: 28 February 2000     

Plant growth-promoting rhizobacteria, Paenibacillus polymyxa and Paenibacillus lentimorbus suppress disease complex caused by root-knot nematode and fusarium wilt fungus

Aim:  To screen and evaluate the biocontrol potential of Paenibacillus strains against disease complex caused by Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici interactions.
Methods and Results:  Paenibacillus strains were collected from rotten ginseng roots. The strains were tested under in vitro and pots for their inhibitory activities, and biocontrol potential against disease complex caused by M. incognita and F. oxysporum f. sp. lycopersici on tomato. In in vitro experiments, among 40 tested strains of Paenibacillus spp., 11 strains showed antifungal and nematicidal activities against F. oxysporum f. sp. lycopersici and M. incognita, respectively. Paenibacillus polymyxa GBR-462; GBR-508 and P. lentimorbus GBR-158 showed the strongest antifungal and nematicidal activities. These three strains used in pot experiment reduced the symptom development of the disease complex (wilting and plant death), and increased plant growth. The control effects were estimated to be 90–98%, and also reduced root gall formation by 64–88% compared to the untreated control.
Conclusion:  The protective properties of selected Paenibacillus strains make them as potential tool to reduce deleterious impact of disease complex plants.
Significance and Impact of the Study:  The study highlights biocontrol potential of Paenibacillus strains in management of disease complex caused by nematode-fungus interaction.     

Effect of Ferric Iron on Siderophore Production and Pyrene Degradation by Pseudomonas fluorescens 29L

The effect of ferric iron [Fe(III)] on pyrene degradation and siderophore production was studied in Pseudomonas fluorescens 29L. In the presence of 0.5 muM of Fe(III) and 50 mg of pyrene per liter of medium as a carbon source, 2.2 mg of pyrene was degraded per liter of medium per day and 25.3 muM of 2,3-DHBA (2,3-dihydroxybenzoic acid) equivalent of siderophores was produced per day. However, the pyrene degradation rate was 1.3 times higher and no siderophores were produced with the addition of 1 muM of Fe(III). Similar trends were seen with 50 mg of succinate per liter of medium as a carbon source, although the growth of strain 29L and the succinate degradation rate were higher. In the absence of siderophore production, pyrene and succinate continued to be biodegraded. This indicates that Fe(III) and not siderophore production affects the hydrocarbon degradation rate. Only 18% of strain 29L mutants capable of growth on pyrene produced siderophores, while among the mutants capable of growth on succinate, only 10% produced siderophores. This indicates that siderophores are not required for pyrene biodegradation. Fe(III) enhances pyrene degradation in Pseudomonas fluorescens 29L but it may be utilized by mechanisms other than siderophores.     

Nitrate Reductase Activity in Calcifugous and Calcicolous Tomatoes as Affected by Iron Stress

Calcicolous plants are generally more Fe-efficient than calcifugous plants, because they respond to Fe stress by releasing H-ions and “reductants” from their roots that causes Fe to become available. The objective of our study was to determine if differential response to Fe stress in calcicolous and calcifugous varieties affects nitrate reductase activity. T3238FER (Fe-efficient) and T3238fer (Fe-inefficient) tomato (Lycopersicon esculentum Mill.) cultivars were grown in nutrient solutions supplied with N as NH₄⁺-N plus NO₃^?-N, and as NO₃^?-N only. The chemical reactions induced by Fe stress concomitantly increased nitrate reductase activity in roots and tops of calcicolous, but not in calcifugous tomato. This nitrate reductase activity decreased, however, when Fe was made available to the plants. When Fe stress was eliminated by adding Fe, nitrate reductase activity was comparable in the two cultivars.     

Relationship Between Fe2+-Induced Ethylene Production from Husk and Growth of Fe2+-Stimulated Rice Seedling

Ferrous iron might induce ethylene production from rice (Oryza sativa L. ) husk. The induction was a non-enzymic chemical process, in which probably a non-gaseous precursor could be converted to ethylene by the action of Fe2+. Fe2+-stimulated seedling growth was probably mediated by the induced ethylene based on the following evidences: ( Ⅰ ) The induction and the stimulation were synchronous in time within one day only. Once seedling growth was initiated renewal of Fe2+ solution, even on the following day, exerted no more effect neither on ethylene production nor on seedling growth; ( Ⅱ ) Ferrous iron was neither capable to stimulate the ethylene production of rice caryopsis, nor able to stimulate the seedling growth of germinated caryopsis.     

Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture

The optimum dilution rate for 2, 3-butanediol (BDL) production by Paenibacillus polymyxa was 0.2 h1 and the optical purity of BDL remained above 98 % at all dilution rates. With decreasing culture pH, ethanol and BDL production increased, whereas the optical purity of BDL decreased to 94 % at pH 5.7. In the chemostat culture at pH 6.3 and a 0.1 h1 dilution rate, the optimum air supply for BDL production was 200 ml min–1 in which the O2 uptake rate was 6.7 mmol l–1 h–1. Under this condition, the optical purity of BDL decreased to 93 %. © Rapid Science Ltd. 1998     

Induction of Ferric Reductase Activity and of Iron Uptake Capacity in Chlorococcum littorale Cells under Extremely High-CO2 and Iron-Deficient Conditions

The marine green alga, Chlorococcum littorale, accumulated ironin its cells and showed high activity of plasma membrane ferricreductase under high-CO₂ and iron-deficient conditions. Theseactivities disappeared upon exposure to ordinary air and byadding excess FeSO₄. The iron uptake had high affinity for theFe(II) form (K_m of 0.13 µM). Carbonylcyanide m-chlorophenylhydrazoneand N,N-dicyclohexylcarbodiimide significantly suppressed theiron uptake, suggesting that the Fe(II) uptake was driven byATPase. These results indicate that high CO₂ and iron deficiencycooperatively induce the Fe(II) uptake and cell-surface ferricreductase activity. (Received October 20, 1997; Accepted January 29, 1998)     

House Production by Oikopleura dioica (Tunicata, Appendicularia) Under Laboratory Conditions

We examined the generation time and the house renewal rate ofOikopleura dioica under various conditions. Animals were fedtwo flagellates, Isochrysis galbana and Tetraselmis sp., withconcurrent determination of the carbon contents of body andhouse to estimate house production. The generation time was6 days at 15°C, 4 days at 20°C and 3 days at 25°Cat both 25 and 30 p.s.u. with a food concentration of 4 x 10⁴cells ml^–1. The carbon content of newly secreted housesranged from 0.5 to 0.8 µg, corresponding to 15.3 ±4.8% of body carbon. The house renewal rates increased withincreasing temperature and decreasing salinity. Food concentrationsranging from 100 to 16 x 10⁴ cells ml^–1, body size andlight condition had no effect on house renewal rate. Cloggingof the inlet filter by adding the large diatom Ditylum sol causedan increase in house renewal rates. The total number and carboncontent of houses during an animal's lifetime ranged from 46to 53 houses and from 6.5 to 10.4 µg, respectively. Sincedaily house production calculated for the O. dioica populationcorresponded to 130–290% of its biomass and daily discardedhouse materials corresponded to 490–1100% of the biomass,this organism must play an important role as a producer of macroscopicaggregates.