Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2

Zinc concentrations ranging between 0.1 and 1 mm only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mm zinc and could not be repressed by iron even at a concentration of 100 m. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.     

基于基因组的一株土壤固氮菌分离菌株鉴定及其促生作用

[目的] 为获得高效固氮菌株,充分研究利用土壤固氮菌资源。[方法] 选取固氮能力较高的紫色土发育水稻土,采用富集纯化法分离固氮微生物菌株。通过16S rRNA基因系统发育分析和全基因组相关指数比较对新分离菌株进行物种鉴定。采用乙炔还原法和¹⁵N₂示踪法定量测定新分离菌株的固氮能力,通过培养特性和接种效果初步研究固氮菌株的促生作用。[结果] 从紫色土发育水稻土中分离得到1株可在无氮培养基上快速生长的菌株P208。基于16S rRNA基因和基因组92个核心基因的系统发育分析结果表明,新分离菌株P208与Azotobacter chroococcum IAM 12666^T（=ATCC 9043^T）系统发育距离最近（16S rRNA基因相似度为99.79%）。菌株P208与A.chroococcum ATCC 9043^T的基因组平均核苷酸一致性（ANI）、平均氨基酸一致性（AAI）和数字DNA-DNA杂交值（dDDH）高于物种分类阈值（ANI>95%-96%,AAI>95%-96%,dDDH>70%）,最大唯一匹配指数（MUMi）低于物种分类阈值（<0.33）,得出新分离菌株P208为褐球固氮菌（A.chroococcum）。A.chroococcum P208固氮活性为模式菌株A.chroococcum ATCC 9043^T的2.61倍。除固氮能力外,A.chroococcum P208具有IAA生成、溶磷活性和铁载体生成等促进植物生长潜力的培养特性,室内培养条件下接种A.chroococcum P208能够促进水稻、小麦幼苗根系的生长。[结论] 从固氮能力较强的水稻土中分离纯化得到1株具有较强固氮、促生潜力的固氮菌,具有潜在的开发应用价值,可为研究利用生物固氮提供微生物资源。     

Overproduction of pyoverdins by Fur mutants of Pseudomonas aeruginosa strains PAO1 and Fe10 in stirred bioreactors

Fur mutants FPA12 and FF13 of strains Pseudomonas aeruginosa PAO1 and Fe10, respectively, were prepared and their production of pyoverdin evaluated. The strains were cultivated in stirred bioreactor in iron-deficient and iron-supplemented medium containing Casamino acids (CA) or succinate as a source of carbon and energy. When the pyoverdin production rate reached its maximum, the demand of iron-depleted cultures for O₂ was decreased. Mutant FF13 overproduced pyoverdin in both iron-depleted (862 mg l^–1) and iron-supplemented (428 mg l^–1) CA medium and could also be used to produce pyoverdin when grown in a conventional stirred tank fermenter.     

Fur mutants of Pseudomonas aeruginosa PAO1: phenotypic characterization in terms of iron-dependent pattern of deregulation of pyoverdin synthesis

Fur mutants (Mn^r) of Pseudomonas aeruginosaFe10 (FF13 and FF28) and PAO1 (FPA12) were evaluated for the pattern of deregulation of pyoverdin synthesis in iron-replete medium with Casamino acids [CAM(Fe)] or succinate [SM(Fe)]. With respect to siderophore synthesis, we found in CAM(Fe) medium two Fur phenotypes: FurA (full deregulation, FF13) and FurB (partial deregulation, FF28 and FPA12). Fur mutants compared to parental strains grew with slower specific growth rates on SM(0) and CAM(0) media in a stirred bioreactor. Fur mutants grew in SM(0) with about half of that in CAM(0).     

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.     

The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO

Summary The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA ⁺-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R plasmids.     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

A new bio-formulation containing plant growth promoting rhizobacterial mixture for the management of sheath blight and enhanced grain yield in rice

Three plant growth promoting rhizobacterial (PGPR) strains, PF1,FP7 and PB2, were tested alone and in combinations for suppression ofrice sheath blight disease and promotion of plant growth underglasshouse and field conditions. The mixture of PGPR strainssignificantly reduced the sheath blight incidence when applied as eitherbacterial suspension through seed, root, foliar and soil application inglasshouse conditions, or as talc-based formulation under fieldconditions, compared to the respective individual strains. The averagemean of disease reduction was 29.2% for single strains and45.1% for mixtures. In addition to disease suppression, treatmentwith mixture of PGPR strains promoted plant growth in terms of increasedplant height and number of tillers, and ultimately grain yield. Theaverage increases in yield for single strains were 17.7%, and25.9% in case of mixture. Mixture of three PGPR strains reduceddisease and promoted growth to a level equivalent to two strainmixtures. Though seed treatment of either single strain or strainmixtures alone could reduce the disease, subsequent application to root,leaves or soil further reduced the disease and enhanced the plantgrowth. The mixture consisting of PF1 plus FP7 was the most effective inreducing the disease and in promoting plant growth and grainyield.     

Effect of plant growth and addition of plant residues on the phosphatase activity in soil

Summary The phosphatase activity of the soil amended with roots and tops of clover (Trifolium repens) plant material (0.1% by weight) remained essentially constant in the absence of growing plants but changed considerably in the presence of plants (Avena sativa) grown for 10 weeks. There was a significant relationship between the phosphatase activity and organic and inorganic P in the soil solution only in the presence of growing plants. The differences in phosphatase activity between roots and tops amended soil were attributed to total C as well as differences in the degree of availability of C added through plant materials. This may also apply to the carpet grass (Axonopus affinis) amended soil.     

Desulfurization of light gas oil by a novel recombinant strain from Pseudomonas aeruginosa

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa NCIMB 9571 by using a transposon vector. Resting cells of the recombinant strain, PAR41, desulfurized 63 mg sulfur l^–1 of light gas oil (LGO) containing 360 mg S l^–1. The desulfurization activity for LGO by the resting cells of strain PAR41 grown with n-tetradecane (50% v/v) was much higher (1018-fold) than in glucose-grown cells. P. aeruginosa NCIMB 9571 is able to take up water-insoluble compounds from an oil phase which is enhanced by n-alkane.     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

Summary In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60±0.64 M) almost ten times greater than that of the wild type strain (Km=0.39 M). There was, however, no change in the V_max for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium resulting in an 18%–35% decrease in growth when compared with the wild type.     

Selective plant growth suppression by shoot application of soil bacteria

Selected rhizosphere bacterial isolates, previously determined as plant growth deleterious, were tested for their ability to suppress plant growth after foliar spray applications, for selectivity with regard to plant species, and in pilot field experiments for their potential as weed biocontrol agents. Inundative foliar applications of aqueous bacterial suspension were performed on a range of weed and crop species. Plant symptoms after spraying ranged from rapid necrosis and wilting to an overall growth suppression or stunting. Significant and selective reductions in biomass of up to 90% fresh weight, as well as large reductions in plant survival and plant height were recorded in greenhouse pot experiments. However, monocotyledonous plants were affected weakly or not at all by two isolates extensively tested. Effects of these were dose- and plant age-dependent, and were for some plants enhanced by high relative humidity. For one isolate, A153, effects were also expressed in cell-free culture filtrates pointing to involvement of specific metabolites. In pilot field experiments, strong growth suppression was observed on broad-leaved plants, while barley crop plants were unaffected.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

两种云杉种子萌发和幼苗生长对环境因子的适应性

青海云杉是我国沙区通过种子育苗引种成功的物种。通过对不同储藏时间青海云杉和沙地云杉种子生活力测定、种子萌发对温度、光照、水盐胁迫以及幼苗生长对水盐胁迫和沙埋的响应实验,比较两种种子萌发和幼苗生长对环境因子适应性,为沙地云杉在我国沙区广泛引种提供参考。结果表明:1)在2年的储藏过程中,沙地云杉和青海云杉种子生活力分别由79%和72%下降了19%和5%; 2)沙地云杉和青海云杉种子适宜萌发温度分别为15—30℃和10—30℃,最适萌发温度分别为25℃(72%)和25/15℃(69%),除10℃和10/30℃外,两种种子萌发率在各温度下没有显著差异; 3)沙地云杉种子萌发光照条件为14 h光照/8 h黑暗交替(67%),青海云杉为24 h光照(61%)或24 h黑暗(61%); 4)水势在-2.7—0 MPa时,2种云杉的IGR(初始萌发率)、RGR(恢复萌发率)、ISL(初始幼苗长度)和RSL(恢复幼苗长度)均没有显著差异; 5) NaCl浓度在200 mmol/L和250 mmol/L,青海云杉种子IGR显著大于沙地云杉,NaCl浓度在0—450 mmol/L,青海云杉和沙地云杉种子RGR没有显著差异,当NaCl浓度为50 mmol/L和100 mmol/L,青海云杉ISL显著大于沙地云杉; 6)在0.5—2.0 cm沙埋深度时,青海云杉出苗率显著高于沙地云杉,沙地云杉最适沙埋深度0.5 cm,青海云杉为0.5—1.5 cm。因此,青海云杉种子萌发和幼苗生长比沙地云杉有更强的环境适应性,但只要采取合理的播种时间、播种深度和水分管理等措施,沙地云杉会和青海云杉一样在我国沙区大面积引种育苗。     

Isomerization and biodegradation of beta-cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production

Pseudomonas aeruginosa CH7, isolated from activated sludge, was able not only to isomerize and degrade beta-cypermethrin but also to utilize it as the sole source of carbon and energy for growth and produce biosurfactant. The strain effectively degraded beta-cypermethrin with inocula biomass of 0.1-0.2 g L⁻¹ at 25-35 °C, pH 6-9, and a final concentration of beta-cypermethrin 25-900 mg L⁻¹. Via response surface methodology analysis, we found the optimal condition was 29.4 °C, pH 7.0, and inocula biomass of 0.15 g L⁻¹; under these conditions, about 90% of the beta-cypermethrin could be degraded within 12 days. Noticeably, biosurfactant was detected in the MSM culture of strain CH7, suggesting that the biosurfactant (rhamnolipid) could potentially enhance the degradation of beta-cypermethrin by promoting the dissolution, adsorption, and absorption of the hydrophobic compounds. Therefore, CH7 may serve as a promising strain in the bioremediation of wastewater and soil polluted by beta-cypermethrin.     

Increased root length and branching in cotton by soil application of the plant growth regulator PGR-IV

Field and growth chamber studies were used to determine the effect of in-furrow application of PGR-IV on root and shoot development, and yield of cotton. In the field study, an in-furrow application of PGR-IV @ 73 mL ha^–1 at planting increased yield by 18% compared to the untreated control, and by 11% compared to 2-foliar applications of 292 mL/ha^–1 each at pinhead square stage of flower development and at first flower appearance. Growth chamber studies revealed that the in-furrow applications of PGR-IV @ 1.131L/plant dramatically increased root length (+47%), root dry weight (+29%), number of lateral roots per plant (+75%), and nutrient uptake one week after planting. These differences were still apparent five weeks later at pinhead square but to a lesser degree. The yield enhancement from the foliar applications was associated with increases in leaf growth, nutrient uptake, and boll number, whereas the yield enhancement from the soil application was associated with enhanced root growth and nutrient uptake. The positive effect of PGR-IV on root growth and accelerated early-season growth could have very substantial benefits in cotton production.     

Selection and preliminary characterization of a Pseudomonas aeruginosa strain mineralizing selected isomers in a branchedchain dodecylbenzenesulphonate mixture

A bacterium able to grow at the expense of some isomers in a commercial surfactant preparation consisting of branched-chain dodecylbenzenesulphonate was isolated (W51), and it was identified as a Pseudomonas aeruginosa strain. A faster growing derivative was selected (W51D) after enrichment in batch culture under microaerobic conditions, using the surfactant as the sole source of carbon and energy. Strain W51D is the first microorganism reported to degrade at least 70% of a branched-chain alkylbenzenesulphonate mixture and to be resistant to high concentrations of this surfactant. The ability to degrade the surfactant was shown to be transferred by conjugation to other P. aeruginosa strains and to an Escherichia coli strain.G. Soberón-Chávez and J. Campos are with the Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Apdo Postal 510-3, Cuernavaca, Mor. 62250, México.A. Hädour and L. Ramos are with Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas, Protesor Albareda 1, Granada 18008, España. J. Ortigoza is with Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Apdo. Postal 42-186. México D.F. 11340. México.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.