The ppuI-rsaL-ppuR quorum-sensing system regulates biofilm formation of Pseudomonas putida PCL1445 by controlling biosynthesis of the cyclic lipopeptides putisolvins I and II

Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C(10)-N-acyl homoserine lactone (3-oxo-C(10)-AHL) or 3-oxo-C(12)-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.     

Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.     

不同营养条件下斑玉蕈菌丝生长及产酶特性

分析测定了不同营养条件下斑玉蕈菌丝形态特征、生长速度及产酶规律。低碳氮盐培养基上菌丝生长速度最快,但其菌丝非常稀疏,边缘不整齐,在整个生长阶段酶活力(包括漆酶、锰过氧化物酶、木素过氧化物酶、木聚糖酶、纤维素酶)较低,营养不足对该菌菌丝生长速度影响不明显,但对菌丝形态和酶活有很大的影响;低氮条件下最先产生木质素过氧化物酶,说明限氮条件可以刺激木质素过氧化物酶的产生;高无机盐条件下最先产生漆酶和锰过氧化物酶,但菌丝生长速度较慢,酶活性比较低,浓度过高会影响菌丝生长。结果表明,不同的营养条件对斑玉蕈的菌丝生长及多种酶活性有很大影响,这为斑玉蕈改变营养条件调节菌丝生长速度、菌丝形态以及基质降解提供了理论依据,同时对斑玉蕈栽培过程中基质的高效利用、缩短生产周期、降低生产成本具有重要的指导意义。     

Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267

Aims: Plant growth‐promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping‐off of cucumber evaluated. Methods and Results: The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping‐off of cucumber, since both wild type strain 267 and its biosurfactant‐deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC‐MS and MS‐MS analyses. Conclusions: The biosurfactants produced by Ps. putida 267 were identified as putisolvin‐like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping‐off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study: Pseudomonas putida 267 suppresses Phy. capsici damping‐off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin‐like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici.     

lux-Marking and application of carbofuran degrader Burkholderia cepacia PCL3

A luxAB-mutant of the carbofuran degrading bacterium Burkholderia cepacia PCL3 was successfully constructed with the capability to emit a luminescence signal of 1.6×10(-3)RLUcfu(-1). The mutant has a growth pattern and carbofuran degradation ability similar to PCL3 wild-type. The luminescent emission by PCL3:luxAB1 directly correlated with the metabolic activity of the cells. The optimal pH, temperature and n-decanal concentration for luminescence emission are 7.0, 35°C and 0.01%, respectively. PCL3:luxAB1 was used to assess the toxicity of carbofuran and carbofuran phenol in basal salt medium (BSM) in which the different sensitivity of the cells is dependent on the biomass concentration. With the luciferase system, the degradative fraction of the augmented PCL3:luxAB1 and the difference between the active augmented PCL3:luxAB1 and indigenous microorganisms at the contaminated site could be indicated.     

甘蓝型油菜苗期生长阶段对NaCl胁迫的生理响应

以甘蓝型油菜自交系2205(强耐盐型)、487(中耐盐型)和1423(敏盐型)为材料,采用土培+水培方法于五叶期研究了0(无盐胁迫,CK)、60 mmol·L-1(低盐)、120 mmol·L-1(中低盐)、180 mmol·L-1(中盐)、240mmol·L-1(高盐)NaCl胁迫后的叶片MDA、可溶性糖、甜菜碱和叶绿素含量的变化特征,为油菜耐盐性评价提供理论依据。结果显示:(1)叶片MDA含量在低盐和中低盐胁迫下降低且显著低于CK,在中盐和高盐胁迫下显著升高,并以品系2205含量最低且升幅最小,品系1423最高且升幅最大。(2)叶片可溶性糖含量在低盐和中低盐胁迫下减少(但2205在中低盐胁迫下显著高于CK),在中盐和高盐胁迫下升高且显著高于CK,并以品系2205含量最高增幅最大且显著高于品系487和1423,品系1423含量最低且增幅最小。(3)叶片甜菜碱含量随NaCl浓度升高增加,品系2205和487在中低以上盐浓度胁迫下显著升高,品系1423仅在高盐胁迫下显著升高,并以品系2205含量最高且增幅最大,1423含量最低且增幅最小。(4)叶片叶绿素含量在低盐和中低盐胁迫下显著增加,且以2205叶片叶绿素含量最高且增幅最大,但在中盐和高盐胁迫下显著减少,并以品系1423含量最低且降幅最高。研究表明,120mmol·L-1以下NaCl胁迫对油菜苗期生长可能有促进作用,180mmol·L-1以上NaCl胁迫则有明显抑制作用,且NaCl浓度越高油菜受伤害越重;油菜苗期生长阶段,NaCl胁迫浓度在120~180mmol·L-1时各生理指标发生显著变化,可能是鉴定耐盐性强弱的适宜浓度;综合分析认为,品系2205具有强的耐盐性,品系1423耐盐性最差,这与之前萌芽期和幼苗期鉴定结果一致。     

Growth responses and root porosity of rice in relation to temperature,light intensity and aeration

Summary Experiments were conducted under which rice plants were grown under three energy input levels (temperature and light) and two solution-oxygen concentrations. The rice variety was Colusa. Increasing the energy input increased total top growth. There was no significant effect of solution-oxygen concentration on top growth. The highest number of tillers developed under medium energy input followed by high energy input and the least under low energy input. There was a trend towards more tiller production under low oxygen conditions as compared to high oxygen. Decreasing the oxygen supply increased the dry weight production of roots under the two higher energy input levels, but had no significant effect under the low energy input. The highest amount of root growth occurred under high and medium energy input and least under lower energy input. Increasing the energy input increased the percent root porosity, but the oxygen level had no significant effect on root porosity. None of the treatments had a significant effect on nitrogen concentration in the tops. The amount of water necessary per unit dry matter production had a trend towards increase with increased energy input level and also significantly higher under low oxygen as compared to high oxygen conditions. Contribution of the Department of Soils and Plant Nutrition, University of California, Riverside. Supported in part by NSF Grant No. GB-5733X1.     

REGULATION OF GAMETOGENESIS IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE)1

The effects of nutrients, temperature and light on gametogenesis in Scenedesmus obliquus (Turp.) Kütz, were studied in culture. Concentrations of nitrogen in the medium employed showed a marked influence on gamete production. Gametogenesis is inhibited by N excess but is not a response to N starvation or depletion. A drop in N level from that of the growth medium is not required, nor does it per se trigger gametogenesis. The N concentration satisfying growth requirements insufficiently low to permit sexual differentiation. Nitrogen level in the growth medium has no effect on subsequent N to maintain a typical culture. Number of gametes present at maximum production time is inversely related to N concentration, but neither time of onset of gametogenesis, nor time of maximum gamete production is affected by N concentration. Cultures incubated at 15 C in medium lacking N take a minimum of 20—24 h to develop cells irreversibly committed to gamete formation. At the concentrations tested, no medium component other than the N-containing salt affected gametogenesis. Temperature influences both time of maximum production and numbers present at maximum production time. Time of maximum production is inversely related to incubation temperature; a 15 C incubation temperature yielded highest gamete production. Light enhances gametogenesis but gamete formation can occur in absence of light. Achievement of a light-saturated response is dependent upon illumination given at two critical periods: one occurs shortly after N withdrawal; the other occurs later, when cells are becoming irreversibly committed to gamete formation. Ability to produce gametes diminished with prolonged laboratory culture.     

Effect of culture conditions on lactic acid production of Tetragenococcus species

AIMS: To investigate the effects of the salt concentration, incubation temperature and initial pH of the medium on the fermentative ability of the halophilic lactic acid bacteria, Tetragenococcus muriaticus and T. halophilus. METHOD AND RESULTS: The growth, lactic acid production and pH reduction ability of five strains of T. muriaticus and T. halophilus in MRS broth medium under various culture conditions such as salt concentration (3, 7, 15 and 23% NaCl), temperature (20, 30 and 40 degrees C), and initial medium pH (5.8, 6.5 and 7.5) were investigated. Those of T. halophilus were seriously affected by a high salinity (23% NaCl); in contrast, those of T. muriaticus were affected by a low initial pH (5.8). CONCLUSIONS: The results indicate that high saline concentrations and low pH values have significant impact on the growth, lactic acid production and pH reduction ability of T. halophilus and T. muriaticus, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study appears to be important in biopreservation during the manufacture of fermented food products. Both T. muriaticus and T. halophilus may support each other in reducing pH in hypersaline or low pH environment. To our knowledge, this is the first report on the fermentation ability of T. muriaticus.     

盐胁迫对四种基因型冬小麦幼苗Na+、K+吸收和累积的影响

以4种不同基因型冬小麦品种为试验材料，研究了盐胁迫下小麦幼苗的生长及Na^＋、K^＋和Cl^-的吸收、累积规律。结果表明，盐胁迫下小麦吸水困难，幼苗生长受抑；幼苗含水量、生物量及干物质量明显下降；Na^＋、Cl^-含量和单株累积量显著增加。K^＋含量和单株累积量则明显降低。Na^＋／K^＋比值随介质中的盐浓度的增加而升高。盐胁迫下各基因型冬小麦幼苗Na^＋、K^＋和Cl^-的单株累积量及其在地上部分和根系中的含量变化较大，说明小麦根系对Na^＋、K^＋和Cl^-的吸收存在基因型差异。盐处理下，暖型小麦NR9405对K^＋的选择吸收能力强，对Na^＋的吸收和累积少，植株体内的K^＋浓度较高，Na^＋／K^＋比值小；幼苗的生物量较大，耐盐性强。冷型小麦RB6对K^＋的选择能力差，对Na^＋的吸收和累积量大，幼苗的Na^＋／K^＋比值大，生物量小，耐盐性较差。低盐浓度下，Na^＋可作为渗透调节物质维持植物体内渗透平衡。高盐浓度下，Na^＋的过度吸收和累积可能是盐害的主要原因。维持体内较低的Na^＋水平和Na^＋／K^＋比值是小麦耐盐性的一个重要特征。     

A <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> PCC 7942 mutant with a higher tolerance toward the herbicide bentazone also confers resistance to sodium chloride stress

Following a N-methyl-N'-nitro-N-nitrosoguanidine-based mutagenesis of Synechococcus elongatus PCC 7942 wild type, we were able to select several mutants with an enhanced tolerance toward the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). Mutant Mu1 has in part been previously characterized. In the present paper we report on another mutant, called Mu2, which also has a higher tolerance toward bentazone. Since Mu2 showed a better growth than WT when cultivated with elevated NaCl concentrations in the growth medium and since S. elongatus WT has previously been classified to be low salt tolerant, we were especially interested in the identification of the modifications conferring this higher salt tolerance to mutant Mu2. Immunoblot analyses provided evidence that Mu2 had a constitutively higher expression of PsbO and of IsiA. In addition, in Mu2 a significantly higher concentration of IdiA was detected under salt stress as compared to WT. These three proteins most likely contribute to a better protection and/or stabilization of photosystem II. Moreover, Mu2 had a higher amount of the photosystem I reaction center proteins PsaAB under salt stress than WT. In addition, the amount of the ferredoxin:NADP+ oxidoreductase and also of the ATP synthase was constitutively higher in Mu2 than in WT. In contrast to WT the latter two proteins did not decrease under salt stress in Mu2. Therefore, it can be assumed that Mu2 could maintain a high cyclic electron transport activity around photosystem I under salt stress. It can be assumed that the combination of these modifications of the electron transport chain cause a better protection of photosystem II against oxidative damage and cause an increase of cyclic electron transport activity around photosystem I with ATP synthesis. Thus, the overall cellular energization in Mu2 relative to WT is improved. Together with putative other not yet identified modifications this seems to enable Mu2 to energize its cytoplasmic membrane-localized ion pumps more effectively than WT and, as a consequence, to keep the intracellular NaCl concentration low.     

Effect of chloramphenicol and actinomycin D on extracellular alkaline RNAse biosynthesis by Bacillus intermedius

By means of chloramphenicol it was found that biosynthesis of alkaline exocellular RNAase was repressed in Bacillus intermedius by inorganic phosphate. Actinomycin D at a low concentration stimulates RNAase biosynthesis in a medium with a minimal phosphorus concentration in model experiments with washed cells and in the batch culture. As a result, the activity of RNAase rises 2-4 times. The stimulating effect of actinomycin D decreases when phosphorus concentration in the medium is increased The effect of actinomycin D is maximal if the antibiotic is added to the medium when the specific growth rate of the bacterium falls down and the rate of RNAase biosynthesis rises.     

The influence of culture conditions on carotenogenesis in Streptococcus faecium UNH564P.

The growth of Streptococcus faecium UNH564P and its production of triterpenoid carotenoids under a variety of culture conditions were examined. Total extractable cell lipid and carotenoid levels increased with culture age and paralleled the growth curve of the bacterium. Variations of the medium glucose concentration produced significant changes in both cell growth and carotenoid production, with the xanthophyll content decreasing at high glucose concentrations. Carotenoid degradation products were found in highly aerated cultures although a high glucose concentration appeared to have a sparing effect on oxidative degradation. Culture age appeared to have little effect on carotene:xanthophyll ratios. The significance of the production of total and individual carotenoids under the various culture conditions is discussed and related to a postulated scheme of triterpenoid carotenoid biosynthesis in the organism.     

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific

Halophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K⁺/Na⁺ ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.     

Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.     

Correlation between phosphatidylcholine labeling and hormone receptor levels in alveolar type II epithelial cells: effects of dexamethasone and epidermal growth factor

Phosphatidylcholine labeling was studied in freshly isolated adult rat alveolar type II epithelial cells exposed to dexamethasone and epidermal growth factor. Dexamethasone at a medium concentration of 10^?8m, enhanced phosphatidylcholine labeling in type II cells by about 25%. In lung fibroblast controls, dexamethasone had no effect. Phosphatidylcholine secretion into the culture medium was not observed in either cell type. Quantitation of dexamethasone receptors revealed a twofold greater number of receptors in type II cells than in control fibroblasts. In contrast, the addition of epidermal growth factor to the medium of type II cells or lung fibroblasts had no effect on phosphatidylcholine labeling or secretion into culture medium. Lung fibroblasts were found to have 11-fold more surface receptors for epidermal growth factor than isolated type II cells. These results indicate that dexamethasone significantly increases phosphatidylcholine synthesis in type II cells and thus, may also effect the production of surfactant by these cells.     

Improved production of curdlan with concentrated cells ofAgrobacterium sp.

The addition of a limited concentration of yeast extract to a minimal salt medium (MSM) enhanced cell growth and increased the production of curdlan whereas nitrogenlimitation was found to be essential for the higher production of curdlan byAgrobacterium sp. ATCC 31749. As the amount of the inoculum increased, the cell growth as well as the production of curdlan also increased in the MSM without a nitrogen source. The cell growth and production of curdlan increased as the initial pH of the medium decreased as low as 5.0. The conversion rate and concentration of curdlan from 2% (w/v) glucose in the MSM with concentrated cells under nitrogen deletion was 67% and 13.4 g/L, respectively. The highest conversion rate of curdlan under the conditions optimized in this study was 71% when the glucose concentration was 1% (w/v).     

1, 3-丙二醇发酵过程中盐浓度的胁迫作用

通过对克雷伯氏菌(Klebsiella pneumoniae)甘油发酵生产1, 3-丙二醇(1, 3-PD)过程的研究发现, 盐浓度对 1, 3-PD发酵有胁迫作用。盐浓度较低时, 菌体生长和产物生成均维持较高速率; 盐浓度较高时会导致菌体生长减慢, 1, 3-PD最终浓度, 甘油到1, 3-丙二醇的转化率降低, 同时1, 3-丙二醇氧化还原酶受到抑制。在5 m3罐中控制合适的盐浓度可以提高1, 3-PD的发酵水平, 使1, 3-PD的最终浓度达到64 g/L, 转化率61%, 生产强度2.1 g/(L·h)。