K253R和N184V点突变对葡萄糖异构酶热稳定性的影响

用人工合成的寡核苷酸突变引物,以双引物法于重组Ｍ１３ｍｐ１９载体上进行定点突变,分别得到了葡萄糖异构酶的突变体ＧＩＫ２５３Ｒ和ＧＩＮ１８４Ｖ。测定它们的热稳定性,并将之与野生型酶比较,结果表明：（１）ＧＩＫ２５３Ｒ于７０℃、８０℃下的热稳定性小于野生型,但在７０℃、１ｍｏｌ／ＬＬ－鼠李糖中,两者的失活速度相近。另外;ＧＩＫ２５３Ｒ的比活是野生型的１．５倍。（２）ＧＩＮ１８４Ｖ的比活和热稳定性都远低于野生型,Ａｓｎ１８４为酶活性中心构象的维持所必需．本文还根据动力学数据和分子结构模型对以上结果作了初步分析．     

Diversity of antifungal and plant-associated Serratia plymuthica strains

A total of 21 plant-associated Serratia plymuthica strains were characterized phenotypically by their nutritional patterns, susceptibility to antibiotics, antifungal and haemolytic properties, and genotypically by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA, PCR fingerprints using BOX primers (BOX-PCR) and pulsed-field gel electrophoresis (PFGE) after digestion with SpeI. All of the investigated strains demonstrated antifungal activity in vitro against fungal pathogens while only six strains produced the antifungal antibiotic prodigiosin. Haemolytic activity and antibiotic resistance patterns were investigated to assess the risk associated with the use of isolates in plant protection. The strains were haemolytic at human-relevant temperatures. The level of resistance to antibiotics was low. This work shows that BOX-PCR and PFGE are useful fingerprinting methods to characterize Ser. plymuthica strains, although the discriminatory effect between the two methods differed. Computer-assisted analysis of phenotypic and genotypic features demonstrated relationships between the origin of isolation, the production of prodigiosin and the molecular fingerprint.     

普城沙雷氏菌splI及spsI基因突变株的构建

普城沙雷氏菌(Serratia plymuthica)G3分离自小麦内茎,是一种可产生多种抗菌因子和植物激素的内生细菌。目前对S.plymuthica G3的两个群体感应系统splI/splR与SpsR/SpsI的了解仍十分有限。构建了2个N-乙酰基高丝氨酸内酯信号合成酶编码基因splI和spsI突变菌株及互补菌株,并检测了其生物表型。结果发现spsI、splI突变后,对细菌信号分子合成有一定的影响,蛋白酶活性明显降低。通过对突变体和互补菌株的泳动性分析发现,splI负调控G3的运动性,而spsI正调控G3的运动性。     

Complete genome sequence of Serratia plymuthica strain AS12

A plant-associated member of the family Enterobacteriaceae, Serratia plymuthica strain AS12 was isolated from rapeseed roots. It is of scientific interest because it promotes plant growth and inhibits plant pathogens. The genome of S. plymuthica AS12 comprises a 5,443,009 bp long circular chromosome, which consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome was sequenced within the 2010 DOE-JGI Community Sequencing Program (CSP2010) as part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens".     

Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K_m was 39.9 mM, the V_max was 638 U mg⁻¹, and the K_cat/K_m was 1.79 × 10⁴ M⁻¹ s⁻¹, compared to a K_m of 76.0 mM, a V_max of 423 U mg⁻¹, and a K_cat/K_m of 0.62 × 10⁴ M⁻¹ s⁻¹ for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.     

Quorum-sensing effects in the antagonistic rhizosphere bacterium Serratia plymuthica HRO-C48

The rhizosphere-associated bacterium Serratia plymuthica HRO-C48 is not only able to suppress symptoms caused by soil-borne pathogens but is also able to stimulate growth of plants. Detailed knowledge about the underlying mechanisms and regulation are crucial for the application in biocontrol strategies. To analyse the influence of N -acyl homoserine lactone (AHL)-mediated communication on the biocontrol activity, the AHL-degrading lactonase AiiA was heterologously expressed in the strain, resulting in abolished AHL production. The comparative analysis of the wild type and AHL negative mutants led to the identification of new AHL-regulated phenotypes. In the pathosystem Verticillium dahliae –oilseed rape, the essential role of AHL-mediated signaling for disease suppression was demonstrated. In vitro , the regulatory function of AHLs in the synthesis of the plant growth hormone indole-3-acetic acid is shown for the first time. Additionally, swimming motility was found to be negatively AHL regulated. In contrast, production of extracellular hydrolytic enzymes is shown to be positively AHL-regulated. HRO-C48 emits a broad spectrum of volatile organic compounds that are involved in antifungal activity and, interestingly, whose relative abundances are influenced by quorum sensing (QS). This study shows that QS is crucial for biocontrol activity of S. plymuthica and discusses the impact for the application of the strain as a biocontrol agent.     

First report of Serratia plymuthica causing onion bulb rot in Poland

Specific bacterial disease symptoms were observed on onion bulbs in almost all regions in Poland. For the purpose of identification of agents causing disease, bacteria were isolated from the symptomatic plants. Their pathogenicity was confirmed by using pathogenicity test on onion scales. These bacteria were identified biochemically and molecularly as Serratia plymuthica.     

Pleiotropic effects of chitinase gene from Serratia plymuthica in transgenic potato

We estimate the influence of transgenic bacterial chitinase from soilborne Serratia plymuthica onto agronomical important traits of potato, such as productivity and non-specific resistance. Transgene has been delivered into potato variety Delfin via Agrobacterial transformation with pGreen0229 and pBI121-based vectors. Growth inhibition of chitin-containing pathogenic fungi was shown. However, over 40% oftransgenic lines demonstrated decreased non-specific resistance to late blight (down to 62% compared to control genotype), as well as 40-60% productivity drop.     

Pleiotropic effects of the chitinase gene from Serratia plymuthica in transgenic potato

The pleiotropic effects of transgenesis includes different consequences of the insertion of a transgene that are not related to the direct action of its product. It is necessary to evaluate the outlook for the application in selection of the transgenic potato strain containing the bacterial chitinase gene chiA we created for studying the possible nonspecific influence of the introduction of the transgene on the phenotypical properties of the transgenic lines. In the present investigation we will consider the effect of the introduction of the chitinase transgene on such agronomically important characteristics as yield and nonspecific resistance.     

G138P定点突变对葡萄糖异构酶热稳定性的改善

通过分子设计,确定Ｇｌｙ１３８为改善葡萄糖异构酶（ＧＩ）热稳定性的目标氨基酸。用双引物法对ＧＩ基因进行体外定点突变,构建了ＧＩ突变体Ｇ１３８Ｐ。含突变体的重组质粒ｐＴＫＤ－ＧＩＧ１３８Ｐ在Ｅ．ｃｏｌｉＫ３８菌株中表达。ＧＩＧ１３８Ｐ与野生型ＧＩ比较实验表明：（１）ＧＩＧ１３８Ｐ的热失活半衰期约是野生型ＧＩ的２倍;（２）ＧＩＧ１３８Ｐ的最适反应温度提高了１０￣１２℃;（３）ＧＩＧ１３８Ｐ的比活与野生     

Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica

Abstract The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans ( S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).     

Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9

Serratia plymuthica are plant-associated, plant beneficial species belonging to the family Enterobacteriaceae. The members of the genus Serratia are ubiquitous in nature and their life style varies from endophytic to free-living. S. plymuthica AS9 is of special interest for its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The genome of S. plymuthica AS9 comprises a 5,442,880 bp long circular chromosome that consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome is part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI Community Sequencing Program (CSP2010).     

Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48

The chitinolytic rhizobacterium Serratia plymuthica HRO-C48 was previously selected as a biocontrol agent of phytopathogenic fungi. One endochitinase (E.C. 3.2.1.14), CHIT60, and one N-acetyl-beta-1,4- D-hexosaminidase (E.C. 3.2.1.52), CHIT100, were purified and characterized. The endochitinase CHIT60, with an apparent molecular mass of 60.5 kDa, had a N-terminal amino acid sequence highly similar to that of chitinases A from Serratia liquefaciens and Serratia marcescens. The enzyme activity had its peak at 55 degrees C and pH 5.4, and increased by more than 20% in the presence of 10 mM Ca(2+), Co(2+) or Mn(2+). Activity was inhibited by 80% in the presence of 10 mM Cu(2+). CHIT100 appeared to be a monomeric enzyme with a molecular mass of 95.6 kDa and a pI of 6.8. Optimal activity was obtained at 43 degrees C and pH 6.6, and decreased by more than 90 % in the presence of 10 mM Co(2+) or Cu(2+). CHIT100 (100 microg ml(-1)) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively. With CHIT60 (100 microg ml(-1)), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation.     

Draft genome sequence of the antagonistic rhizosphere bacterium Serratia plymuthica strain PRI-2C

Serratia plymuthica strain PRI-2C is a rhizosphere bacterial strain with antagonistic activity against different plant pathogens. Here we present the 5.39-Mb (G+C content, 55.67%) draft genome sequence of S. plymuthica strain PRI-2C with the aim of providing insight into the genomic basis of its antagonistic activity.     

Identification of environmental Serratia plymuthica strains with the new combo panels type 1S

Automated systems are required when numerous samples need to be processed, offering both high through put and test of a multiple simultaneously. This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Neg Panels Type 1S with conventional biochemical methods for identifying ten environmental Serratia plymuthica strains. High correlation between both methods were observed for all the 21 tests evaluated, and the MicroScan system was found capable of correctly identifying all S. plymuthica strains tested. In all tests, the percentage of correlation was 100%, except in raffinose test (91%).     

Field performance of a weed-suppressing Serratia plymuthica strain applied with conventional spraying equipment

In previous glasshouse experiments, the soilbacterium Serratia plymuthica, strainA153, showed strong growth-suppressingactivities against a range of broad-leavedweeds after foliar spraying. In field tests ofthis strain in spring wheat, spring barley andpotatoes, variable effects were achieved on arange of weeds including Chenopodiumalbum, Stellaria media, Polygonumconvolvulus and Galeopsis speciosa. Atone site, good suppression of C. albumwas observed when the strain was applied in atank mix with another bacterial isolate or withreduced doses of a herbicide. Effects on weedsappeared to be independent of the applicationvolume (1000, 600, 500 l ha^–1), but weedswere in some cases more strongly suppressed athigher bacterial doses. Barley yields weresomewhat reduced by the bacterial application,but wheat yields were less affected. AlthoughS. plymuthica suppressed certain weedswhen applied in the field in a simple aqueousformulation and with conventional sprayingequipment, the level of weed suppression wasunsatisfactory from a practical standpoint.     

Analysis of Oxidation Sensitivity of Maleate cis-trans Isomerase from Serratia marcescens

The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.