联合固氮菌Klebsiella oxytoca SA2和水稻幼苗的相互作用

从内蒙流沙地先锋植物沙竹（Psammochloa mongokca）内分离到一株内生细菌，经鉴定定名为Klebsiella oxytoca SA-2 K.oxytoca SA-2兼性厌氧固氮，NH4+抑制其固氮酶合成。部分抑制固氮酶活性;N03 -抑制其固氮酶的合成和活性。 60℃灭活K.oxytoca SA-2整体菌免疫兔子得到抗血清。免疫印迹表明此抗血清具K.oxytoca SA-2种特异性。石蜡切片免疫金银染色结合显微观察发现K.oxytoca SA-2定殖于沙竹叶鞘薄壁细胞和叶片的薄壁细胞内。 K.oxytoca SA-2在半固体培养基中接种水稻幼苗，限菌培养21天，根内重新分离的数量达l06 cfu/g．FWroot，但K.oxytoca SA-2在富养的土壤中生长良好，表现为兼性内生菌。 限菌培养水稻（Oryza sativa）幼苗，石蜡切片免疫金银染色结合显微观察研究了的K.oxytoca SA-2的侵染特性．K.oxytoca SA-2可以通过侧根发生处和表皮细胞胞间层进入根内，在皮层薄壁细胞间隙大量定殖，在解体和看似完整的薄壁细胞内也有定殖，在根和茎中柱内K.oxytoca SA-2进入了木质部导管。在根基，K.oxytoca SA-2大量侵入了已解体的内皮层和中柱鞘细胞，植物细胞在K.oxytoca SA-2侵入后解体，可能表现为严格的局部超敏反应。 接种K.oxytoca SA-2 21天，水稻地上苗部分没有发现肉眼和显微可见的病症。与对照相比，接种K.oxytoca SA-2显著促进限氮培养水稻幼苗的生长。由于K.oxytoca SA-2在限碳限氮培养基和水稻幼苗共培养时能分泌NH4+和植物激素，它可能通过向水稻幼苗提供氮素和分泌植物激素促进植物生长。而且用固氮酶铁蛋白抗血清进行免疫金银染色发现定殖在根基皮层薄壁细胞胞间层和细胞间隙，木质部导管和茎基木质部导管的K.oxytoca SA-2可以表达固氮酶，固氮参与了K.oxytoca SA-2在水稻幼苗中的内生。 培养基内碳源（苹果酸）和培养温度对K.oxytoca SA-2和水稻幼苗相互作用的影响也进行了研究。 研究表明，K.oxytoca SA-2作为兼性内生固氮菌，能够和植物紧密联合，并在植物体内开拓一个有利的生态位固氮，而且K.oxytoca SA-2可以分泌NH4+和植物激素，在和植物相互作用中使植物受益。

Diversity and abundance of ammonia-oxidizing bacteria in eutrophic and oligotrophic basins of a shallow Chinese lake (Lake Donghu)

Classical cultivation and molecular methods based on the ammonia monooxygenase gene (amoA) were used to study the abundance and diversity of beta-proteobacterial ammonia-oxidizing bacteria (AOB) in lake sediments. The eutrophic and oligotrophic basins of a Chinese shallow lake (Lake Donghu), in terms of ammonium (NH4+) concentrations, were sampled. The AOB number was significantly lower in the oligotrophic basin, but significantly higher in the eutrophic basin. In addition, using restriction fragment length polymorphism targeting the amoA, ten restriction patterns including six unique ones were found in the eutrophic basin, while five patterns were observed in the oligotrophic basin with only one unique restriction group. Phylogenetic analysis for AOB revealed that Nitrosomonas oligotropha- and Nitrosomonas ureae-related AOB and Nitrosospira-affiliated AOB were ubiquitous; the former dominated in the eutrophic basin (87.2%), while the latter dominated in the oligotrophic basin (65.5%). Furthermore, Nitrosomonas communis-related AOB was only detected in the eutrophic basin, at a small proportion (3.2%). These results indicate significant selection and adaptation of sediment AOB in lakes with differing trophic status. (C) 2009 Elsevier Masson SAS. All rights reserved.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Comparing the Performance of Biomimetic Pattern Recognition with W8 and SVM on Prediction of Horizontal Gene Transfers in Bacteria Genomes

National Natural Science Foundation of China 60753001

Establishment and characterization of dual-species biofilms formed between a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities

In this study, the possibility of establishing a dual-species biofilm from a bacterium with a high biofilm-forming capability and a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading bacterium, Comamonas testosteroni A3, was investigated. Our results showed that the combinations of strain A3 with each of five strains with a high biofilm-forming capability (Pseudomonas sp. M8, Pseudomonas putida M9, Bacillus cereus M19, Pseudomonas plecoglossicida M21 and Aeromonas hydrophila M22) presented different levels of enhancement regarding biofilm-forming capability. Among these culture combinations, the 24-h dual-species biofilms established by C. testosteroni A3 with P. putida M9 and A. hydrophila M22 showed the strongest resistance to 3,5-DNBA shock loading, as demonstrated by six successive replacements with DMM2 synthetic wastewater. The degradation rates of 3,5-DNBA by these two culture combinations reached 63.3-91.6% and 70.7-89.4%, respectively, within 6 h of every replacement. Using the gfp-tagged strain M22 and confocal laser scanning microscopy, the immobilization of A3 cells in the dual-species biofilm was confirmed. We thus demonstrated that, during wastewater treatment processes, it is possible to immobilize degrader bacteria with bacteria with a high biofilm-forming capability and to enable them to develop into the mixed microbial flora. This may be a simple and economical method that represents a novel strategy for effective bioaugmentation.