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.

Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

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.

石油烃污染对稻田土壤微生物生态系统的影响

由于含油污水灌溉、原油储存运输和石油冶炼过程中导致的泄漏事故等原因，石油类物质已经成为环境中的主要污染物之一。石油烃污染物对土壤微生物遗传多样性和群落结构的影响在国外已有一些报道，但有关于石油烃污染对稻田土壤微生物生态系统的影响一直以来一直没有一个全面和系统的认识。本论文首次采用传统微生物培养方法、常规生化分析方法与PC树DGGE等现代分子生态学研究方法相结合的手段系统评价了长期石油污水灌溉对中国最大的石油类污水灌区-沈抚灌区的稻田土壤微生物种群数量、细菌群落组成、多样性及代谢活性的影响。结果表明，总石油烃（Total Petloleum hydrocarbon／TPH）在灌区干渠和支渠中的积累和分布趋势大体上是上游地区较严重，下游地区较轻，并且与土壤中有机质含量呈显著正相关。在目前的污染程度下，石油污水能够刺激土壤好氧异养细菌（Aerobic heterotrophic bacteria／AHB）和真菌的生长，其数量与TPH含量呈显著正相关。细菌基因多样性与TPH含量呈显著负相关，细菌群落中的优势菌群为变形细菌β-亚群和γ-亚群的菌种。土壤脱氢酶、过氧化氢酶、多酚氧化酶活性和土壤底物诱导呼吸（substrate-induced respiration／SIR）与土壤中TPH含量呈显著正相关，而土壤脉酶活性则与之呈显著负相关。实验室110天不同浓度石油烃胁迫模拟试验结果表明，当石油烃胁迫浓度低于1000mgk扩时可以被定义为轻度污染，土壤中AHB大量增殖，细菌多样性和群落结构可在处理第30天和第50天得到恢复；当石油烃胁迫浓度为5000mg·kg~(-1)-10000mg·kg~(-1)时可以被定义为中度污染，AHB数量显著增加，放线菌和真菌数量显著降低，土壤细菌多样性在培养前巧天显著降低，至培养结束时有逐渐恢复的趋势，细菌群落结构发生明显改变，土壤脱氢酶、多酚氧化酶、脉酶的活性及土壤SIR受到一定程度的抑制，但能够显著刺激土壤过氧化氢酶活性的提高。当石油烃胁迫浓度为10000mg·kg~(-1)-50000mg·kg~(-1)时可以被定义为重度污染，对AHB生长的刺激作用更为显著，土壤细菌多样性在培养前15天显著降低并不可恢复，群落组成与对照差异很大，所有土壤酶和SIR均受到严重抑制。从沈抚灌区上游地区旱田土壤、灌渠底泥和实验室高浓度石油烃胁迫土壤中筛选出了4株TPH生物降解率在60％以上的高效石油烃降解菌并鉴定其归属，这些菌株均能够利用正构烷烃、单环和多环芳烃及环烷烃。