Phylogeny of γ-proteobacteria inferred from comparisons of 3’ end 16S rRNA gene and 5’ end 16S-23S ITS nucleotide sequences

The phylogeny of γ-proteobacteria was inferred from nucleotide sequence comparisons of a short 232 nucleotide sequence marker. A total of 64 γ-proteobacterial strains from 13 Orders, 22 families, 40 genera and 59 species were analyzed. The short 232 nucleotide sequence marker used here was a combination of a 157 nucleotide sequence at the 3’ end of the 16S rRNA gene and a 75 nucleotide sequence at the 5’ end of the 16S-23S Internal Transcribed Spacer (ITS) sequence. Comparative analyses of the 3’ end of the 16S rRNA gene nucleotide sequence showed that the last 157 bp were conserved among strains from same species and less conserved in more distantly related species. This 157 bp sequence was selected as the first part in the construction of our nucleotide sequence marker. A bootstrapped neighbor-joining tree based on the alignment of this 157 bp was constructed. This 157 bp could distinguish γ-proteobacterial species from different genera from same family. Closely related species could not be distinguished. Next, an alignment of the 16S-23S ITS nucleotide sequences of alleles from same bacterial strain was performed. The first 75 bp at the 5’ end of the 16S-23S ITS was highly conserved at the intra-strain level. It was selected as the second part in the construction of our nucleotide sequence marker. Finally, a bootstrapped neighbor-joining tree based on the alignment of this 232 bp sequence was constructed. Based on the topology of the neighbour-joining tree, four major Groups, Group I to IV, were revealed with several sub-groups and clusters. Our results, based on the 232 bp sequence were, in general, in agreement with the phylogeny of γ-proteobacteria based on the 16S rRNA gene. The use of this 232 bp sequence as a phylogenetic marker presents several advantages over the use of the entire 16S rRNA gene or the generation of extensive phenotypic and genotypic data in phylogenetic analyses. First, this marker is not allele-dependant. Second, this 232 bp marker contains 157 bp from the 3’ end of the 16S rRNA gene and 75 bp from the 5’ end of the 16S-23S ITS. The 157 bp allows discrimination among distantly related species. Owing to its higher rate of nucleotide substitutions, the 75 bp adds discriminating power among closely related species from same genus and closely related genera from same family. Because of its higher percentage of nucleotide sequence divergence than the 16S rRNA gene, the 232 bp marker can better discriminate among closely related γ-proteobacterial species. Third, the method is simple, rapid, suited to large screening programs and easily accessible to most laboratories. Fourth, this marker can also reveal γ-proteobacterial species which may appear misassigned and for which additional characterization appear warranted.

基于PCR-DGGE技术分析生物絮团的细菌群落结构

在草鱼养殖过程中添加碳源(葡萄糖)维持水体C∶N为20∶1以培养生物絮团,通过对生物絮团细菌群落构成进行种类鉴定来评价生物絮团中功能微生物的组成。采用PCR-DGGE(变性梯度凝胶电泳)技术分析生物絮团形成第5、10和15天的细菌群落结构。DGGE指纹图谱结果分析表明:第5天和第10天的相似性最高,达67.4%;第5天和第15天相似性系数最低仅为40.5%。第10天时微生物多样性最高,第15天时多样性最低。对DGGE指纹图谱特征条带进行回收、克隆测序,结果表明,生物絮团培养过程主要微生物类群隶属于以下6个纲:α-变形菌纲(Alphaproteobacteria)、γ-变形菌纲(Gammaproteobacteria)、β-变形菌纲(Betaproteobacteria)、放线菌纲(Actinobacteria)、芽孢杆菌纲(Bacilli)、拟杆菌纲(Bacteroidetes),其中α-、β-及γ-变形菌占据主要位置。α-proteobacteria为3个阶段的共有优势菌,第5天时特异菌包括食酸菌属(Acidovorax)、气单胞菌属(Aeromonas)、土壤杆菌属(Agrobacterium),第10天和15天分别为芽孢杆菌属(Bacillus)与红球菌属(Rhodococcus)。研究首次发现,生物絮团应用于淡水养殖系统时细菌的组成和多样性都极其丰富,通过结合分析这些微生物的功能特点,为生物絮团技术在实际养殖生产中的进一步应用奠定基础。

秸秆还田后土壤微生物群落结构变化的初步研究

通过实验室条件下的小麦秸秆粉和油菜秸秆粉的还土试验,结合常规的分析手段和基于DNA的分子技术(变性梯度凝胶电泳(DGGE)及测序技术),初步研究了秸秆粉还田后土壤物理化学特性及生物学特性的变化。结果表明,培养60d后,秸秆还田土壤的肥力明显提高,其纤维素酶活性明显增强。DGGE图谱表明,对照土壤(S)以及处理土壤(SW和SR),在培养过程中,β-Proteobacteria类细菌组成都在发生变化。对其中一个样品的DGGE条带进行了割胶测序,测序结果表明,大部分条带代表的微生物是未培养的或不可培养的。采用传统分离培养技术,从秸秆还田土壤中分离了2株纤维素降解菌。以上结果说明,秸秆还田具有良好的土壤综合效应。

环境因素对甲基叔丁基醚生物降解的影响研究

采用β-Proteobacteria PM1完整细胞降解甲基叔丁基醚(MTBE),研究了金属离子、pH值、细胞浓度等因素对MTBE降解速率的影响.结果表明,K+和Ba2+对MTBE降解有明显的促进作用,较适宜的pH值为7.0;细胞在BaCl2溶液中基本上处于静息状态;细胞浓度越高,降解MTBE的速率就越快;降解过程需要有氧气参与;PM1完整细胞降解叔丁醇(TBA)的速率比降解MTBE更快,MTBE的存在对TBA的降解有抑制作用,但TBA的存在不影响MTBE的降解;静息细胞法降解MTBE的过程中没有检测到TBA等中间产物;PM1静息细胞降解MTBE的米氏常数Km为0.73mmol·L-1,最大反应速率Vmax为0.14mmol·L-1·h-1.

硒超积累植物壶瓶碎米荠的根际微生物特征研究

对湖北恩施的硒超积累植物——壶瓶碎米荠的根际微生物特征进行16S rRNA基因文库分析,结果显示其根际微生物相较于非根际土壤微生物具有更高的丰度和更低的复杂度,而且主要由α-变形菌纲(15%~22%)、β-变形菌纲(10%~16%)、放线菌纲(10%~18%)、酸杆菌纲(8%~15%)、γ-变形菌纲(5%~16%)等组成;此外,根际微生物还存在很多特异性微生物,如:硝化螺旋菌纲(2%~5%)、芽单孢菌纲(2%~5%)、疣微菌纲(2%~4%)、浮霉菌纲(1%~2%)、其他(丰佑菌纲、鞘脂杆菌纲、芽孢杆菌纲、梭菌纲)(3%~4%)。代表性的根际微生物α-变形菌纲和硝化螺旋菌纲可能在壶瓶碎米荠对硒的吸收、积累过程中扮演了重要的作用。

α-变形菌纲新的保守特征性插入缺失

在基于16S rRNA基因的分类系统中α-变形菌被定为变形菌门的一个纲。本文根据系统发育基因组学分析,首次发现并鉴定了α-变形菌纲普遍存在的5个保守特征性插入缺失（CSIs）。23S rRNA基因具有1个98-105 nt的特征性缺失和1个5-7 nt的特征性插入,DNA复制解旋酶（DnaB）具有1个17-29 aa的特征性插入,精氨酰-tRNA合成酶（ArgRS）具有1个15 aa的特征性插入,DNA促旋酶A亚基（GyrA）具有1个27-63 aa的特征性插入。α-变形菌纲的这些CSIs使得该纲与包括变形菌门其他纲在内的其他细菌显著区分开来,为该群细菌系统发育及系统分类研究提供了可靠手段。

Aerobic degradation of methyl tert-butyl ether by a Proteobacteria strain in a closed culture system

The contamination of methyl ten-butyl ether （MTBE） in underground waters has become a widely concerned problem all over the world. In this study, a novel dosed culture system with oxygen supplied by H2O2 was introduced for MTBE aerobic biodegradation. After 7 d, almost all MTBE was degraded by a pure culture, a member of β-Proteobacteria named as PMI, in a closed system with oxygen supply, while only 40% MTBE was degraded in one without oxygen supply. Dissolved oxygen （DO） levels of the broth in closed systems respectively with and without H2O2 were about 5-6 and 4 mg/L. Higher DO may improve the activity of monooxygemase, which is the key enzyme of metabolic pathway from MTBE to tert-butyl alcohol and finally to CO2, and may result in the increase of the degrading activity of PM1 cell. The purge and trap GC-MS result of the broth in closed systems showed that tea-butyl alcohol, isopronol and acetone were the main intermediate products.

辛基酚的好氧生物降解及微生物群落特征

利用典型的活性污泥实验了辛基酚(octylphenol,OP)的生物降解性能。研究了水力停留时间、OP浓度和碳氮比对OP处理效率的影响,通过实时PCR和PCR-DGGE方法分别分析了微生物群落的总体密度和多样性变化。通过30d的启动,OP在序批式活性污泥典型好氧条件下出现有效生物降解。在OP浓度50～150μg/L整个实验过程中,OP去除率(84%～99%)基本保持稳定。在实验范围内,较长的水力停留时间(12 h)和碳氮比(2∶1)有利于OP生物降解。通过实时PCR方法测定得到系统微生物平均密度约为1.22 g/L,但富碳状态下生物密度增长上升。通过DGGE分离到17种优势基因型,包括了α-proteobacteria(6种)、β-proteobacteria(5种)、γ-proteobacteria(2种)、Actinobacteria(2种)和δ-proteobacteria(1种)群落。α-proteobacteria和δ-proteobacteria菌群密度呈现增长,β-proteobacteria和Actinobacteria菌群中的部分种类密度出现减少,OP在活性污泥中的好氧生物降解可能与α-proteobacteria中的特定类别相关。