Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes

The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp., Arthrobacter sp., and a bacterium of uncertain affiliation. dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonas sp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified as R. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8) dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil.

本研究针对土壤中含硫烃类的选择性效应——即其对细菌群落结构变化、脱硫生物与基因的筛选作用——展开探究。研究分析了两类土壤样品：一类采自受污染农田土壤（A土壤），沿含硫原油浓度梯度布设采样点；另一类为经含二苯并噻吩（dibenzothiophene，DBT）原油处理的土壤微宇宙（FSL土壤）。分析内容涵盖总细菌与DBT降解菌的平板计数、基于土壤DNA的聚合酶链式反应-变性梯度凝胶电泳（PCR-DGGE）分子群落图谱分析，以及编码烃类脱硫酶的基因（即dszA、dszB与dszC）的检测。对A土壤的分析结果显示，原油浓度梯度对两种培养基上的可培养细菌数量均无显著区分效应；总体而言，DBT降解菌的计数结果较总可培养细菌数低1~2个数量级。然而，PCR-DGGE分析表明，分子群落图谱中检测到的条带数量随土壤原油含量升高而减少。对高污染土壤样品对应的图谱中3条优势条带的序列分析显示，其对应的宿主菌分别与放线菌属（Actinomyces）、节杆菌属（Arthrobacter）及一株分类地位未定的细菌具有亲缘关系。所有A土壤样品中均检出dszA、dszB与dszC基因，而多份未受污染的土壤样品在该检测中呈阴性结果。对FSL土壤的研究结果显示，原油-DBT处理对可培养细菌总数的影响微弱，但对DBT利用菌群具有显著调控作用；未处理FSL土壤的分子群落图谱随时间推移基本保持稳定，而经含DBT原油处理的样品则随时间呈现渐进性的群落变化。直接PCR检测结果显示，未处理的FSL土壤中存在与dszB同源的信号，且原油-DBT处理可筛选出与dszA、dszC同源的序列。将PCR-DGGE应用于以A土壤与处理后FSL土壤为接种源、在含DBT的无硫基础盐培养基中进行的连续富集培养，结果筛选出至多10条不同的条带。对其中部分条带的测序结果显示，存在与恶臭假单胞菌（Pseudomonas putida）、假单胞菌属（Pseudomonas）某菌种、嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）及红平红球菌（Rhodococcus erythropolis）亲缘相关的菌属。从A土壤与FSL土壤的以DBT为唯一硫源的琼脂平板上分离得到的52株菌落中，有若干株的条带迁移位置与富集培养中筛选得到的条带一致。在12株菌株中检出了dszB基因，而仅分别在7株与6株菌株中发现dszA与dszC基因；携带dszA或dszC的菌株多数被归类为红平红球菌（R. erythropolis）相关类群，且所有此类菌株均具备DBT脱硫能力。对从A土壤、FSL土壤、上述土壤的富集培养物及分离菌株中通过PCR扩增得到的37条dszA序列进行比对，结果显示所有序列均与标准序列（红平红球菌IGTS8菌株的dszA序列）高度相似，且内部保守性较强；所获得的37条序列可划分为3个簇：包含30条序列的第一组与IGTS8序列的同源性至少为98%，包含2条序列的第二组差异略大，包含5条序列的第三组同源性为95%；前两组序列均来自两类土壤与富集培养物（包括分离菌株），而第三组序列仅直接取自污染的A土壤。