Mechanisms on the Biosynthetic and Catabolic Diversity of Gordonia Spp. and Their Application in Bioremediation

汙染土壤之生物復育是近年來受到重視的課題，如何利用合適的菌種與復育策略快速降解環境中的有機污染物已成為目前主要的研究方向。戈登氏(Gordonia)放線菌具有分解脂肪族與芳香族碳氫化合物的能力、在環境中穩定存在、細胞壁含有可與油類物質親合的黴菌酸成分、可產生界面活性劑等特徵，是可應用於污染場址整治上極佳的目標微生物。本研究旨在研究戈登氏放線菌之生合成及代謝機制，並探討不同菌種間之表現型及基因型多樣性，以開發可應用於生物復育上之優良戈登氏菌株。計畫中將利用核酸分析法快速偵測及解析戈登氏菌在trehalose lipid、mycolic acid 生合成與脂肪族及芳香族碳氫化合物氧化酵素相關基因，並利用代謝產物分析及酵素活性分析進行不同戈登氏菌種之表現型特性研究，開發多功能之本土戈登氏菌株。此外將針對普遍存在環境中且成分複雜的有機污染物如柴油及重油，利用經表現型及基因型特性篩選後具代表性的戈登氏菌群進行降解試驗。以生物添加方式接種戈登氏菌群至污染土壤中進行孵育，於不同孵育時間內分析污染物濃度變化，同時利用終端限制片段長度多型性分析技術監測汙染土壤中戈登氏菌族群變化，以評估戈登氏菌群在污染土壤中進行生物復育之成效。Bioremediation gains much attention in the removal of organic contaminants from soilsin the recent years. How to inoculate suitable microorganisms to degrade organic pollutantsin a rapid way have become main research topics. Members of the genus Gordonia showabilities to degrade aliphatic and aromatic hydrocarbons, exist stably in soil environment,contain mycolic acid in their cell wall which contributes to their hydrophobicity nature andproduce biosurfactant render them superior microorganisms used in the contaminated soils.The present studies will be undertaken to clarify the mechanisms of their biosynthesis andcatabolism involved in the degradation of hydrocarbons. Besides, the phenotypic andgenotypic diversity will be explored to develop superior Gordonia consortium used in theremediation of contaminated soils. The nucleic acid based technology will be used to detectand resolve genes associated with trehalose lipid and mycolic acid production, oxygenase foraliphatic and aromatic hydrocarbon degradation. By adopting cell metabolites analyses andenzyme activity assay the phenotypic characteristics of various Gordonia species will beinvestigated systematically. The superior Gordonia strains with multiple functions will beused as inoculants in the diesel oil or heavy oil contaminated soils. During the incubationperiod the concentration of residual oil and Gordonia populations will be monitored toassess the bioremediation efficiency of Gordonia members

Molecular Detection, Classification and Identification of Members of the Genus Gordonia

The importance of the emerging genus Gordonia in industrial and environmental biotechnology is well evidenced from recently increasing publications and patents. But, due to the limitations with isolation or detection techniques, explorations of the valuable Gordonia members are restricted. This motivated us to design genus-specific oligonucleotides primer pairs which can assist in rapid detection of species of the genus Gordonia by means of PCR-specific amplification. Members of the metabolically diverse genus Gordonia were isolated from various biotopes including pristine and polluted sites around Taiwan. Identification, comparison and diversity assessment based on gyrB gene were carried out using a newly developed primer pair for gyrB gene and 16S rRNA gene was also sequenced for comparison. The Gordonia specific 16S rDNA fragment (829 bp or 436 bp) was successfully amplified for all the reference Gordonia species with the designed primer pair G268F/G1096R or G699F/G1134R whereas, no amplification was noted for closely-related species from other genera. The genus specificity was validated with 47 strains including wild type isolates. Preliminary screening of soil samples with Gordonia-specific primers was successful in the rapid detection of the presence of 36 Gordonia wild-type isolates. A 1.2 kb fragment of the gyrB gene of 17 Gordonia strains including type strains were determined by direct sequencing of PCR amplified fragments. Total 25 strains (8 were retrieved from public database) of genus Gordonia form a distinct phyletic line in the gyrB-based tree and are separated from other closely-related species of genera to suborder Corynebacterineae. Sequence similarity of gyrB sequence from twelve Gordonia type strains ranged from 79.3% to 97.2%, corresponding to 270-41 nucleotide differences while there was only 0.3-3.8% difference in 16S rRNA gene sequence similarity at the interspecies level. Phylogenetic analysis based on gyrB gene is consistent with those of 16S rDNA tree and provided a better discrimination within the species of Gordonia compared to 16S rRNA gene. The present study demonstrates that gyrB gene analysis will aid in describing novel species belonging to the genus Gordonia. The detection and analyses of Gordonia populations within complex microbial communities were achieved precisely and rapidly by PCR-DGGE method and provide better assessment of the species diversity of Gordonia in the environment.Gordonia (戈登氏菌)菌屬是近年來逐漸受到環境污染處理的重視，且廣泛被研究的一屬放線菌。此屬之微生物具有降解多環類芳香族碳氫化合物的特性，在生物復育上扮演重要角色。篩選Gordonia分離株不但可開發新放線菌資源，並且了解此類放線菌之分布及在生態中的角色，此外研究Gordonia屬內菌種之親緣關係，有助於了解各菌種之演化地位，並提供土壤中可靠且快速之鑑定結果。本研究為加速環境中Gordonia菌種之篩選，利用16S rRNA基因作為標的，設計Gordonia菌屬特異性引子，並配合聚合酶連鎖反應(PCR)之快速偵測技術鑑定此類放線菌。此外為提高對Gordonia 16S rDNA序列相似度極高菌種間(大於99%)之鑑別能力，本研究首度設計針對Gordonia放線菌gyrB基因之PCR增殖及定序引子，探討演化標誌基因16S rRNA與gyrB基因在Gordonia菌種間與不同分離株間之差異性。研究結果顯示，利用G268F/G1096R及G699F/G1134R兩組Gordonia菌屬特異性引子，可有效區別Gordonia菌屬與其他放線菌菌屬，並且利用此偵測技術已從台灣分離出36株Gordonia分離株。經多序列分析比對軟體計算並比較gyrB基因序列，可知Gordonia菌屬與7種鄰近放線菌的gyrB基因序列相似度介於72.8-82.1%，而在16S rDNA序列相似度則介於90.0-95.4%，gyrB基因可明顯區分Gordonia菌屬與其他屬之放線菌。在Gordonia菌種間之差異研究中則發現12種Gordonia gyrB基因序列相似度介於79.3%-97.2%，16S rDNA的序列相似度為96.2%-99.7%，可知Gordonia放線菌的16S rDNA保留性極高，較難利用此基因序列進行菌種間之區分。gyrB基因之變異程度無論是在菌屬、菌種甚至各分離株間均較16S rRNA基因大。本研究之結果將可利用gyrB基因作為Gordonia菌種快速鑑定之依據，若兩菌株之16S rDNA序列相似度大於99.7%、gyrB基因相似度大於97.2%，則判定兩菌株為相同菌種。本研究利用上述兩基因序列相似度初步判定4株本土Gordonia新菌種之分類地位，其中分離株CC-AB07經由生理生化特性及化學組成分析，已確認為本土Gordonia新菌種。此外利用菌屬特異性引子配合變性梯度凝膠電泳(DGGE)，成功應用至泡沫污水中Gordonia族群之分析，未來將探討Gordonia在環境中之族群多樣性。摘要 I Abstract III 目錄 V 表次 VII 圖次 VIII 前言 1 前人研究 4 一、 細菌之分類與Gordonia菌屬之分類現況 4 (一) 細菌之分類歷程 4 (二) 細菌之分類現況、鑑定及命名 6 (三) Gordonia菌屬之分類現況 9 二、 細菌分類學上之特徵與研究方法 10 (一) 細菌分類學上之特徵 11 (二) 數值分類 13 三、 細菌之分子偵測、分子演化與種系發生 18 (一) 細菌之分子偵測 18 (二) 細菌之分子演化與種系發生 19 (三) 種系發生樹之重建方法 23 (四) 種系發生樹之可靠性評估 30 四、 Gordonia菌屬放線菌之特性及其在生物復育應用之潛力 33 (一) Gordonia菌屬的分布、代謝及生化合成之多樣性 33 (二) Gordonia菌屬對碳氫化合物降解及脫硫之機制 35 (三) 細菌應用於污染物之降解 37 (四) Gordonia菌屬在生物復育之潛在應用性 38 五、 土壤微生物之多樣性與其研究策略 42 (一) 土壤微生物多樣性 42 (二) 微生物多樣性研究的基礎：分子親緣分析 47 (三) 利用培養法的研究策略 48 (四) 利用不須培養的研究策略 51 研究目的 56 材料與方法 58 一、 試驗菌株及培養條件 58 (一) 參考菌株 58 (二) 分離菌株 58 (三) 菌株培養及保存條件 58 二、 Gordonia菌屬之快速偵測 63 (一) 細菌染色體DNA分離純化 63 (二) Gordonia菌屬特異性引子設計 66 (三) Gordonia菌屬特異性引子靈敏度測試 69 (四) Gordonia菌屬特異性引子專一性測試 70 (五) 利用環境分離菌株測試Gordonia菌屬特異性引子 71 三、 Gordonia菌種及本土分離株之分類鑑定 74 (一) 16S rDNA序列分析 74 (二) gyrB基因序列分析 78 (三) 基因序列相似度分析及種系發生樹之重建 80 (四) 生理生化特性分析 81 (五) 分子指紋圖譜之建立 89 (六) 抗生物質感受性試驗 89 四、 環境中Gordonia菌種多樣性分析 91 (一) 樣品來源及採集 91 (二) 藉由培養之研究策略 92 (三) 不須培養之研究策略 93 結果與討論 97 一、 Gordonia菌屬快速偵測方法之建立 97 (一) Gordonia菌屬特異性引子設計 97 (二) Gordonia菌屬偵測方法之靈敏度及專一性測試 104 (三) 環境中Gordonia分離株之篩選 115 二、 Gordonia菌種及本土分離株之分類鑑定 120 (一) 分子親緣分析-16S rRNA基因序列分析 120 (二) 分子親緣分析-gyrB基因序列分析 130 (三) 分子序列差異性比較與本土分離株之鑑定 146 (四) 本土Gordonia分離株BOX-PCR基因型分析 162 (五) 本土Gordonia新菌種之多相策略分析 167 (六) Gordonia菌株與鄰近放線菌對抗生物質之感受性試驗 169 三、 Gordonia菌種多樣性研究方法之建立 176 (一) 利用培養法篩選分離環境中的Gordonia分離株 176 (二) 利用PCR-DGGE圖譜分析環境中的Gordonia族群 178 結論 191 參考文獻 19

Rapid detection and identification of the metabolically diverse genus Gordonia by 16S rRNA-gene-targeted genus-specific primers

The importance of the emerging genus Gordonia in industrial and environmental biotechnology is evidenced by the recent increase in associated publications and patents. But, investigations into potentially valuable Gordonia members are restricted by the limitations of current isolation and detection techniques. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Gordonia by means of PCR-specific amplification. The Gordonia-specific 16S rDNA fragment (829 bp) was successfully amplified for all the reference Gordonia species with the designed primer pair G268F/G1096R. No amplification was noted for closely related species from other genera. The genus specificity was validated with 47 strains including wild-type isolates. Interestingly, two strains assigned earlier as Gordonia nitida (DSM 777) and Gordonia rubripertinctus (ATCC 21930) failed to produce a Gordonia-specific fragment with this primer pair. Further analysis of these two isolates based on 16S rDNA sequencing and phylogenetic analysis classified them to the genus Rhodococcus. Preliminary screening of soil samples with the Gordonia-specific primers was successful in terms of the rapid detection of nine Gordonia wild-type isolates. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved

Rapid detection and identification of the free-living nitrogen fixing genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers

The modern agricultural practice utilizing plant growth promoting rhizobacteria (PGPR) has brought great benefits in the promotion of crop growth. Among PGPR, Azospirillum is considered as an important genus which is not only closely-associated with plants but also shows potential in the degradation of organic contaminants. However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification. The sensitivity and specificity of the newly designed primer pair Azo494-F/Azo756-R were tested against 12 Azospirillum type strains and other closely-related genera. The Azospirillum-specific 16S rRNA gene fragment (263 bp) was successfully amplified for all the reference Azospirillum species with the designed primer pair. No amplification was noted for closely-related species from other genera. The genus specificity was validated with 18 strains including environmental isolates. Interestingly, two strains assigned earlier as Azospirillum amazonense (DSM 2787(T)) and Azospirillum irakense (DSM 11586(T)) failed to produce an Azospirillum-specific fragment with this primer pair. Further phylogenetic analysis of these two isolates based on 16S rRNA gene sequences shows that these two strains might belong to other genera rather than Azospirillum. Preliminary screening of isolates and soil samples with the Azospirillum-specific primers was successful in terms of the rapid detection of Azospirillum isolates. By using real-time PCR analysis the minimum limit of Azospirillum detection was 10(2) CFU g(-1) in the seeded soil sample. The newly designed primers can be used to study the diversity of Azospirillum in ecosystems and aid in the exploration of novel species

鑑定固氮螺旋菌之引子對、套組及其鑑定方法

一種用於鑑定固氮螺旋菌屬之引子對，其係選自下列引子對及彼等之退化性引子(degenerate primer)以進行鑑定：正向引子，其具有5’-GGCCYGWTYAGTCAGRAGTG-3’之序列；反向引子，其具有5’-GCTAGACGYYGGGGTGCATGCACTT-3’之序列；並藉由添加相關試劑即可應用於微生物製劑，如生物肥料產品中固氮螺旋菌屬之快速檢測、碳氫化合物或重金屬汙染土壤生物復育可行性評估之依據

鑑定固氮螺旋菌之引子對、套組及其鑑定方法

一種用於鑑定固氮螺旋菌屬之引子對，其係選自下列引子對及彼等之退化性引子(degenerate primer)以進行鑑定：正向引子，其具有5’-GGCCYGWTYAGTCAGRAGTG-3’之序列；反向引子，其具有5’-GCTAGACGYYGGGGTGCATGCACTT-3’之序列；並藉由添加相關試劑即可應用於微生物製劑，如生物肥料產品中固氮螺旋菌屬之快速檢測、碳氫化合物或重金屬汙染土壤生物復育可行性評估之依據

The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-A174

Burkholderia cepacia CC-Al74 with a high ability for solubilizing tricalcium phosphate (TCP) was used to study the P-solubilization mechanism. We collected filtrates able to solubilize TCP from the cultures of strain CC-Al74 and demonstrated that the P-solubilization increased from 0 mu g ml(-1) to 200 mu g ml(-1) during exponential growth, when the pH decreased from 8 to 3. HPLC-analysis revealed that the solubilization of TCP was mainly caused by the release of 16.3 mM gluconic acid. At this concentration, gluconic acid was capable of solubilizing 376 mu g ml(-1) of TCP whereas water at pH 3 only solubilized 35 mu g ml(-1). The difference is due to the final H+ concentrations which were 13.5 mM and 1 mM in 16.3 mM gluconic acid and deionized water, respectively at pH 3. (c) 2005 Elsevier Ltd. All rights reserved

Functional diversity and dominant populations of bacteria in banana plantation soils as influenced by long-term organic and conventional farming

A three-year field study (2012–2014) of functional diversity and dominant populations of bacteria in banana plantation soils after long-term organic and conventional farming was conducted. The pH and water content were significantly higher in 17-year and 7-year organic farming soils than that in 39-year conventional farming soils, while organic matter content and viable numbers of four bacterial groups were also slightly higher. Both organic farming soils harbored diverse functional potential and metabolic activity of fast-growing heterotrophs, as can be seen from higher substrate richness, average well color development value and diversity index. Distinct carbon source utilization patterns between organic and conventional farming soils were demonstrated through principal component analysis. A limited number of heterotrophs were isolated during 2012–2013 sampling period to obtain culturable populations, and 16S rDNA sequence analyses assigned them mainly to the phylum Firmicutes. As for 2014-collected samples more diverse uncultured phyla were revealed by clone library-based method, and members belonging to the phylum Proteobacteria were more prevalent in banana plantation soils. The predominance of copiotrophic members affiliated with β-proteobacteria and fewer clones belonging to oligotrophic Acidobacteria and α-Proteobacteria especially in 7-year organic farming soils may contribute to higher functional diversity of fast-growing heterotrophs. The present study addressed functional diversity and dominant populations of bacteria in banana plantation soils and provided insight into the influences caused by long-term organic and conventional farming

Identification and kinetic characteristics of an indigenous diesel-degrading Gordonia alkanivorans strain

An indigenous strain Gordonia alkanivorans CC-JG39 was isolated from oil-contaminated sludge of a local gas station located in central Taiwan. The bacterial isolate was able to grow on diesel-containing Bushnell-Haas medium and also tolerate various chemical additives frequently used in petroleum products (e.g. BETX, methyl-tert-butyl ether, and naphthalene). Kinetics of diesel-limited cell growth and biodegradation of diesel followed a Monod-type model. The kinetic constants for cell growth (mu(max) and K(S,G)) were 0.158 h(-1) and 3196 mg/l, respectively, while those for biodegradation of diesel (v(max, diesel) and K(S,D)) were 3.59 mg/h/mg cell and 2874 mg/l, respectively. G. alkanivorans CC-JG39 produced extracellular surface-active material, leading to a low surface tension of nearly 33 mN/m. The CC-JG39 strain also possessed the ability to float towards the oil/water interface. These features might play some roles in enhancing the mass transfer efficiency between oil substrate and the bacterial cells. Therefore, G. alkanivorans CC-JG39 may have potential applications in bioremediation of oil pollution sites