聚氧乙基醇類界面活性劑對格蘭氏陰性菌 Pseudomonas nitroreducens TX1 之影響：原子力顯微鏡型態與脂肪酸組成變化

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林駿晏(Chun-Yen LIN) 查詢紙本館藏

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論文名稱

聚氧乙基醇類界面活性劑對格蘭氏陰性菌 Pseudomonas nitroreducens TX1 之影響：原子力顯微鏡型態與脂肪酸組成變化
(Effect of ethoxylate surfactants on cell envelope of Gram-negative bacterium Pseudomonas nitroreducens TX1 examined by atomic force microscopy and fatty acid profiling)

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摘要(中)

細菌與界面活性劑的交互作用在許多領域皆有其研究價值，肺囊狀纖維化常見感染菌株綠膿桿菌會利用肺泡產生之界面活性蛋白作為碳源生長、腸道菌群與具有界面活性之膽汁和膳食中之乳化劑不時產生交互作用、環境菌群亦對排放在環境中之界面活性劑進行生物轉化，將其降解為可被再利用之小分子、亦或轉化成更具毒性之化合物。為瞭解細菌與界面活性劑之交互作用，本研究從非離子性界面活性劑對細菌最外層，即細菌外膜之影響開始。研究選用了數個含有聚氧乙基醇鏈的非離子性界面活性劑，如辛基苯酚聚氧乙基醇（octylphenol polyethoxylates, OPEOn）、辛乙烯二醇單十二烷基酯（ octaethylene glycol monododecyl ether）、PEG 400 與 PEG 1000、Tween 60 ＆ 80。本研究使用格蘭氏陰性 Pseudomonas nitroreducens 菌株 TX1 作為模式菌株。它與重要之人類病原 Pseudomonas aeruginosa 在親緣關係上接近，除此之外 TX1菌株已在本研究團隊先前研究得知其能夠快速降解工業界面活性劑 OPEOn。研究中亦使用兩個轉位子突變株 ΔcbrA 與 ΔcbrB 作為材料，此二基因與細菌對碳源之利用和活動力相關，希望能藉此進一步瞭解菌株 TX1 如何利用 OPEOn，與細菌和界面活性劑交互作用之生理學。液態培養時，濁度計與光學顯微發現同濁度下，相較於 OPEOn，生長在含琥珀酸控制組為唯一碳源之 TX1 菌液有較高細胞濃度，且琥珀酸生長的細胞亦會形成 30 ~ 兩三百細胞之聚合體，然而 OPEOn 實驗組菌液內，直至晚對數期（late log phase）依然沒有發現聚合體。在固態、含 OPEOn 培養基上，TX1 菌體對彼此和培養基表面的粘性均增加，明顯的難以從培養基表面將菌落刮除。此現象可能有助於基礎微生物生理學了解界面活性對於綠膿桿菌之感染如何抑制、乳化劑存在時對腸道菌群間的交互作用、或腸道粘膜之貼附作用、以及其對免疫之影響。電子與原子力顯微鏡沒有發現聚氧乙基醇類界面活性劑生長之細胞在表面型態有不同處。然而原子力顯微鏡在細菌大小上的統計顯示，對數生長期 (log phase) 時，相較於琥珀酸控制組， OPEOn 的存在會使菌體變得較短且細。而在靜止期（stationary phase）時，野生株之 TX1 不論培養基，其長度皆會縮短。如此一來，OPEOn 生長之菌體會有更大的表面積對體積比例，從 12 至 14。另外，OPEOn 培養之菌體切面較接近圓形，而琥珀酸之菌體為相對扁平之橢圓形。在野生株與突變株上進行的脂肪酸成分與組成分析使用了六種聚氧乙基醇類界面活性劑，並以唯一碳源或外加葡萄糖之方式培養。分析發現菌株 TX1 生長在聚氧乙基醇類界面活性劑時，其含有 OH 官能基之脂肪酸會增加，例如 10:0 3OH、12:1 3OH、14:0 3OH 。也因此，聚氧乙基醇類界面活性劑使菌株 TX1 之飽和脂肪酸增加，以 OPEOn 來說，其飽和脂肪酸為 48％，而琥珀酸實驗組為 37 % ，此增加應會提高膜之硬度。本研究是第一個發現在 OPEOn 生長之格蘭氏陰性菌體會較琥珀酸生長之菌體來的接近圓形；且此現象與其飽和脂肪酸成分相關，飽和脂肪酸越多，菌體越接近圓形。菌株 TX1 在切面形狀之改變與其表面積對體積比值之增加，可能使細胞與環境之物質交換更迅速。而相應較飽和、較堅硬之細胞膜亦可能為菌株對抗界面活性劑之機制。

摘要(英)

Interaction of surfactants with bacteria has immense implication on various aspects. Pulmonary surfactants were used as carbon sources by Pseudomonas aeruginosa in cystic fibrosis infected patients; enteric microbiota are in constant contact with surface-active bile acid and ingested dietary emulsifiers. Biodegradation of discharged man-made surfactants renders them harmless again, or even more harmful. To gain understanding on bacterial-surfactant interactions, this study begins with effects of non-ionic surfactants on the outer most part of bacteria, the outer membrane. Ethoxylate surfactants chosen were octylphenol polyethoxylate (OPEOn), octaethylene glycol monododecyl, polyethylene glycol (PEG) 400 and 1000, Tween 60 and 80. The model strain used in this study is a Gram-negative bacteria, Pseudomonas nitroreducens strain TX1. It is a close relative to P. aeruginosa, an important human pathogen. Strain TX1 is a highly efficient degrader of industrial surfactants such as octylphenol polyethoxylates, Triton X-100. In addition to the wild type strain, two transposon insertion mutants, ΔcbrA & ΔcbrB, whose functions closely relate to carbon source utilization and motility were used to shed light on how TX1 interact and catabolize OPEOn. In liquid culture, calculation of relation of turbidity and light microscopy cell count found that, at the same turbidity, succinate-grown wildtype cultures had higher cell numbers under light microscopy compared with OPEOn-grown cultures. Coupled with the observation that succinate-grown culture forms aggregate, while that of OPEOn-grown culture did not, it was suggested that surfactant such as OPEOn prevents cells from forming aggregates. On solid media, old colonies that became hyper-adherent were observed when TX1 grows on OPEOn. Cells clings to together, as well as on to the agar surface, rendering it difficult for it to be scraped from the agar surface without leaving messy residues. The hyper-adherent phenomena of TX1 grown on non-ionics surfactant may help elucidate how the Pseudomonas species switch between motile and a sessile, biofilm-forming stage, which is of great bacterial physiological implication regarding pathogenesis of P. aeruginosa in cystic fibrosis infection and the interaction among gut bacteria and that with mucus membrane in the presence of ethoxylate surfactants. To further observe morphology, scanning electron microscopy and atomic force microscopy (AFM) were used. Although not much difference on the cell surface were noticed with both methodologies, cell dimension statistics by AFM showed OPEOn-containing medium resulted in shorter and thinner cells at log phase. In stationary phase, wildtype cells were always shortened in succinate and OPEOn media. As a result, in log phase, surface area to volume ratio of succinate and OPEOn grown cells increase from 12 to 14, respectively, when surfactant is present. An intriguing discovery was that, the OPEOn-grown cells had a rounder cross-section as observed by AFM, while succinate-grown cells are flatter. Since fatty acids were the major constituents of membrane lipids which in turn make up the cell envelope, total cell fatty acid contents and compositions were analyzed for wildtype and the two mutants grown on individual ethoxylate surfactants as sole carbon source or supplemented along side with glucose. It was shown that raise in hydroxylated fatty acids, such as 10:0 3OH, 12:1 3OH, 14:0 3OH was a general finding from strain TX1 grown in the presence of ethoxylate surfactants with or without glucose. As a result, when cell grows on ethoxylate surfactants such as OPEOn, saturated fatty acids in total fatty acid increase to 48%, from 37% of succinate-grown cells, suggesting increase in membrane rigidity. This is the first study to demonstrate higher saturated fatty acid content could be used to correlate with a rounder, rather than flatter Gram-negative bacterial cell cross-section. In conclusion, increase in surface-area-to-volume ratio and a rounder cell cross-section may facilitate material exchange with the extracellular environment, while general increase in saturated fatty acids counteract against the membrane-disrupting action of surfactant by granting rigidity to cell membrane.

關鍵字(中)

★ 原子力顯微鏡
★ Pseudomonas
★ 辛基苯酚聚氧乙基醇
★ 脂肪酸組成
★ Pseudomonas nitroreducens TX1
★ ΔcbrA
★ ΔcbrB
★ 格蘭氏陰性菌
★ 聚氧乙基醇類界面活性劑

關鍵字(英)

★ atomic force microscopy (AFM)
★ Pseudomonas
★ octylphenol polyethoxylates
★ fatty acid profile
★ Pseudomonas nitroreducens TX1
★ ΔcbrA
★ ΔcbrB
★ Gram-negative bacteria
★ ethoxylate surfactants

論文目次

Abstract ii
Acknowledgement xi
1 Introduction 1
1.1 Cell envelope of Gram-negative bacteria 1
- Introduction to cell envelope 1
- Lipids of Gram-negative bacteria 2
1.2 Pseudomonas nitroreducens TX1 3
- Pseudomonas nitroreducens TX1 3
1.3 Ethoxylate surfactants 3
- Effect on bacteria 3
- Octylphenol polyethoxylate, polyethylene glycol and Tweens 5
1.4 Research aims and study outline 7

2 Materials & Methods 9
2.1 Bacterial strain and media 9
- Strains 9
- Media 9
2.2 Light microscopy 12
- Culture and pretreatment 12
- Observation 12
2.3 Scanning Electron Microscopy 13
- Sample preparation 13
2.4 Atomic force microscopy 13
- Sample preparation and observation 13
2.5 Fatty acid profiling 15
- Fatty acid methyl ester preparation 15

3 Results 17
3.1 Growth curve 17
- Strain TX1 17
3.2 Light microscopy 17
- Relation of cell density and turbidity 17
3.3 Scanning electron Microscopy 18
- Surface roughness 18
3.4 Atomic force microscopy 21
- Dimensional changes of cells 21
- Cell aggregation 27
- Virus-like particle/Membrane vesicle 28
3.5 Fatty acids profiling 28
- Fatty acids profiles of TX1 wildtype, mutants and K-12 28
- Individual effects of ethoxylate surfactants 33
- Lipolytic activity and incorporation of fatty acid 36
- The overloading peak 37
- Decreased percentage of 16:1 w9c in presence of glucose 39
3.6 Strains showing hyper-adherence on solid media 40

4 Discussions 41
4.1 Morphological effect 41
- Presence of virus-like particle 41
- Physiological impact of dimensional changes 41
- Cause of cell shape change 42
4.2 Fatty acid change and effect 45
- Increase of hydroxylated fatty acids 45
- Saturation of membrane fatty acid correlates with cell shape 48
- Bacterial response of fatty acid profile to organic compounds 49
- Implication of tuberculostearic acid, if true 51
4.3 Incorporation of fatty acids into cell 52

5 References 53
6 Suggestions 54

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2016-8-31

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