碳源對大腸桿菌細胞分裂基因 ( ftsZ, minC, minD ) 的影響 - 生長速率與大腸桿菌細胞分裂之關係

6.3 生長速率與大腸桿菌細胞分裂之關係

6.3.1 碳源對大腸桿菌細胞分裂基因 ( ftsZ, minC, minD ) 的影響

由Fig 8 可發現在批次培養下 ftsZ 基因表現是以葡萄糖為碳源時較高，由於 FtsZ 具有 GTPase 活性，會水解 GTP 產生 GDP 與 Pi 將 FtsZ 單體聚合成 Z ring，先前文獻也提出 FtsZ 會受能量狀態調控（Rueda et al., 2003），由

ftsZ 表現量較高，此外菌體分裂速度也在葡萄糖為碳源時分裂較快，此現象與 ftsZ 基因表現較高相一致。 minD 基因的表現也是以葡萄糖為碳源時較高，由 於 MinD 具有 ATPase 活性，會水解 ATP 形成雙聚體分子後與細胞膜內側結合以抑制 Z ring 形成，因此 MinD 也受能量影響（Lackner et al., 2003）；由上述結果可知在葡萄糖為碳源時，大腸桿菌體內 ftsZ 與 minD 等細胞分裂基因表現量都較高，而 ATP 含量與 ATP/ADP 比例也比以醋酸為碳源時來得高，顯示 ftsZ 與 minD 等基因的表現受能量所調控，也證實細胞分裂會受能量狀態影響。因 minC 基因是以醋酸為碳源時表現量較高，已知生長速率上升時，菌體的直徑大小會呈正比增加（Frederick et al, 1996），此外 Lackner 等學者說明 minC 基因的表現會受細胞大小影響（Lackner et al., 2003），在本實驗中觀察到以醋酸為碳源的菌體大小比葡萄糖為碳源時來得小（Fig 10），我們推測若細胞兩端的間隔愈大，相對地 minC 基因表現量隨之減少，因此以葡萄糖為碳源時 minC 基因表現量較低。

6.3.2 ppGpp 對大腸桿菌細胞分裂基因（ftsZ, minC, minD）與菌體型態之影響

當大腸桿菌處於營養素貧乏時，例如胺基酸、碳源或磷酸缺乏，菌體內會立刻停止許多 RNA 的轉錄，先前文獻提出 ppGpp 會影響細胞分裂，推測菌體處於靜止生長期（stationary growth phase）時， ppGpp 會刺激 rpoS 基因轉錄以增加 σ^S（sigma factor）合成，σ^S 進而活化 ftsQAZ 操縱子的 ftsQ1p 啟動子以促進其轉錄，因此在菌體靜止生長期時， ppGpp 會正向調控細胞分裂

（Joseleau-Petit et al., 1999）。

在本論文批次培養中，探討 ppGpp 對細胞分裂基因之研究皆是收取對數生長期的菌體，立即進行碳源貧乏培養後加以分析。我們也觀察到先前文獻所提出

之結果，發現 ppGpp 會促進 ftsZ 基因表現（Fig 9）（Joseleau-Petit et al., 1999），這是由於 ftsQAZ 操縱子的上游有六個啟動子，分別為 ftsQ1p、ftsQ2p、

ftsAp、ftsZ2p、ftsZ3p 與 ftsZ4p ，這些啟動子個別受到不同因子調控，其中 ftsQ2p 啟動子在對數生長期時大量表現，而 ftsQ1p 啟動子則在靜止生長期大量 表現，可知不同生長時期會活化不同的啟動子表現，而使得 ftsZ 基因有不同的 表現量（Navarro et al., 1998）。在菌體進行碳源貧乏培養時菌體隨即誘發 ppGpp 合成， ppGpp 會活化 ftsQ1p 啟動子以增加 ftsZ 基因表現，並且使得 Z ring 形成的數量增加，而長絲狀的菌體型態也擁有較多的 Z ring ，因此我們由 Fig 11 的菌體型態也得知碳源貧乏時會使菌體呈現長絲狀，此一推論與 Khattar 學者所提出長絲狀細胞 Z ring 形成較多的觀點一致（Khattar et al., 1997），也得知 ppGpp 不會抑制細胞隔板的形成，相反地會增加細胞隔板的形成（Schreiber et al., 1995）。

此外也發現當菌體完全缺乏 ppGpp 時會使 minC 與 minD 等基因表現量上升（Fig 9），表示 ppGpp 對 minC 與 minD 基因進行負向調控，已知 ppGpp 含量隨生長速率上升而下降，由此推測菌體在生長緩慢狀態下， ppGpp 含量較高而降低 minC 與 minD 基因的表現，也表示 Z ring 的抑制者濃度下降，而使得 Z ring 可在細胞內形成的機率提高，因此也證實上述論點 ppGpp 會使得 Z ring 的產生增加，並且由 Fig 11 觀察到 ppGpp 會使菌體型態呈現長絲狀。

由細菌型態來觀察，不論是大腸桿菌野生株或 △relA △spoT 雙突變株中，

菌體的型態都是短桿狀，但菌體大小會隨碳源不同而改變，這是由於菌體大小會隨生長速率加快而增加，生長速率愈快時生物質量（biomass）愈多，因此造成菌體大小產生差異，可知生長速率與細胞質量兩者間呈現正比的關係（Frederick et al., 1996）。我們發現在批次培養條件下，大腸桿菌野生株與 △relA △spoT

△relA △spoT 雙突變株的菌體大小又比野生株來得大些，由 Table 2 可知

△relA △spoT 雙突變株中由於缺乏 ppGpp 合成，使得細胞生長速率變得略快，

且細胞大小會隨生長速率加快而增加，因此觀察到 △relA △spoT 雙突變株的細胞大小較野生株大。

另外也觀察到碳源貧乏的環境會使大腸桿菌菌體型態趨向長絲狀，並呈現鍊狀結構（chain-formatiom）（Fig 11），推測原因可能是由於細胞停止生長時

（growth arrest），也同時停止巨分子之合成，使得細胞分裂趨於緩慢而呈現長絲狀的型態（Chang et al., 2002）。同時菌體型態也可能與細胞分裂基因的表現有關，由Fig 11 觀察到 △relA △spoT 雙突變株經碳源貧乏培養後， ftsZ 基因表 現降低，而 minC 與 minD 基因則上升，細胞隔板無法形成使得細胞呈現長絲狀，也因此在Fig11 觀察到當菌體完全缺乏 ppGpp 時，其型態呈現長絲狀的情形更為明顯。

在Fig 10A 與 10C 中，我們發現 starvation 時菌體誘發 ppGpp 產生會影響菌體型態，推測 ppGpp 透 σ^S 或 RpoS regulon 直接或間接地影響了特定蛋白質表現使細菌型態改變（Xiao et al., 1991）。 Khattar 等學者發現與細胞分裂有關基因之一 ftsW 的調控是 relA -depedent，且 FtsW 與細胞延長（elongation）以及維持桿狀型態有密切關係，並會幫助 Z ring 形成（Khattar et al., 1997），由此可知 ppGpp 有可能透過影響細胞分裂基因的表現而改變細胞大小以及菌體型態，推測 ppGpp 雖會抑制蛋白質的合成卻不會抑制阻止隔板形成，推測長絲狀細胞中可能存在不只一個以上的 Z ring ，但缺乏分裂的能力，另外也觀察到 ppGpp 突變株細胞有形成鍊狀的型態，這種改變可能與 Z ring 形成有多寡有關。

Table 1. Effect of carbon source on energy state of E. coli in batch culture.

carbon source (2.25 mM)

generation time (min)

ATP

(pmol/ 10⁷ cell)

ADP

(pmol/ 10⁷ cell)

ATP/ADP ratio acetate 72 ± 3 07.4 ± 1.1 03.1 ± 1.1 2.4 ± 0.3 glucose 50 ± 2 64.8 ± 1.7 17.1 ± 1.7 3.8 ± 0.6 glycerol 60 ± 4 31.7 ± 1.4 05.1 ± 1.4 6.2 ± 0.4 succinate 65 ± 3 22.5 ± 0.9 03.1 ± 0.9 7.3 ± 0.7

Table 2. Effect of starvation and ppGpp on energy state of E. coli in batch culture.

strains carbon source (2.25 mM)

generation time (min)

ATP^a (pmol/ 10⁷ cell)

ADP^a (pmol/ 10⁷ cell)

ATP/ADP ratio^a acetate 72 ± 3 7.1 ± 0.5 2.0 ± 0.5 3.5 ± 0.4 K12 glucose 50 ± 2 68.3 ± 0.8 12.6 ± 0.8 5.4 ± 0.6

acetate 57 ± 5 47.3 ± 1.1 12.4 ± 1.1 3.8 ± 0.7 K12 △relA

△spoT glucose 46 ± 3 49.3 ± 0.9 9.3 ± 0.9 5.3 ± 0.6

a. Wild-type K12 and relA spoT double mutant separately incubated in acetate or glucose minimal medium at 37 to OD600 0.4~0.45, then starvation for 30 ℃ mins to collect cell pellets for detection of ATP and ADP concentrations.

Table 3. The changes of ATP generating gene expression in various carbon medium.

carbon source Acetate Glucose Glycerol Succinate concentration

(mM) 2.25 40 2.25 40 2.25 40 2.25 40

pgk 3 25 1 3 20 50 6 15

pyk 15 6 9 1 37 94 11 65

acKA 21 3 31 1 78 92 22 48

sucCD 122 140 23 92 33 63 114 22

atpI 43 69 55 45 60 77 40 48

The boldface indicated the lower or higher expression of ATP generating genes. pgk: phosphoglycerate kinase, pyk: pyruvate kinase, ackA:

acetate kinase, sucCD: succinate thiokinase, atpI: ATPase

Fig 1. Effect of carbon source on intracellular ATP of E. coli in batch culture. The E. coli. cells were cultured in 2.25 mM minimal medium with different carbon sources at 37 . ace: acetate. glc: glucose. gly: glycerol. suc℃ : succinate.

0 20 40 60 80

0 20 40 60 80 100

Intracellular ATP (pmol/ 107 cell )

Generation Time (min) glc

gly suc

ace

: Ace Glc Gly Suc

ATP/ADP : 2.4 3.8 6.2 7.3

Fig 2. Effect of carbon source on DNA superocoiling of E. coli in batch culture. Chloroquin-agarose gel electrophoresis showing differences in DNA supercoiling of pBR322 from strains grown in different carbon sources. The wild-type K12 strain were cultured in 2.25mM acetate ( lane 1 ), glucose ( lane 2 ) , glycerol ( lane 3 ) and succinate ( lane 4 ) minimal medium.

carbon sources

Strain : K12 △relA K12

△spoT

carbon : Glc Ace Glc Ace ATP/ADP : 5.3 3.8 5.4 3.5

Fig 3. Effect of starvation and ppGpp on DNA superocoiling at different carbon sources in E. coli in batch culture. The relA spoT double deletion mutant ( lane1 and 2 ) and wild-type K12 strain ( lane 3 and 4 ) were cultured in 2.25mM glucose or acetate minimal medium, then starvation for 30 mins.

( A )

Fig 4. Effect of carbon source and growth rate on the expressions of topoisomerase genes ( gyrA, gyrB, topA ) in E. coli. The wild-type K12 strain was cultured in carbon-limited chemostates at 37 and specific growth ℃ rates were 0.24/h or 0.96/h. （A）Northern blot analysis of gyrA, gyrB and topA.

（B）The quantitative northern blot analysis of gyrA, gyrB and topA. Indicated

glucose acetate 0.96

relative gene expression level

Ace

Fig 5. Effect of carbon source on the expressions of ATP generating genes ( pgk, pyk, ackA, sucCD, atpI ) in E. coli. （A）Northern blot analysis of pgk, pyk, ackA, sucCD and atpI. （B）The internal controls of total RNA. The upper bands were 23S rRNA, and the under bands were 16S rRNA. （I）pgk,

（II）pyk, （III）ackA, （IV）sucCD,（V）atpI.

（A）

pgk pyk ackA sucCD atpI

2.25mM Ace

pgk pyk ackA sucCD atpI

40mM Ace 40mM Glc 40mM Gly 40mM Suc

Fig 6. The quantitative northern blot analysis of pgk, pyk, ackA, sucCD and atpI. The carbon sources were used in acetate, glucose, glycerol and succinate minimal medium at 37 . The different carbon concentrations were used in 2.25 ℃ mM (A) or 40 mM (B). Indicated were calculated mRNA profiles as determined by

relative gene expression level relative gene expression level

Fig 7. The metabolic pathways of glycolysis, tricarboxylic acid cycle and electron transfer chain. The green arrows and letters represented glycerol as carbon source entry into glycolysis. The abbreviations were shown. aceyl-P:

acetyl-phosphate, BPG: bisphosphoglycerate, DHAP: dihydroxyacetone phosphate, G3P: glycerate-3-phosphate, OAA: oxaloacetate. 3PG: 3-phosphoglycerate, PEP:

phosphoenolpyruvate, α-KG: α-ketoglutarate, pgk: phosphoglycerate kinase. The ATP generating genes were shown pyk: pyruvate kinase, ackA: acetate kinase, sucCD: succinate thiokinase, atpI: ATPase

G3P BPG 3PG

（A）

Fig 8. Effect of carbon source on the expressions of cell division genes（ftsZ, minC, minD） of E. coli. The wild-type K12 strain were cultured in 40 mM glucose or acetate minimal medium. （A）Northern blot analysis of ftsZ, minC and minD. （B）

The quantitative northern blot analysis of ftsZ, minC and minD.

minD

relative gene expression level

（A）

Fig 9. Effect of starvation and ppGpp on the expressions of cell division genes（ftsZ, minC, minD）at different carbon sources in E. coli. The relA spoT double deletion mutant ( lane1 and 2 ) and wild-type K12 strain ( lane 3 and 4 ) were cultured in 40 mM glucose or acetate minimal medium, then starvation for 30 mins. （A）Northern blot analysis of ftsZ, minC and minD. （B）The quantitative northern blot analysis of ftsZ, minC and minD.

relative gene expression level

△relA△spoT 40 mM Ace

△relA△spoT 40 mM Glc

（A）K12 in acetate minimal medium （B ） △relA △spoT mutant in minimal medium

（C）K12 in glucose minimal medium （D ） △relA △spoT mutant in minimal medium

Fig 10. Morphology of wild-type K12 and ppGpp mutant under different carbon sources in minimal medium. Cells were stained with gram’s stain and observed by light microscopy using an oil immersion objective. Wild-type K12 and relA spoT mutant were grown in acetate minimal medium (A) (B), and grown in glucose minimal medium (C) (D). The bar represented 20 µm.

（A ） K12 starved in acetate minimal medium

（B ） △relA △spoT mutant starved in acetate minimal medium

（C ） K12 starved in glucose minimal medium

（D ） △relA △spoT mutant starved in glucose minimal medium

Fig 11. Morphology of wild-type K12 and relA spoT mutant in carbon-starved minimal medium. Cells were stained with gram’s stain and observed by light microscopy using an oil immersion objective. Wild-type K12 and relA spoT mutant were starved in acetate minimal medium (A) (B), and starved in glucose minimal medium (C) (D). The bar represented 20 µm.

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在文檔中碳源與生長速率對大腸桿菌DNA 超螺旋結構、ATP生合成基因與細胞分裂基因調控之研究 (頁 69-95)