Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824
Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824
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DOI：
10.1128/jb.184.13.3586-3597.2002
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发表时间：
2002-07-01
影响因子：
3.2
通讯作者：
Papoutsakis, ET
中科院分区：
文献类型：
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作者：
Harris, LM;Welker, NE;Papoutsakis, ET
克隆乙酰丁酸梭菌ATCC 824 spo0A基因，得到spo0A失活菌株（SKO1）和spo0A过表达菌株[824(pMPSOA)]两个重组菌株。SKO1是通过能够双交叉染色体整合的复制质粒进行靶向基因失活而开发的，这是一种以前从未用于溶剂源性梭菌的技术。SKO1在溶剂形成方面严重不足：与亲本菌株产生的92 mM丙酮和172 mM丁醇相比，SKO1仅产生2 mM丙酮和13 mM丁醇。在固体培养基上生长72 h后，SKO1形成了杆状细胞的长丝，但不能分离。SKO1细胞从未达到亲本菌株典型的肿胀梭状菌形式，也没有形成内生孢子。SKO1中未检测到spo0A转录本，而两个溶剂形成操纵子（aad-ctfA-ctfB和adc，其启动子区域均含有0A盒）的转录有限。菌株824（pMSPOA）比对照菌株[824(pIMP1)]产生更高的丁醇浓度，显著提高spo0A转录水平，并表现出与枯草芽孢杆菌相似的spo0A转录双峰模式。显微镜下的研究表明，与824（pIMP1）和亲本菌株相比，spo0A的过表达增强和加速了孢子的形成。与此一致的是，菌株824（pMSPOA）比质粒对照更早表达了关键溶剂形成基因（aad-ctfA-ctfB和adc）和3个产孢特异性基因（spoIIGA、sigE和sigG）。这些数据支持了Spo0A是一种转录调节因子的假设，它积极控制孢子形成和溶剂的产生。它对溶剂形成的影响是调节孢子形成与溶剂基因表达的平衡行为：它的过度表达显然打破了平衡，有利于加速和增强孢子形成，以牺牲整体溶剂产生为代价。
The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration-a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B. subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.