Jennert Katrin C B, Tardif Chantal, Young Danielle I, Young Michael
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Penglais SY23 3DD, UK1.
Université de Provence, Marseille, France3.
Microbiology (Reading). 2000 Dec;146 Pt 12:3071-3080. doi: 10.1099/00221287-146-12-3071.
Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CCE:I, is an isoschizomer of MSP:I (HPA:II). Electro-transformation was employed to establish plasmids containing the replication functions of pAMss1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BSU:FI methylase in vivo or MSP:I methylase in vitro. Plasmids based on the pAMss1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10(2) transformants per microg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10(5) and 10(7) transformants per microg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.
尽管人们对细菌纤维小体及其各种蛋白质成分已有很多了解，但目前仍无法评估它们对细菌在纤维素上生长以及体内纤维素分解过程的贡献。为了弥补这一点，作者开发了针对解纤维梭菌ATCC 35319的基因转移技术。首先，利用粪肠球菌供体实现了Tn1545的转移。其次，也实现了IncP介导的从大肠杆菌供体质粒的接合转移。在这两种情况下，接合子的产量都很低，这可能受到寡营养型解纤维梭菌与富营养型供体共培养时必须采用的次优生长条件的限制。在解纤维梭菌的粗提物中检测到一种限制性内切酶。这种酶名为CCE:I，是MSP:I（HPA:II）的同裂酶。采用电转化法在解纤维梭菌中构建了含有pAMss1（粪肠球菌）、pIM13（枯草芽孢杆菌）、pCB102（丁酸梭菌）、pIP404（产气荚膜梭菌）和pWV01（乳酸乳球菌乳脂亚种）复制功能的质粒。只有当DNA在5'-CCGG-3'序列的外部C上使用BSU:FI甲基转移酶在体内或MSP:I甲基转移酶在体外进行适当甲基化时，才能获得转化子。基于pAMss1和pIM13复制子的质粒比基于pCB102复制子的质粒更稳定地维持。在固体培养基上选择转化子导致明显的转化效率较低（约每微克DNA有10²个转化子），这可能部分反映了该生物体的低平板效率。在液体培养基中选择转化子导致转化子的明显产量较高（每微克DNA有10⁵至10⁷个转化子）。这里开发的方法将为体内各种纤维小体成分的功能分析铺平道路。
