Gene transfer to Clostridium cellulolyticum ATCC 35319
Gene transfer to Clostridium cellulolyticum ATCC 35319
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DOI：
10.1099/00221287-146-12-3071
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发表时间：
2000-12-01
期刊：
影响因子：
2.8
通讯作者：
Young, M
中科院分区：
文献类型：
--
作者：
Jennert, KCB;Tardif, C;Young, M
尽管人们对细菌纤维素体及其各种蛋白质成分了解很多，但目前还无法评估它们对纤维素上细菌生长和体内纤维素分解过程的贡献。为了解决这一问题，作者开发了溶纤维梭菌ATCC 35319的基因转移技术。首先，已使用粪肠球菌供体获得Tn 1545的转移。其次，IncP介导的接合动员质粒从大肠杆菌供体也已实现。在这两种情况下，接合转移体的产量都很低，可能是受次优生长条件的限制，而次优生长条件必须用于寡营养C.纤维素酶与共养供体的混合物。在C.纤维素分解菌这种酶被命名为CceI，是MspI（HpaII）的异构体。电转化用于建立含有pAM β 1复制功能的质粒（En.粪球菌）、pIM 13（枯草芽孢杆菌）、pCB 102（丁酸梭菌）、pIP 404（产气荚膜梭菌）和pWV 01（乳酸乳球菌乳酸亚种）。cremoris）在C.纤维素分解菌只有当DNA在序列5 ′-CCGG-3 ′的外部C上使用体内BsuFI甲基化酶或体外MspI甲基化酶适当甲基化时，才获得转化体。基于pAM β 1和pIM 13复制子的质粒比基于pCB 102复制子的质粒更稳定地维持。在固体培养基上选择转化体导致低的表观转化效率（约10%）。10（2）转化体/mug DNA），这可能部分反映了生物体的低平板接种效率。在液体培养基中选择转化体导致更高的转化体表观产量（每μ g DNA 10（5）至10（7）个转化体）。这里开发的方法将铺平道路，在体内的各种纤维素酶体成分的功能分析。
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 CceI, is an isoschizomer of MspI (HpaII). Electrotransformation was employed to establish plasmids containing the replication functions of pAM beta1 (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 BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAM beta1 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 mug 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 mug DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.