通过构建单一合成卷曲纤维操纵子工程化黏附性细菌

摘要

描述了一种合成操纵子的设计，该操纵子编码纤维素分泌装置以及纤维素结构单体。E. coli底盘细胞过表达这些淀粉样蛋白和黏附性多聚物后，可测得其黏附能力显著增强¹。文中还介绍了可视化和定量黏附能力的简便方法。

摘要

本文所述方法通过将最少数量的卷曲菌毛基因置于强效且可被金属过量诱导的启动子控制下，重新设计大肠杆菌（E. coli）的黏附特性，并对细菌黏附能力的提升进行可视化与定量分析。该方法应用了合成生物学中抽象化与标准化的适当工程学原理，最终构建出Biobrick标准组件BBa_K540000（iGEM 2011年度最佳新型Biobrick器件，经工程化设计）。

第一步是设计合成操纵子，使其能够响应金属离子并促进纤维素的过量表达，从而增强野生型菌株的黏附能力。原始的纤维素操纵子经过计算机模拟（in silico）改造，以优化其转录和翻译信号，并摆脱纤维素表达的“天然”调控机制。该方法成功验证了我们目前对纤维素生成机制的理解。此外，通过将内源性的复杂启动子（已鉴定出超过10种转录调控因子）替换为简单的金属离子调控启动子，使纤维素的表达调控变得更加简便易控。

第二步包括通过实施简单方法对黏附能力进行定性和定量评估。这些方法适用于多种黏附性细菌，而不论其生物膜形成过程中涉及的生物结构如何。在24孔聚苯乙烯板中进行的黏附试验，可在结晶紫染色后快速初步观察细菌生物膜。该定性试验可通过量化黏附百分比进一步优化。这种方法非常简便，且比仅使用结晶紫染色的方法更为准确¹，具有良好的重复性和可重现性。通过荧光显微镜或激光共聚焦显微镜观察标记GFP的细菌在玻璃片上的情况，可直接观察该现象，从而加强24孔板试验所得结果的可信度。

引言

细菌对非生物载体的黏附在生物修复、生物催化或微生物燃料电池中起着重要作用。生物修复过程利用微生物降解有机物质，或改变金属的分布（固定化、挥发）或形态。这些有益的微生物活动不仅存在于水生和陆地生态系统中，也出现在为处理工业和生活污水而开发的人工系统中。微生物活动的强度和质量取决于理化因素，同时也受微生物生活方式（自由漂浮或形成生物膜）的影响。生物膜的形成伴随着代谢变化，可通过多种机制增强对杀菌剂的抗性。因此，在大多数生物修复过程中，这一现象将被积极促进。此外，通过基因工程改造Escherichia coli细胞以调控生物膜的形成，已成功应用于将全细胞传感器固定在生物芯片上^2-3。

微生物通过多种机制适应高浓度金属环境，例如吸附到胞外基质组分、激活外排泵或能够将金属富集到细胞内的特异性载体。通过基因工程增强这些细菌活性，可在实验室规模实现对金属污染的高效且低成本处理，尤其适用于如Raghu et al. 2008⁴所述的微量高毒性金属。在这种情况下，细菌修复技术相较于使用离子交换树脂的传统化学方法，具有更强的竞争性和成本优势。作者描述了一种经过基因改造的E. coli底盘菌株，首先通过敲除编码外排泵的基因rcnA，使其丧失将钴排出细胞的能力；随后通过转入一个多拷贝质粒，实现对钴具有优先摄取能力的转运蛋白的过量表达。该菌株在处理放射性废水方面表现出替代离子交换树脂的潜力，但该技术面临的一个关键未解难题是在处理过程结束后如何有效回收被污染的细菌⁴。因此，本研究的目标是构建一种可特异性附着于玻璃或塑料等非生物载体表面的定制菌株。

在已鉴定的革兰氏阴性菌的全部黏附素和黏附性菌毛中，我们选择设计一种能够表达卷曲纤维（curli）的系统。卷曲纤维是直径为2–5 nm的纤细且高度聚集的淀粉样纤维，从菌体表面突出 E. coli 和 沙门氏菌 表面作为非晶态且不溶性的基质 ^5-7纤维蛋白也参与非生物表面的定植以及生物膜的形成 ⁸最近研究表明，卷曲菌毛可结合汞离子 ⁹淀粉样蛋白确实已知对金属离子（如 Cu）具有高亲和力²⁺，Zn²⁺ 和 Fe^{3+ 10}这一特性可能进一步提高对金属污染废水的去污效果。 csg 簇负责产生纤毛纤维，由两个反向转录的操纵子组成（图1）。该 csgB, csgA 和 csgC 基因构成感受操纵子，编码两个卷曲菌毛亚基 CsgA 和 CsgB。CsgC 似乎参与卷曲菌毛生物合成系统中的氧化还原反应，并影响 CsgG 孔道的行为。 ¹¹然而，大多数产生curli的细菌中均不存在csgC，这表明其对应的蛋白质仅对curli的生物合成提供次要水平的调控。为了简化系统，我们选择仅使用最少数量的基因进行研究。

基因csgDEFG操纵子编码对CsgA和CsgB调控及其向细胞表面转运至关重要的蛋白质。CsgD是csgBAC操纵子的转录激活因子，在生物膜形成过程中发挥关键作用，通过调控菌毛素（curli）纤毛及其他生物膜组分（如纤维素¹²）的生成，并抑制鞭毛的合成¹³。CsgE、CsgF和CsgG共同构成外膜中一种特异性的菌毛素分泌装置，主要菌毛素亚基蛋白CsgA以此途径以可溶性蛋白形式分泌至胞外。体内条件下CsgA的聚合依赖于膜结合的成核蛋白CsgB（综述见¹⁴）。已有研究表明，多个双组分系统参与的复杂调控通路可调控菌毛素基因的表达^15-16。这些复杂的调控机制使细菌能够响应环境信号，通过产生菌毛素形成厚实的生物膜，但在工业应用中难以精确控制。为了在工业过程中便于回收负载金属的细菌，确实需要通过明确定义的参数来调控细菌在固体载体上的固定。菌毛素的黏附特性与其淀粉样蛋白性质相关¹⁷，可用于改进生物修复工艺，但仍需构建一种更简单且易于控制的系统。

在这7个基因中 ¹⁸，一组合成纤维素所必需的5个核心基因（csgB 和 csgA 编码纤维单体）并输出（csgE csgF 和 csgG 编码卷曲菌毛分泌复合物）被选中用于构建合成操纵子。为了摆脱卷曲菌毛的“天然”调控，构建了一个包含这5个 csg 由强启动子控制且可被钴过量诱导的基因（图2设计并合成了功能性的合成操纵子。详细描述了编码卷曲纤维（curli）区域的逐步分析过程及功能性合成操纵子的设计流程。阐述了两种用于可视化和定量分析细菌在聚苯乙烯和玻璃表面黏附的方法。

方案

1. Biobrick Design and Synthesis of the Curli Operon

1. Determine the genetic organization and localize the endogenous transcriptional and translational signals of the curli genes. These informations are gathered in specialized databases such as RegulonDB^a or EcoGene^b and completed by a careful reading of pertinent publications. Data management and in silico preanalysis were performed with Clone Manager software^c.
2. Select the pertinent coding sequences. A set of five absolutely required genes for curli synthesis (csgB and csgA encoding fiber monomers) and export (csgE csgF and csgG, encoding the curlin secretion complex) were selected to construct the synthetic operon. Extract the chosen sequences from the data base in FASTA format.
   As the csgGFE cluster is antisense in E. coli genome, convert this sequence into its reverse complement counterpart by using the Clone Manager tools Operations> Process molecule>Invert molecule. Paste the csgEFG sequence behind csgBA by using the function "Ligate" (Clone> Ligate).
3. Add the appropriate promoter. By placing the five selected curli genes under control of the promoter Prcn, curli are predicted to be over-produced in presence of cobalt and nickel. The rcn locus encodes an efflux pump responsible for Ni and Co detoxification (rcnA) and its cognate metallo-regulator (rcnR). RcnR controls the expression of rcnA and its own gene in response to Ni and Co ¹⁹. The rcn sequence comprising rcnR CDS, the whole rcn intergenic region plus the 41 first nucleotides of rcnA was placedin front of the csgBAEFG chimerical sequence (Figure 1, the sequence of the whole construct is provided as supplementary data).
4. Optimize the transcriptional signals. A perfect ribosome binding site (or perfect RBS= AAGGAGGTATATA) was added in front of the first ATG of the csgBA DNA sequence. A second perfect RBS was added in front of the csgEFG sequence. Endogenous RBS for the csgB and csgF and csgG genes were conserved.
5. To fit iGEM standards, the device must be flanked by a standard BioBrick prefix and suffix, containing restriction sites for EcoRI, PstI (prefix) and SpeI and XbaI (suffix). Paste the corresponding sequences at each end of the device^d.
6. Eliminate any EcoRI, PstI, SpeI and XbaI recognition site in the device. To facilitate further assembly process, the BioBrick part itself may not contain any of these restriction sites. In silico restriction analysis of the device revealed one PstI site in the csgA sequence, one PstI site in the rcnR sequence and one EcoRI site in the csgE gene. These sites are respectively located in position 80 (Mut1), 1340 (Mut2) and 1830 (Mut3) of the device sequence (supplementary data) and were modified as follow by silent mutations.
   Mut1 PstI site CTGCAG changed in CGTCTG
   Mut2 PstI site CTGCAG changed in CAGCAG
   Mut3 EcoRI site GAATTC changed in GAATT
7. Run through the translation simulation using the Clone Manager software (Operation>Process molecules>Translate molecules). Use the sequence alignment program BLAST to verify the perfect homology between the wild type curli protein and the proteins encoded by the synthetic operon.
8. Order the synthetic operon. Commercial gene synthesis services are available from numerous companies worldwide, our partner was Genecust (Luxembourg). 4 μg of the artificial operon (3165 bp) were received few weeks later and inserted into pUC57 (pIG2).

2. Visualize and Quantify Adherent Bacteria on Polystyrene

1. Fill each well of a 24-well polystyrene plate with 2 ml of M63 minimal medium (glucose 0.2%) and inoculate each well with 106 cells of an overnight culture. Grow bacteria at 30 °C for 18 to 48 hr without shaking. Each column (4 wells) represents a modality. Add the proper amount of cobalt (25 to 100 mM) and antibiotic (ampicillin 100 μg.L^-1) when needed. The 3 first rows of the 24-well plate are used to quantify the adherent bacteria by averaging the 3 repetitions, the last row left is used to visualize the biofilm.
2. For the 3 first rows of the 24-well plate: for each well, recover the supernatant containing the planktonic cells.
3. Carefully rinse each well with 1 ml of M63 and pool the 1 ml wash with the initial supernatant obtained in 2.2). This pool is referred to as swimming cells (=S).
4. Recover the biofilm in 1 ml of M63 by scraping and pipetting up and down (=B). Vortex 15 sec. Estimate the number of surface-attached and swimming bacteria from the optical density at 600 nm (OD600) to give the adherence percentage corresponding to each modality.
5. The percentage of adherence is calculated by using the formula: Bx100/(3xS+B).
6. Only for the last row of the 24-well plate: discard planktonic cells.
7. Rinse with 1 ml of M63 the biofilm which had developed on the bottom of the plate.
8. Dry the open plate for 1 hr at 80 °C.
9. Add 100 μl of 20% crystal violet for 2 min in each well followed by extensive washes with water to visualize the surface-attached bacteria.
   Three independent assays (plates) have to be performed to ensure reproducibility.

3. Visualize Adherent Bacteria on Glass by Microscopy

1. Get a fluorescent chassis. The E. coli SCC1^e strain which constitutively expresses green fluorescent protein (GFP) with no observable difference from the parent strain MG1655 ²⁰ is a suitable host for the plasmid bearing the synthetic curli operon.
2. Transform SCC1 with the pIG2 plasmid (=synthetic curli operon inserted at the EcoRI/PstI site of the pUC57 plasmid) to obtain the S23 strain. Transform SCC1 with a control plasmid (pUC18) to obtain the control strain S24 ²¹.
3. Grow overnight precultures of the S23 and of the control (S24) strains at 30 °C in M63 medium supplemented with glucose (0.2 %) and ampicillin (100 μg.L^-1).
4. Inoculate 15 ml of the same medium in Petri dishes with 100 μl of the precultures. Add the appropriate concentration of cobalt (i.e. 25 μM) and don't forget the negative control without cobalt. Introduce 3 rectangular glass coverslip in each Petri dish. Incubate overnight at 30 °C without shaking.
5. Remove the coverslip from the Petri dish and carefully drain it. Carefully clean the lower face with a small cotton stuff impregnated with 70 ° ethanol by wiping off every bacterial residue. For confocal observation, clean both upper ends on a few millimeters to allow further fixation of the coverslip.
6. Adherent bacteria can be visualized directly but quickly under fluorescence microscope, but carefully avoid drying of the sample.
7. For confocal observation, deposit the coverslip on a glass slide with the upper face covered by the biofilm placed face up. The biofilm is then covered with a larger coverslip, which is fixed to the glass slide with varnish. Invert the setup so that the biofilm will now be on the lower face.
8. Place the setup under the confocal laser microscope. A right Axioplan2 LSM510 (Zeiss) confocal laser microscope was used at the Platim platform^f.
9. Setting. Excite GFP at 488 nm, and collect the bacterial fluorescence in the range 500 to 600 nm. Use 40x oil immersion objective for acquiring images in laser scanning confocal mode. Scan the overall three-dimensional structures of the biofilms from the solid surface to the interface with the growth medium, using a step of 1 μm.
10. Perform three-dimensional projections with IMARIS software (Bitplane, Zürich, Switzerland). Determine biofilm thickness by the analysis of the average z value along statistical longitudinal sections. Quantification of biofilm biovolumes can be extracted from confocal z-stacks as described elsewhere ²².

^aRegulonDB provides mechanistic information about operon organization and their decomposition into transcription units, promoters and their sigma type, genes and their ribosome binding sites, terminators, binding sites of specific transcriptional regulators,as well as their organization into regulatory phrases. http://regulondb.cs.purdue.edu/index.jsp

^bThe EcoGene database contains updated information about the E. coli K-12 genome and proteome sequences, including extensive gene bibliographies. A major EcoGene focus has been the re-evaluation of translation start sites. http://ecogene.org/

^chttp://www.scied.com/dl_cmp9d.htm

^dhttp://partsregistry.org/wiki/index.php?title=Help:BioBrick_Prefix_and_Suffix

^eGift of Chun Chau Sze, Nan Yang Technical University, Singapore.

^fPlatim microscopic platform UMS3444 BioSciences Gerland - Lyon Sud.

结果

in silico 对与转录和翻译信号优化相关的 E. coli K12 野生型 csg 序列进行注释，从而设计出如图3所示的单一合成卷曲纤维操纵子 Prcn-csg（完整序列见补充数据）。采用方案2和方案3来观察和量化与卷曲纤维产生相关的黏附能力。通过在24孔聚苯乙烯板上进行结晶紫染色（方案2），可快速观察到每个孔底部的生物膜形成情况，结果显示野生型菌株的黏附能力弱于工程菌株，表现为紫色染色强度的差异（图4A）。该定性结果通过定量测定得到进一步支持，定量结果显示工程菌株的黏附率高出1.5倍（图4B）。此外，定量方法的精确性使得能够在钴离子浓度递增条件下检测到黏附能力的显著增强。事实上，在钴离子浓度分别为0 μM、25 μM和50 μM的培养基中，黏附细胞的比例分别达到25%、30%和40%（图4B）。还可通过在载玻片上培养表达GFP标记的细菌并结合显微镜观察来比较黏附能力。落射荧光显微镜观察结果（黑色背景上的绿色细菌）显示，工程菌...

讨论

关键步骤

在该合成生物学方法中，最关键的步骤是基因设计。合成基因的设计必须严谨细致，以确保系统能够高效生产。本研究将编码纤维单体的两个基因，以及编码其分泌系统相关蛋白的三个基因，与一个强效且金属可诱导的启动子组装在一起，构建出一个用于新应用的功能单元：核废液的生物净化。正如预先设计和预测的那样，该装置可显著增强curli的高产量表达，且随着培养基中钴浓度的增加，表达进一步增强。这一成功得益于所选启动子（PrcnA）中包含适当的转录信号，以及在每组curli基因（csgBA 和 csgEFG）前方添加的高效翻译信号（见图3）。此外，在使用多拷贝质粒时，需特别注意参与启动子调控的调控因子的拷贝数。在此，所用启动子（PrcnA）的主要调控蛋白RcnR也由同一质粒提供多个拷贝（图3）。

局限性及可能的改进方案

为确保完全可重复性，黏附实验必须始终使用相同...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
pIG2			含有一个3165 bp EcoRI/PstI片段的pUC57（pMB1 ori，2710 bp），该片段包含合成的Prcn-csgBAEFG操纵子；Ampr
pUC18			多拷贝质粒（pMB1 ori，2686 bp），Ampr
S23			SSC1（= 带GFP标签的MG1655，C.C. Sze惠赠）/pIG2
S24			SSC1/pUC18
CoCl2	Sigma		0.1 M 储存液，室温保存
M63			29
24孔板	Nunc	55429	聚苯乙烯24孔板