抽动运动功能的可视化和表征的角色的<em> PilG</em>在<em>叶缘焦枯病菌</em
Summary
在这项研究中，纳米微流体流动室被用于可视化和功能上表征叶缘焦枯病菌，导致皮尔斯病在葡萄细菌的抽搐蠕动。
Abstract
叶缘焦枯病菌是革兰氏阴性非鞭 毛的细菌引起的若干植物的经济上重要的疾病。抽搐蠕动提供X. fastidiosa长距离内厂运行和殖民化，手段在十朝贡献致病fastidiosa，X的运动性抽动fastidiosa是由IV型菌毛操作。 叶缘焦枯病菌的IV型菌毛由pilG，在弼CHP操纵子编码的蛋白质趋化性调节器所涉及的信号转导通路调节。为了阐明pilG的角色X的抽动运动fastidiosa，一个pilG缺陷型突变体Xf为 ΔpilG及其互补株XfΔpilG-含有C本地pilG被开发。与时间推移图像记录系统集成的微流控室，观察抽搐蠕动在XfΔpILG，XfΔpilG-C及其野生型菌株。 利用这种记录系统中，其允许聚合的长期的空间和时间的观察，个体细胞，并通过抽动蠕动细菌种群的迁移。X. fastidiosa野生型和互补XfΔpilG-C株显示，微流体流室直接观察到典型的抽动运动特征，而突变XfΔpliG展出抽搐缺陷表型。这项研究表明，pilG有助于X的运动性抽动fastidiosa。微流体流动室被用作用于观察抽动运动的装置。
Introduction
叶缘焦枯病菌是革兰氏阴性非鞭 毛，病原菌引起许多经济上重要的作物疾病，包括皮尔斯在葡萄病（ 葡萄属）1,2，3条。这种细菌是有限的导水木质部船只。葡萄感染引起木质部导管和水分胁迫的结果和营养不足3堵塞。成功定植取决于细菌的从感染的初始位点移动到植物3的其余部分的能力。抽搐蠕动是鞭毛无关细菌移动的装置通过扩展，附件，和极性IV型菌毛4已特征在于X的缩回fastidiosa 5,6,7。
抽搐运动已由激光镊子和原子力显微镜（AFM）8,9,10观察。使用这些技术，叔由N型菌毛IV产生的魔力motilities 淋球菌和P.由FL uorescently标签菌毛和显微镜捕捉他们的动作绿脓杆菌进行了表征。虽然这两种方法有详细的个体细菌的粘合力，该程序是复杂和耗时9,10。微FL uidic室，观察单个细胞的长途迁徙，以及细菌细胞5,6小聚集。这些室被设计成与隔时图像记录系统11,12,13,14一体的板一个微制造纳米通道。微FL uidic室装置用于研究细菌的运动行为和细胞 – 细胞相互作用提供几个优点：（i）其提供了具有多个信道功能的集成平台; （ii）其可以检查运动和单细胞的细菌的纳米级特征的聚合; （三）它允许直接米细菌细胞和时间推移分析icroscopic图像记录，（ⅳ）它提供了一种在微环境的细菌的个体和/或群体的长期的空间和时间的观测; （五）培养基中的信道的流量可以精确控制和培养基（ⅵ）仅有一个非常小的体积（1ml）中，需要对每个实验。
最近，微FL uidic溢流系统已被用于研究在各种微环境14,15,16细菌细胞的行为。的密合性和E的表面附着大肠杆菌 15，X。 fastidiosa 16和敏捷食酸citrulli 14到玻璃表面用微FL uidic室评估。由敏捷食citrulli的IV型菌毛介导的聚集和生物膜的形成进行了分析14。另外，A的运动citrulli下溢流Ç观察onditions表明，IV型菌毛可能在殖民化中发挥重要作用，传播的A. citrulli在木质部导管在边溢流条件。 铜绿假单胞菌和X的抽搐motilities fastidiosa细胞在微加工流动室5,6,17针对流体当前成功观察。 IV型菌毛短少X的pilB和pilQ突变fastidiosa被发现深刻地改变微FL uidic装置5,6,18的溢流条件下抽搐运动的速度。在微FL uidic器件细菌粘附和运动进行的研究表明，微FL uidic室是特别适合于在体外分析菌毛介导的细菌的抽搐运动和迁移。这些结果可以解释这有利于细胞间的连接，聚集和殖民内的抽动介导的迁移机制主机，最终导致全身感染。
X的弼热电联产操纵子fastidiosa包含pilG，霹雳，pilJ，丸剂，chpB和CHPC其中编码信号转导通路20。跨膜化学感受器结合化学刺激在周质结构域和在其细胞质部分以最终控制细菌抽搐蠕动激活信号级联。在X的弼CHP操纵子fastidiosa，一个磷酸化蛋白班车是PilG同源到崔泰源。在E.大肠杆菌和P.假单胞菌，崔泰源是在与该鞭毛马达蛋白19，21交互趋化系统的响应调节。虽然弼CHP操纵子在X的贡献对毒力fastidiosa最近检查20，pilG在趋操纵子的作用，响应于环境信号及X的调节/马达IV型菌毛fastidiosa是UNC李尔。为了阐明X的抽动运动的活动趋调节pilG的洞察力fastidiosa，微FL uidic室被用来评估X的抽搐蠕动fastidiosa。 十fastidiosa的pilG的特征在于，通过比较一个缺失突变体 Xf为 ΔpliG，互补应变XfΔpliG-C及其野生型在体外 的表型 。结果突出pilG在X的抽动运动中的作用fastidiosa。
Protocol
Representative Results
Discussion
在这项研究中，我们的特点X的运动行为fastidiosa PilG突变Xf为 ΔpilG和新设计的多个并行的纳米通道微FL uidic室互补XfΔpilG-Ç株。新设计的微FL uidic腔室可以具有100微米的纳米通道多达四个平行腔室宽度相比仅与单个50微米宽的通道18早期的设计。改进的更宽纳米通道便于引入细菌细胞与媒体的流动。此外，该微流体室1）简单的构建和组装; 2）相对便宜;?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国农业部农业研究服务支持。本出版物中的商品名称或商业产品只是提到提供具体信息之目的，并不由农业部美国农业部暗示推荐或认可。美国农业部是一个平等机会提供者和雇主。
Materials
| Biology materials | |||
| X. fastidiosa (Xf) Temecula wild type | Costa, H. S., et al., 2004 22 | ||
| pilG deletion mutant XfΔpliG | Shi, X. Y., et al., 2007 26 | ||
| pilG complementary strain XfΔpliG-C | Davis, M. J., wt al. 1998 23 | ||
| Physical materials and equipment | |||
| Disposable inoculating loops | VWR international, Radnor, PA | #22-363-607 | quantitative procedures such as bacterial collection |
| Polydimethylsiloxane (PDMS) | Dow Corning Corporation | #0002709226 | Sylgard 184 silicone Elastomeric Kits |
| AmScope MD2000 digital camera | AmScope, Irvine, CA | SE305R-AZ-E | Image, video recording and measurement |
| Tubes line | Edgewood, NY | #T4300 | Connected to the syringe and microfluidic chamber |
| Plastic luer connectors | Edgewood, NY | Connected to the syringe and microfluidic chamber | |
| Syringe pumps | Pico Plus, Harvard Apparatus, MA | #702209 | The flow rate can be adjusted while the pump is running. |
| Syringes | Gastight, Hemilton Company, Reno, NV | #1005 | Provide the flowing broth |
| Inverted Olympus IMT-2 microscope | Olympus | IMT-2 FLuoro PHase | Image observation and recording |
| SPOT-RT digital camera | Diagnostic Instruments, Inc., MI | RT230 | Image, video recording and measurement |
| Microscope Shutter | The UNIBLITZ, US | #LS2T2 | Control camera’s exposure time |
| Microscope Shutter Control system | The UNIBLITZ, US | VCM-D1 | VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver |
| MetaMorph Image software | Universal Imaging Corp., PA | Real-time super-resolution image processing |
References
- Purcell, A. H., Hopkins, D. L. Fastidious xylem-limited bacterial plant pathogens. Annu. Rev. Phytopathol. 34, 131-151 (1996).
- Purcell, A. H. Xylella fastidiosa, a regional problem or global threat. J. Plant Pathology. 79, 99-105 (1997).
- Hopkins, D. L. Xylella fastidiosa: Xylem-limited bacterial pathogen of plants. Annu. Rev. Phytopathol. 27, 271-290 (1989).
- Mattick, J. S. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56, 289-314 (2002).
- Meng, Y., et al. Upstream migration of Xylella fastidiosa via pilus-driven twitching motility. J. Bacteriol. 187, 5560-5567 (2005).
- Li, Y., et al. Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell-cell aggregation. Microbiology. 153, 719-726 (2007).
- Simpson, A. J. G., et al. The genome sequence of the plant pathogen Xylella fastidiosa. Nature. 406, 151-157 (2000).
- Maier, B., Potter, L., So, M., Long, C. D., Seifert, H. S., Sheetz, M. P. Single pilus motor forces exceed 100 pN. Proc. Natl. Acad. Sci. USA. 99, 16012-16017 (2002).
- Touhami, A., Jericho, M. H., Boyd, J. M., Beveridge, T. J. Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy. J. Bacteriol. 188, 370-377 (2006).
- Skerker, J. M., Berg, H. C. Direct observation of extension and retraction of type IV pili. Proc. Natl. Acad. Sci. USA. 98, 6901-6904 (2001).
- Brown, D. C., Larson, R. S. Improvements to parallel plate flow chambers to reduce reagent and cellular requirements. BMC Immunol. 2, 9 (2001).
- Thomas, W. E., Nilsson, L. M., Forero, M., Sokurenko, E. V., Vogel, V. Shear-dependent ‘stick-and-roll’ adhesion of type 1 fimbriated Escherichia coli. Mol. Microbiol. 53, 1545-1557 (2004).
- Thomas, W. E., Trintchina, E., Forero, M., Vogel, V., Sokurenko, E. V. Bacterial adhesion to target cells enhanced by shear force. Cell. 109, 913-923 (2002).
- Bahar, O., Fuente, D. L., Burdman, S. Assessing adhesion, biofilm formation and motility of Acidovorax citrulli using microfluidic flow chambers. FEMS Microbiol. Lett. 312, 33-39 (2010).
- Thomas, W. E. Using a laminar flow system to explain shear-enhanced bacterial adhesion. Proceedings of ICMM2005, Third International Conference on Microchannels and Mini-channels. , 751-759 (2005).
- Fuente, D. L., et al. Assessing adhesion forces of type I and type IV pili of Xylella fastidiosa bacteria by use of a microfluidic flow chamber. Appl. Environ. Microbiol. 73, 2690-2696 (2007).
- DeLange, P. A., Collins, T. L., Pierce, G. E., Robinson, J. B. PilJ localizes to cell poles and is required for type IV pilus extension in Pseudomonas aeruginosa. Curr Microbiol. 55, 389-395 (2007).
- Fuente, D. L., Burr, T. J., Hoch, H. C. Mutations in type I and type IV pilus biosynthetic genes affect twitching motility rates in Xylella fastidiosa. J. Bacteriol. 189, 7507-7510 (2007).
- Ferandez, A., Hawkins, A. C., Summerfield, D. T., Harwood, C. S. Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response. J. Bacteriol. 184, 4374-4383 (2002).
- Cursino, L., et al. Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa. Mol. Plant Microbe Interact. 10, 1198-1206 (2011).
- Hazelbauer, G. L., Falke, J. J., Parkinson, J. S. Bacterial chemoreceptors: High-performance signaling in networked arrays. Trends Biochem. Sci. 33, 9-19 (2008).
- Costa, H. S., et al. Plant hosts of Xylella fastidiosa in and near southern California vineyards. Plant Dis. 88, 1255-1261 (2004).
- Shi, X. Y., Dumenyo, C. K., Hernandez-Martinez, R., Azad, H., Cooksey, D. A. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU. Appl. Environ. Microbiol. 73, 6748-6756 (2007).
- Matsumoto, A., Young, G. M., Igo, M. M. Chromosome-Based Genetic Complementation System for Xylella fastidiosa. Appl. Environ. Microbiol. 75, 1679-1687 (2009).
- Davis, M. J., Purcell, A. H., Thomson, S. V. Isolation Media for the Pierce’s Disease Bacterium. Phytopathology. 70, 425-429 (1980).
- Xia, Y. N., Whitesides, G. M. Soft lithography. Annu. Rev. Mater. Sci. 28, 153-184 (1998).
- Chaudhury, M. K., Whitesides, G. M. Direct measurement of interfacial interactions between semispherical lenses and flat sheets of poly-(dimethylsiloxane) and their chemical derivatives. Langmuir. 7, 1013-1025 (1991).
- Cruz, L. F., Parker, J. K., Cobine, P. A., De La Fuente, L. Calcium-enhanced twitching motility in Xylella fastidiosa is linked to a single PilY1 homolog. Appl. Environ. Microbiol. 80, 7176-7196 (2014).
