细菌中无机聚磷酸盐的检测
Summary
我们描述了一种简单的方法, 快速定量无机聚磷酸盐在不同的细菌, 包括革兰氏阴性, 革兰氏阳性, 和分枝杆菌物种。
Abstract
无机聚磷酸盐 (聚磷) 是一种生物聚合物, 存在于来自生命各个领域的细胞中, 是许多细菌的毒力和应激反应所必需的。生物材料中的聚磷有多种定量方法, 其中许多方法要么是劳动密集型的, 要么是不敏感的, 限制了其用途。我们在这里提出了一种简化的方法, 聚乙二醇定量在细菌中, 使用硅膜柱提取优化快速处理多个样品, 消化聚磷与多 p 特异性外聚磷酸酶 scppx, 并检测通过敏感的抗坏血酸为基础的比色法, 可获得游离磷酸盐。此过程简单、价格低廉, 并允许在不同的细菌物种中进行可靠的聚子定量。我们提出了具有代表性的多 p 定量从革兰氏阴性菌 (大肠杆菌), 革兰氏阳性乳酸菌 (乳酸菌) 和分枝杆菌物种 (分枝杆菌 smegmatis)。我们还包括一个简单的协议, 镍亲和力纯化毫克数量的 scppx, 这是目前不可用的商业。
Introduction
无机聚磷酸盐 (聚磷) 是一种线性生物聚合物的磷酸酐连接磷酸盐单位, 存在于生命的所有领域 1,2,3。在不同的细菌中, 聚磷对于应激反应、动力、生物膜形成、细胞周期控制、抗生素耐药性和毒力 4、5、6、7、8 至关重要 , 9,10,11。因此, 对细菌中的多磷代谢的研究有可能对细菌在不同环境中引起疾病和茁壮成长的能力产生基本的洞察。然而, 在许多情况下, 细菌细胞中的聚磷定量方法是这些研究中的一个限制因素。
目前有几种方法用于测量生物材料中的多磷水平。这些方法通常涉及两个不同的步骤: 提取聚 p 和量化这些提取物中存在的聚磷。目前的金标准方法, 开发酵母酿酒酵母 12 , 提取聚 p与 dna 和 rna 使用苯酚和氯仿, 其次是乙醇沉淀, 治疗脱氧核糖核酸酶 (dnase) 和核糖核酸酶 (rnase), 并消化由此产生的纯化聚苯与酿酒酵母聚 p 降解酶外聚磷酸酶 (scppx)13产生游离磷酸盐, 然后用孔雀石绿基比色法。此过程是高度定量的, 但劳动密集型, 限制了可在单个实验中处理的样品数量, 并且没有针对细菌样本进行优化。另一些人报告说, 使用硅珠 (“玻璃牛奶”) 或硅膜色谱柱6、14、15、16、17从各种细胞和组织中提取聚乙二醇. 18岁这些方法不能有效地提取 12、14、15的短链聚磷 (小于60个磷酸盐单位),尽管这对细菌的关注较少, 而细菌通常被认为主要是合成长链聚乙二醇3。使用强酸19、20 提取聚磷的旧方法不再被广泛使用, 因为聚乙二醇在酸性条件下不稳定12。
还有各种报告的定量聚电的方法。其中最常见的是 4 ‘, 6-二胺-2-苯丙二醇 (dapi), 一种荧光染料更常用于染色 dna。dapi-polyp 复合物的荧光激发和发射最大值与 dapi-dna 复合物21、22 不同, 但其他细胞成分 (包括 rna、核苷酸和肌醇) 的干扰相当大磷酸盐12、15、16、23, 降低了使用该方法进行的多聚子测量的特异性和敏感性。或者, 聚磷和二磷酸腺苷 (adp) 可以转换为三磷酸腺苷 (atp) 使用纯化的大肠杆菌多 p 激酶 (ppk) 和由此产生的 atp 量化使用荧光素酶 14,17 ,18。这样就可以检测到非常少量的聚磷, 但需要两个酶反应步骤, 以及荧光素和非常纯的 adp, 它们都是昂贵的试剂。scppx 专门将聚磷消化成游离磷酸盐6,12,13, 24,这可以用更简单的方法检测, 但 scppx 是由 dna 和 rna12抑制,需要 dnase 和 rnase 处理含有多 p 的提取物。ppk 和 scppx 都不是商业上可用的, ppk 纯化相对复杂25,26。
细胞裂解物或提取物中的聚磷也可以通过 dapi阴性染色 27,28, 29, 30 在聚丙烯酰胺凝胶上显示, 这种方法确实允许对链长进行评估, 但低吞吐量和低量化。
我们现在报告一种快速、廉价、中等吞吐量的聚 p 检测方法, 可快速定量于不同细菌种类中的多磷水平。该方法首先在95°c 下在 4 m 鸟嘌呤异硫氰酸酯 (gitc)14中裂解细菌细胞, 以灭活细胞磷酸酶, 然后进行硅膜柱提取, 以快速处理多个样品。然后用大量过量的 scppx 消化生成的含有多普的提取物, 从而无需 dnase 和 rnase 处理。我们包括一个协议, 直接镍亲和力纯化毫克数量的 scppx。最后, 用简单、灵敏、抗坏血酸的比色法24和归一化为总细胞蛋白的多 p 衍生游离磷酸盐进行量化。该方法简化了细菌细胞中聚苯的测量, 并证明了其在革兰氏阴性菌、革兰氏阳性菌和分枝杆菌中的代表性种类中的应用。
Protocol
1. 纯化酵母外聚磷酸酶 (scppx) 用电穿孔32或化学转化33法将大肠杆菌蛋白过度表达菌株 BL21(DE3)31 与质粒 pScPPX26转化. 接种含有 100μg ml-1 氨匹液的溶酶体肉汤 (lb), 在 2升的无菌瓶中, 单菌落 BL21(DE3) 含有 pscppx2, 在37°c 孵育过夜, 吸收率约为600纳米 (a600)0.3, 在分光光度计中测量。 开始?…
Representative Results
该协议的关键步骤在图 1中以简化的形式绘制。 为了证明该协议在革兰氏阴性菌中的使用, 在37°c 的 lb 富培养基中 , 野生大肠杆菌mg1655 39 在晃动 (200 转/分) 的情况下生长到中原木阶段, 然后冲洗和孵育2小时含有4克 l-1 葡萄糖和 0.1 mm k2 hpo 4 的最低培养基40 , 已知?…
Discussion
这里描述的协议简化并加速了不同细菌中聚电水平的定量, 一组典型的24个样本的时间约为1.5 小时, 可进行完全加工。这允许快速筛选样品和分析突变库, 并简化了测量聚乙二醇随时间积累的动力学实验。我们已经证明, 该协议有效地工作的三个不同的植物的代表: 蛋白藻, 红机和放线菌, 这是臭名昭著的其弹性, 难以裂解细胞壁 49。
用 scppx 消化检测聚乙二醇比…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目得到了阿拉巴马大学伯明翰微生物学系启动基金的支持, 国家卫生研究院向 mjg 拨款 R35GM124590 (mjg), 国家卫生研究院向 fw 拨款 r01ai121364 (向 fw)。
Materials
| E. coli BL21(DE3) | Millipore Sigma | 69450 | |
| plasmid pScPPX2 | Addgene | 112877 | available to academic and other non-profit institutions |
| LB broth | Fisher Scientific | BP1427-2 | E. coli growth medium |
| ampicillin | Fisher Scientific | BP176025 | |
| isopropyl β-D-1-thiogalactopyranoside (IPTG) | Gold Biotechnology | I2481C | |
| HEPES buffer | Gold Biotechnology | H-400-1 | |
| potassium hydroxide (KOH) | Fisher Scientific | P250500 | for adjusting the pH of HEPES-buffered solutions |
| sodium chloride (NaCl) | Fisher Scientific | S27110 | |
| imidazole | Fisher Scientific | O3196500 | |
| lysozyme | Fisher Scientific | AAJ6070106 | |
| magnesium chloride (MgCl2) | Fisher Scientific | BP214-500 | |
| Pierce Universal Nuclease | Fisher Scientific | PI88700 | Benzonase (Sigma-Aldrich cat. # E1014) is an acceptable substitute |
| Model 120 Sonic Dismembrator | Fisher Scientific | FB-120 | other cell lysis methods (e.g. French Press) can also be effective |
| 5 mL HiTrap chelating HP column | GE Life Sciences | 17040901 | any nickel-affinity chromatography column or resin could be substituted |
| nickel(II) sulfate hexahydrate | Fisher Scientific | AC415611000 | for charging HiTrap column |
| 0.8 µm pore size cellulose acetate syringe filters | Fisher Scientific | 09-302-168 | |
| Bradford reagent | Bio-Rad | 5000205 | |
| Tris buffer | Fisher Scientific | BP1525 | |
| Spectrum Spectra/Por 4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO | Fisher Scientific | 08-667B | other dialysis membranes with MWCO < 30,000 Da should also work |
| hydrochloric acid (HCl) | Fisher Scientific | A144-212 | for adjusting the pH of Tris-buffered solutions |
| potassium chloride (KCl) | Fisher Scientific | P217500 | |
| glycerol | Fisher Scientific | BP2294 | |
| 10x MOPS medium mixture | Teknova | M2101 | E. coli growth medium |
| glucose | Fisher Scientific | D161 | |
| monobasic potassium phosphate (KH2PO4) | Fisher Scientific | BP362-500 | |
| dibasic potassium phosphate (K2HPO4) | Fisher Scientific | BP363-500 | |
| dehydrated yeast extract | Fisher Scientific | DF0886-17-0 | |
| tryptone | Fisher Scientific | BP1421-500 | |
| magnesium sulfate heptahydrate | Fisher Scientific | M63-50 | |
| manganese sulfate monohydrate | Fisher Scientific | M113-500 | |
| guanidine isothiocyanate | Fisher Scientific | BP221-250 | |
| bovine serum albumin (protease-free) | Fisher Scientific | BP9703100 | |
| clear flat bottom 96-well plates | Sigma-Aldrich | M0812-100EA | any clear 96-well plate will work |
| Tecan M1000 Infinite plate reader | Tecan, Inc. | not applicable | any plate reader capable of measuring absorbance at 595 and 882 nm will work |
| ethanol | Fisher Scientific | 04-355-451 | |
| silica membrane spin columns | Epoch Life Science | 1910-050/250 | |
| ethylenediaminetetraacetic acid (EDTA) | Fisher Scientific | BP120500 | |
| 1.5 mL microfuge tubes | Fisher Scientific | NC9580154 | |
| ammonium acetate | Fisher Scientific | A637-500 | |
| antimony potassium tartrate | Fisher Scientific | AAA1088922 | |
| 4 N sulfuric acid (H2SO4) | Fisher Scientific | SA818-500 | |
| ammonium heptamolybdate | Fisher Scientific | AAA1376630 | |
| ascorbic acid | Fisher Scientific | AC401471000 |
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Cite This Article
Pokhrel, A., Lingo, J. C., Wolschendorf, F., Gray, M. J. Assaying for Inorganic Polyphosphate in Bacteria. J. Vis. Exp. (143), e58818, doi:10.3791/58818 (2019).