Fulsundar Shweta, Harms Klaus, Flaten Gøril E, Johnsen Pål J, Chopade Balu Ananda, Nielsen Kaare M
Institute of Bioinformatics and Biotechnology, University of Pune, Pune, India.
Appl Environ Microbiol. 2014 Jun;80(11):3469-83. doi: 10.1128/AEM.04248-13. Epub 2014 Mar 21.
Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10(-6) to 10(-8), with transfer efficiencies of approximately 10(3) and 10(2) per μg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi.
外膜囊泡（OMV）不断地从一系列细菌物种中释放出来。OMV具有多种功能，包括促进水平基因转移（HGT）过程等，这些功能已被提出。在本研究中，我们以革兰氏阴性的拜氏不动杆菌作为模型系统，研究了OMV是否有助于质粒在细菌细胞和物种之间的转移。从细菌培养物中提取OMV，并测试其将基因转移到大肠杆菌和拜氏不动杆菌群体中的能力，包括拜氏不动杆菌天然转化缺陷型突变体。使用抗双链DNA（anti-dsDNA）抗体来确定DNA进入OMV的情况。我们还确定了应激如何影响囊泡化水平和囊泡中的DNA含量。通过测量粒径分布（PSD）和zeta电位对OMV进行进一步表征。使用抗异硫氰酸荧光素（anti-FITC）偶联抗体和anti-dsDNA抗体进行透射电子显微镜（TEM）和免疫金标记，以追踪FITC标记的含DNA的OMV的移动情况。暴露于从含质粒供体细胞中分离出的OMV会导致拜氏不动杆菌和大肠杆菌发生HGT，转移频率范围为10^(-6)至10^(-8)，每微克囊泡DNA的转移效率分别约为10^3和10^2。抗生素应激被证明会影响OMV的DNA含量以及其流体动力学直径和zeta电位。形态学观察表明，拜氏不动杆菌的OMV与受体细胞以不同方式相互作用，这取决于受体物种。有趣的是，PSD测量表明拜氏不动杆菌会释放出不同大小范围的OMV。
