Promoting resuscitation of viable but nonculturable cells of Vibrio harveyi by a resuscitation-promoting factor-like protein YeaZ
Promoting resuscitation of viable but nonculturable cells of Vibrio harveyi by a resuscitation-promoting factor-like protein YeaZ
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DOI：
10.1111/jam.13342
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发表时间：
2017-02-01
影响因子：
4
通讯作者：
Zhang, X.
中科院分区：
文献类型：
--
作者：
Li, Y.;Chen, J.;Zhang, X.
目的研究重组哈维氏弧菌SF-1的复苏促进因子YeaZ的活性。方法与结果从哈维氏弧菌SF-1基因组DNA中克隆了复苏促进因子YeaZ基因。在大肠杆菌中表达该基因，表达产物经Ni ~（2+）亲和层析纯化。利用自杀质粒pNQ 705，通过同源重组，构建了一个yeaZ突变体。在28 ℃的2216 E肉汤中，破坏yeaZ对细胞生长没有显著影响。野生型和突变型活的但不可培养的（VBNC）细胞可以复苏的温度升高的方法。此外，重组YeaZ将可培养计数分别从127 × 10（4）CFU/ml和199 × 10（4）CFU/ml增加到288 × 10（5）CFU/ml和459 × 10（5）CFU/ml。野生型和突变型VBNC细胞在4 ℃下培养120天后，升温法均不能复苏，但加入重组YeaZ后，野生型和突变型VBNC细胞复苏明显，可培养细胞数分别为113 × 10 ~（3）和144 × 10 ~（3）CFU/ml。破坏yeaZ降低了哈维氏弧菌在斑马鱼中的毒力。结论重组YeaZ能有效地促进野生型和突变型哈维氏弧菌细胞从VBNC复苏到可培养状态。该蛋白还促进VBNC野生型和突变型细胞的复苏，这些细胞在4 ℃下保持120天，并且通过温度升高方法不能恢复。YeaZ的破坏降低了哈维氏弧菌在斑马鱼中的毒力。研究的意义和影响在这里，我们展示了哈维氏弧菌复苏促进因子YeaZ的明确证据，以及在VBNC细胞复苏和致病性中的作用。
AimsTo demonstrate the resuscitation-promoting activities of recombinant YeaZ from Vibrio harveyi SF-1.Methods and ResultsThe gene of resuscitation-promoting factor YeaZ was cloned from genomic DNA of V. harveyi SF-1. The gene was expressed in Escherichia coli, and the expressed protein was purified by Ni2+-affinity chromatography. A yeaZ mutant was constructed by using the suicide plasmid pNQ705 with homologous recombination. Disruption of yeaZ did not affect cell growth significantly in 2216 E broth at 28 degrees C. The wild-type and mutant viable but nonculturable (VBNC) cells could be resuscitated by temperature upshift method. In addition, the recombinant YeaZ increased the culturable counts from 127x10(4)CFU per ml and 199x10(4) CFU per ml to 288x10(5)CFU per ml and 459x10(5) CFU per ml, respectively. After the VBNC cells of wild-type and mutant cells were maintained at 4 degrees C for 120days, no resuscitation was obtained by temperature upshift method, but addition of the recombinant YeaZ promoted the resuscitation of the wild-type and mutant cells, with the culturable cell counts of 113x10(3) and 144x10(3) CFU per ml, respectively. Disruption of yeaZ decreased the virulence of V. harveyi in zebrafish. The lethal dose 50% of the yeaZ null mutant was more than 10-fold higher than that of the wild-type cells.ConclusionsThe recombinant YeaZ could efficiently promote resuscitation of the wild-type and mutant cells of V. harveyi from VBNC to culturable state. The protein also promoted resuscitation of the VBNC wild-type and mutant cells, which were maintained at 4 degrees C for 120days and not recovered by temperature upshift method. Disruption of yeaZ decreased the virulence of V. harveyi in zebrafish.Significance and Impact of the StudyHere, we show clear evidence of a resuscitation-promoting factor YeaZ of V. harveyi and the roles in resuscitation of the VBNC cells and its pathogenicity.