藤黄微球菌Rpf因子对土壤细菌可培养物种分离效果的影响
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摘要
通过培养基添加藤黄微球菌Rpf因子对3份土壤样品中的细菌进行分离以了解藤黄微球菌Rpf因子对细菌可培养物种多样性的影响.通过在改良的VL55培养基中添加藤黄微球菌Rpf因子,对采自河北涞水县上港村、青海三江源和云南无量山3个地区的3份土壤样品进行细菌的分离,并对所分离菌株进行16S rRNA基因序列系统发育分析,分析培养基添加藤黄微球菌Rpf因子对土壤中可培养细菌物种分离效果的影响.结果显示:河北土壤中不添加Rpf因子的对照组分离得到5个属,添加Rpf因子的实验组分离得到8个属;青海土壤中对照组分离得到2个属,实验组分离得到6个属;云南土壤中对照组分离得到6个属,实验组分离得到8个属.表明添加藤黄微球菌Rpf因子的实验组分离得到的微生物物种较对照组丰富.其中,青海三江源土壤中分离得到的菌株HBUM200161与多食鞘氨醇杆菌(Sphingobacterium multivorum)IAM14316~T亲缘关系最近,16S rRNA基因序列相似性为97.93%,可能为鞘氨醇杆菌属(Sphingobacterium)潜在的新种.
Bacteria in three soil samples were isolated by adding Rpf factor from Micrococcus luteus to the medium to understand the effect of Rpf factor on the culturable species diversity. By adding the Rpf factor of M. luteus to the modified VL55 medium, the bacteria were isolated from three soil samples collected from Shanggang Village, Laishui County, Hebei Province, Sanjiangyuan, Qinghai, and Wuliangshan, Yunnan. The phylogenetic analysis of the 16 S rRNA gene sequence was carried out on the isolated strains,and the effect of adding Rpf factor to the culture medium on the separation of culturable bacterial species in the soil was analyzed.The results showed that five genera were isolated from the control group in Hebei soil without Rpf factor,and eight genera were isolated from the test group with Rpf factor;two genera were isolated from the control group in Qinghai soil,and six genera were isolated from the test group;in the soil of Yunnan,six genera were isolated from the control group,and eight genera were isolated from the test group.The results show that the microbial species isolated from the test group with M.luteus Rpf added was more abundant than that of the control group.Among them,the strain HBUM200161isolated from the Sanjiangyuan soil in Qinghai has the closest relationship with Sphingobac-bly represents a novel species of the genus Sphingobacterium.
Bacteria in three soil samples were isolated by adding Rpf factor from Micrococcus luteus to the medium to understand the effect of Rpf factor on the culturable species diversity. By adding the Rpf factor of M. luteus to the modified VL55 medium, the bacteria were isolated from three soil samples collected from Shanggang Village, Laishui County, Hebei Province, Sanjiangyuan, Qinghai, and Wuliangshan, Yunnan. The phylogenetic analysis of the 16 S rRNA gene sequence was carried out on the isolated strains,and the effect of adding Rpf factor to the culture medium on the separation of culturable bacterial species in the soil was analyzed.The results showed that five genera were isolated from the control group in Hebei soil without Rpf factor,and eight genera were isolated from the test group with Rpf factor;two genera were isolated from the control group in Qinghai soil,and six genera were isolated from the test group;in the soil of Yunnan,six genera were isolated from the control group,and eight genera were isolated from the test group.The results show that the microbial species isolated from the test group with M.luteus Rpf added was more abundant than that of the control group.Among them,the strain HBUM200161isolated from the Sanjiangyuan soil in Qinghai has the closest relationship with Sphingobac-bly represents a novel species of the genus Sphingobacterium.
引文
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[19]李伦,许崇波,李元.复苏促进因子研究进展[J].生物技术通讯,2009,20(5):703-705.DOI:10.3969/j.issn.1009-0002.2009.05.030.
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[2]岳晨莉.藤黄微球菌Rpf结构域及其突变体基因的克隆、表达及生物活性的初步研究[D].西安:第四军医大学,2008.
[3]MUKAMOLOVA G V,TURAPOV O A,KAZARIAN K,et al.The rpf gene of Micrococcus luteus encodes an essential secreted growth factor[J].Molecular Microbiology,2002,46(3):611-621.DOI:10.1046/j.1365-2958.2002.03183.x.
[4]丁林贤,张萍华,洪华嫦,等.藤黄微球菌Rpf活性蛋白的制取及其对红球菌VBNC菌体的复苏作用[J].微生物学报,2012,52(1):77-82.DOI:10.13343/j.cnki.wsxb.2012.01.015.
[5]柳云帆.复苏促进因子Rpf对淡水浮游细菌可培养性的影响[D].重庆:西南大学,2007.
[6]陈华癸.土壤微生物学[M].上海:上海科学技术出版社,1981.
[7]KAWAI M,MATSUTERA E,KANDA H,et al.16Sribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis[J].Applied and Environmental Microbiology,2002,68(2):699-704.DOI:10.1128/AEM.68.2.699-704.2002.
[8]TSCHECH A,PFENNIG N.Growth yield increase linked to caffeate reduction in Acetobacterium woodii[J].Archives of Microbiology,1984,137(2):163-167.DOI:10.1007/BF00414460.
[9]WIDDEL F,KOHRING G W,MAYER F.Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids-Ⅲ.Characterization of the filamentous gliding Desulfonema limicola gen.nov.sp.nov.,and Desulfonema magnumsp.nov.[J].Archives of Microbiology,1983,134(4):286-294.
[10]EDWARDS U,ROGALL T,Bl9ker H,et al.Isolation and direct complete nucleotide determination of entire genes.characterization of a gene coding for 16Sribosomal RNA[J].Nucleic Acids Research,1989,17(19):7843-7853.DOI:10.1093/nar/17.19.7843.
[11]LANE D J.16S/23SrRNA sequencing[M]//STACKEBRANDT E,GOODFELLOW M.Nucleic Acid Techniques in Bacterial Systematics.New York:Wiley,1991:115-147.
[12]景凤霞.三株原核微生物新属种的多相分类及不同营养因子对微生物新物种分离的影响[D].保定:河北大学,2017.
[13]景凤霞,李云琪,唐灵杰,等.不同营养因子对土壤细菌可培养物种多样性的影响[J].广东农业科学,2018,45(8):65-72.DOI:10.16768/j.issn.1004-874X.2018.08.010.
[14]FELSENSTEIN J.Confidence limits on phylogenies:an approach using the bootstrap[J].Evolution,1985,39(4):783-791.DOI:10.1111/j.1558-5646.1985.tb00420.x.
[15]高晓鹏,苏明权,刘家云,等.结核分枝杆菌Rv1884c和Rv0867c基因的克隆表达及其促生长作用的研究[J].中华检验医学杂志,2008,31(12):1390-1395.DOI:10.3321/j.issn:1009-9158.2008.12.023.
[16]ZHU W,PLIKAYTIS B B,SHINNICK T M.Resuscitation factors from mycobacteria:homologs of Micrococcus luteus proteins[J].Tuberculosis,2003,83(4):261-269.DOI:10.1016/S1472-9792(03)00052-0.
[17]MUKAMOLOVA G V,TURAPOV O A,YOUNG D I,et al.A family of autocrine growth factors in Mycobacterium tuberculosis[J].Molecular Microbiology,2002,46(3):623-635.DOI:10.1046/j.1365-2958.2002.03184.x.
[18]KIM M,OH H S,PARK S C,et al.Towards a taxonomic coherence between average nucleotide identity and16SrRNA gene sequence similarity for species demarcation of prokaryotes[J].International Journal of Systematic and Evolutionary Microbiology,2014,64(2):346-351.DOI:10.1099/ijs.0.059774-0.
[19]李伦,许崇波,李元.复苏促进因子研究进展[J].生物技术通讯,2009,20(5):703-705.DOI:10.3969/j.issn.1009-0002.2009.05.030.
[20]丛延广,胡福泉.细菌复苏促进因子---Rpf[J].生命的化学,2005,25(6):482-484.DOI:10.3969/j.issn.1000-1336.2005.06.017.