摘要
成团泛菌YS19是从水稻"越富"品种中分离的一种优势内生细菌,与宿主水稻互作时具有多种促生作用,其形成的共质体(symplasmata)结构与菌体抗逆及与宿主互作有重要意义.研究发现了一种在YS19共质体形成阶段高表达的差异蛋白,对其用肽指纹图谱进行鉴定,发现其属于周质空间麦芽糖结合蛋白家族.克隆了该蛋白质的基因,重组表达并分离纯化了该蛋白质,发现它是一种兼职功能蛋白,其不仅参与麦芽糖的ABC运输系统,而且在强酸环境下不易发生变性沉淀,并可通过疏水面的显著暴露结合底物蛋白来发挥分子伴侣活性,这些兼职功能构成了菌体抗逆生存适应性的重要分子基础.
Pantoea agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice(Oryza sativa cv.Yuefu) grown in temperate climatic regions in west Beijing(China).The bacterium forms aggregate structures called "symplasmata",in which several(at least two) to hundreds of individual cells tightly bind together.Our previous study revealed that there were two growth stages for YS19,including the single cell stage existing before exponential growth phase and the symplasmata forming stage starting at the end of the exponential growth phase in liquid LB medium.More strikingly,the symplasmata structures contribute to bacterial stress(e.g.,dehydration,heavy metal toxicity,and osmotic shock) resistance and are especially significant for bacterial surviving strategy in suiting an adaptive life to hostile environments.In this research,YS19 was cultivated in LB medium,and the whole cellular protein expression of the cultures sampled at different times(0~12 h) was analyzed by SDS-PAGE.Here,a novel protein differentially expressed at the symplasmata forming stage was captured.The protein was purified and digested by trypsin.The digests were analyzed by MALDI-TOF mass spectrometry and the peptide mass fingerprint of this protein was successfully obtained.Then,database searching with Mascot in the SWISS-Prot database was performed using the recorded peptide mass fingerprint data.It is found that among 53 peptides,there are 14 peptide masses matching to MalE from Enterobacter aerogenes,which is a protein belonging to the periplasmic maltose-binding protein family of the ATP-binding cassette transporters.Then the gene of the MalE protein was cloned from YS19,expressed in the E.coli BL21(DE3),and the recombinant protein was purified to homogeneity.Under stress(acid) treatments,the recombinant protein showed strong anti-aggregation ability,and even exhibited chaperone-like activity.This assay was achieved by comparing the quantity of the substrate proteins remaining in the supernatant under various denaturing acid conditions,where the aggregation of the substrate protein was effectively suppressed in the presence of the recombinant MalE protein.Far-UV circular dichroism spectroscopy examination on the purified MalE proteins under different pHs indicated that the global structures exhibited a sudden transition from a highly ordered conformation to a less ordered one.However,MalE was still capable of maintaining most of its secondary structures under these extreme acidic conditions(pH1~2). Finally,the Bis-ANS fluorescent probe technique was used to explore the effect of acid condition on the hydrophobic surfaces exposing of the protein.It is found that with the decrease of the pH value,the fluorescence intensity of the samples increased gradually,suggesting the MalE protein exposed the hydrophobic surfaces of the molecule to bind the substrates,implying the protein performed its chaperone activity via exposing its hydrophobic surfaces under acidic condition.MalE protein,originally found as a periplasmic maltose-binding protein of the ATP-binding cassette transporters,whose expression in YS19,however,far exceeds the amount being needed for transmembrane transports,is most likely a moonlighting protein for its alternative biological functions as a chaperone protein as revealed by this study.This research provided valuable information on the life styles and survival strategies of microorganisms that forms multicellular aggregates at specific growth stages and also explained the protein basis adopted by YS19 symplasmata-forming cells in surviving the stress conditions.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2012年第3期273-281,共9页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(30870055)~~