title: Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing.
abstract: Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2-DE. The differential 2-DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH-dependent N10 genes using Western blot and real-time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH-dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell-shape mode of pH-dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH-responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic-adaptive mechanism.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Quanhui
authors.initials: Q
authors.lastname: Wang
authors.forename: Huiming
authors.initials: H
authors.lastname: Han
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Xue
authors.forename: Zhong
authors.initials: Z
authors.lastname: Qian
authors.forename: Bo
authors.initials: B
authors.lastname: Meng
authors.forename: Fuli
authors.initials: F
authors.lastname: Peng
authors.forename: Zhuowei
authors.initials: Z
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Tong
authors.forename: Chuanqi
authors.initials: C
authors.lastname: Zhou
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Yonghao
authors.initials: Y
authors.lastname: Guo
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Siqi
authors.initials: S
authors.lastname: Liu
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D020563:Gammaproteobacteria / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000032:analysis*; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010455:Peptides