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.

Membrane proteome of the thermoalkaliphile Caldalkalibacillus thermarum TA2.A1

“…While extracting the membrane fraction from the rest of the cell constituents added a very modest increase in protein hits, adding a membrane solubilisation step significantly increased the detection of membrane proteins (Figures 1A, B). The breadth of solubilisation methods available in the literature (Schuck et al, 2003;Eshaghi et al, 2005;Williams et al, 2007;Wang et al, 2009;Zaparty et al, 2009;Burton and Martin, 2012;Altinisik Kaya et al, 2018) leaves a plethora of options available, but it is well noted that there is no common optimal method. Given our knowledge of membrane proteins, we opted for the use of detergents.…”

“…For alkaliphiles, two studies have been reported. One investigates membrane solubilisation with urea in Alkalihalobacillus marmarensis (Altinisik Kaya et al, 2018), while the other focuses on a few other well-known yet ultimately ineffective detergents for Alkalimonas amylolytica (Wang et al, 2009). None of these studies rely on a comparative approach that assesses the contribution of each step to the protocol.…”

Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica strain N10

“…Recently, proteomic analysis revealed that the expression level of membrane proteins participating in the ion transport in the haloalkaliphilic bacterium Alkalimonas amylolytica strain N10, which grows at NaCl concentrations up to 7% and with an optimum pH of 10 to 10.5 [15] , varies according to changes in pH and salt concentration [16] . This indicated the ion transporters are important for adaptation to high pH and salt in this haloalkaliphile.…”