Profiling and comprehensive expression analysis of ABC transporter solute‐binding proteins of Bacillus subtilis membrane based on a proteomic approach

Abstract

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).

Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore

“…The receptor YclQ was detected in multiple proteomic studies of B. subtilis extracellular proteins from cells grown under various culture and stress conditions, including both iron-poor and iron-rich media, indicating a universal and essential role of this protein in the iron acquisition process (39). Elevated levels of YclQ along with other SBPs were observed in the B. subtilis fur mutant (7).…”

Proteome-wide analysis of the functional roles of bacilysin biosynthesis in Bacillus subtilis

“…With a profiling of ABC-transporter solute-binding proteins, Bunai et al (2004) initiated the work on the B. subtilis membrane protein fraction. Eymann and coworkers (2004) established a technique in which the cytoplasmic membrane is washed in high-salt and alkaline buffers before the membrane proteins are solubilized with the detergent n-dodecyl-β-D-maltoside.…”

Protein Traffic for Secretion and Related Machinery ofBacillus subtilis

“…The predicted amino acid sequences of 1,490 contained more than one definite TMS (certain integral membrane proteins), 635 contained more than one putative TMS (putative membrane proteins), 260 were secretory proteins and lipoproteins with one cleavable TMS in the N-terminal region of their precursor form, and 1,609 lacked a TMS (soluble proteins). 7) Signal peptides for secretory proteins that are transported to the outside of the cell membrane and for lipoproteins that anchor to the membrane via an extended N-terminal hydrophobic lipid can be predicted by the B. subtilis database search using the Signal P algorithm. Such proteins are synthesized as precursors with signal peptides in their N-termini that are frequently removed after arrival at the destination.…”

“…Furthermore, the SOSUI algorithm predicted that approximately 30% of all proteins in 15 single-cell microorganisms including yeast consist of predicted membrane proteins having more than one TMS in their amino acid sequences. 7,77) The proteomes of E. coli membrane proteins have been studied in detail, 78,79) but few experiments have addressed the machinery for protein-targeting to and insertion into the cell membrane. Recent studies of E. coli indicate that SecA functions in the localization of a subset of proteins into the cell membrane.…”

“…Most of the solutebinding proteins were lipoproteins that anchor on the surface of the cell membrane, but only 20 definite integral membrane proteins with over four TMS were identified among the 520 predicted proteins. 7) Based on these proteome analyses of the membrane proteins, we studied the participation of the SecA function in protein localization by whole protein labeling using 15 N-stable isotope. Mass spectrometry can accurately distinguish the molecular masses of 14 N-(light) and 15 N-(heavy) peptides after the proteolysis of a preserved and de novo mixture of a single protein, and it accurately measures the ratios of light and heavy peptides that are equivalent to the abundance of the corresponding light and heavy proteins.…”