Deciphering the iron response in Acinetobacter baumannii: A proteomics approach

Abstract

Iron is an essential nutrient that plays a role in bacterial differential gene expression and protein production. Accordingly, the comparative analysis of total lysate and outer membrane fractions isolated from A. baumannii ATCC 19606 T cells cultured under iron-rich and -chelated conditions using 2-D gel electrophoresis-mass spectrometry resulted in the identification of 58 protein spots differentially produced. While 19 and 35 of them represent iron-repressed and iron-induced protein spots, respectively, four other spots represent a metal chelation response unrelated to iron. Most of the iron-repressed protein spots represent outer membrane siderophore receptors, some of which could be involved in the utilization of siderophores produced by other bacteria. The ironinduced protein spots represent a wide range of proteins including those involved in iron storage, such as Bfr, metabolic and energy processes, such as AcnA, AcnB, GlyA, SdhA, and SodB, as well as lipid biosynthesis. The detection of an iron-regulated Hfq ortholog indicates that iron regulation in this bacterium could be mediated by Fur and small RNAs as described in other bacteria. The iron-induced production of OmpA suggests this protein plays a role in iron metabolism as shown by the diminished ability of an OmpA isogenic deficient derivative to grow under iron-chelated conditions.

Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

“…The biological relevance of this observation is not clear considering that the binding of SecA to the L23 ribosomal protein seems to be the only interaction between this protein and the ribosome that has been described (68). Equally unclear is the apparent association of ribosomal proteins with the outer membrane fraction, a phenomenon we believe is not due solely to fraction contamination since we did not observe it when we used the same experimental approach to study the effect of iron on protein synthesis in the same A. baumannii strain (41). Taken together, our results indicate that although the interruption of the 3= end of secA in A. baumannii ATCC 19606 T is not lethal, it results in a protein homeostatic response to protect cells from different stressors that otherwise could negatively affect overall bacterial viability.…”

“…Considering the role SecA plays in the translocation of different proteins, we examined differences in protein production between the ATCC 19606 T parental strain and the isogenic 2010 secA insertion derivative using the 2-DE mass spectrometry approach that we applied to study the effect of iron on protein production in this pathogen (41). This proteomics study identified 35 protein spots, which represent 27 different proteins that were underproduced or not produced in the 2010 mutant compared with the parental strain (Fig.…”

“…Proteomic analyses of ATCC 19606 T and 2010 cells grown in LB broth were conducted as described before (41). Briefly, five 100-ml culture replicates for each condition were incubated at 37°C for 14 h, the time at which they reached stationary phase (OD 600 between 1.6 and 1.8), pooled, and then centrifuged at 7,000 ϫ g for 10 min at 4°C to collect cells.…”

“…Briefly, five 100-ml culture replicates for each condition were incubated at 37°C for 14 h, the time at which they reached stationary phase (OD 600 between 1.6 and 1.8), pooled, and then centrifuged at 7,000 ϫ g for 10 min at 4°C to collect cells. The inner membrane, outer membrane, and cytoplasmic protein fractions from ATCC 19606 T and 2010 bacteria were prepared and analyzed via two-dimensional gel electrophoresis (2-DE) as described previously (41). Protein spots were visualized by Coomassie blue staining, and gel image analysis was performed using the PDQuest software package (Bio-Rad).…”

“…Differentially produced proteins were identified by peptide mass fingerprinting (PMF) following matrix-assisted laser desorption ionizationtime of flight mass spectrometry (Bruker Ultraflex III MALDI-TOF/TOF mass spectrometer) of trypsin-digested protein spots as reported before (41). PeakErazor software (v 2.01; Lighthouse Data) was used to exclude protein contaminants, and the MASCOT search engine (Matrix Science) was used to match PMFs to bacterial entries in the NCBI nonredundant database.…”