Analysis of membrane–protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native‐PAGE complexome profiling and 2D blue native‐/SDS‐PAGE

Abstract

Sulfate-reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS-reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone-interacting membrane-bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native-PAGE complexome profiling and 2D BN-/SDS-PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN-/SDS-PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na(+) -based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese-like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.

Genome and catabolic subproteomes of the marine, nutritionally versatile, sulfate-reducing bacterium Desulfococcus multivorans DSM 2059

“…Products of four of the sodium/sulfate symporters were identified, and two ( Dmul_C10650/18630 ) were found to be constitutively formed. The latter are reminiscent of the sulfate uptake systems recently dissected by means of BN-PAGE in D. toluolica Tol2 [49] (sequence identities 25–75 %). The product of SulP1 was identified in the membrane protein-enriched fraction of 12 out of the 17 tested substrate conditions, whereas low score detection of SulP2 (Mascot Score 45.8 and 41.4) could indicate the latter to be less relevant for sulfate uptake in D. multivorans .…”

“…It is assumed to link the cellular NADH and ferredoxin pools via bidirectional electron transfer, i.e., (i) oxidation of reduced ferredoxin by RnfB and transfer via RnfDG to RnfC for NAD + reduction coupled to the generation of a Na + -gradient, or (ii), vice versa, from NADH to oxidized ferredoxin driven by the membrane potential [50, 56]. Notably, a different type of the Rnf complex was recently reported to be present in D. toluolica Tol2 [49]. Here, the RnfB subunit involved in ferredoxin oxidation/reduction is larger (~700 amino acids) as compared to characterized RnfB of Clostridium ljungdahlii or Acetobacterium woodii (~300 amino acids) and contains NAD + -binding sites in addition to the 4Fe-4S cluster and ferredoxin-type iron-sulfur binding domain present in all RnfBs.…”

“…Furthermore, also the RnfC subunit of D. toluolica Tol2 differs, lacking the NADH-ubiquinone oxidoreductase and soluble ligand-binding domains necessitating an RnfC electron acceptor/donor other than NAD + /NADH, which has to be identified. Due to these differences, a mode of action different to the classical Rnf complex was suggested [49] that awaits experimental verification.…”

Complementary Metaproteomic Approaches to Assess the Bacterioplankton Response toward a Phytoplankton Spring Bloom in the Southern North Sea

“…The nanoLC effluent was continuously analyzed by an online-coupled iontrap mass spectrometer (amaZon speed ETD; Bruker Daltonik GmbH, Bremen, Germany) using an electrospray ion source (Captivespray; Bruker Daltonik GmbH) operated in positive ion mode as described in detail before (Wöhlbrand et al, 2016). …”

Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

“…This stability may be attributed to the lower protein diversity of the D. toluolica proteome coupled to a higher dynamic range of protein abundance as compared to P. inhibens . Apparently, D. toluolica forms proteins essential for growth under given conditions in large amounts, while less important proteins remain lowly abundant [Wöhlbrand et al, , 2016. In contrast, P. inhibens forms a large number of different proteins with rather homogenous abundance [Zech et al, 2013].…”