Subcellular location of the coupling protein TrwB and the role of its transmembrane domain

Abstract

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

“…The biological relevance of these findings remains to be evaluated, but it should be noted that several studies have identified functions of the various T4CP domains that could contribute-directly or indirectly-to substrate binding. The NTDs, for example, are unlikely to directly bind substrates, but studies have identified several NTD functions, including binding interactions with other channel subunits, stimulatory effects on T4CP oligomerization and catalytic activity, and contributions to T4CP spatial positioning in the cell (17,(64)(65)(66)(67), that could indirectly affect substrate docking. By virtue of their possible accessibility to secretion substrates in the cytoplasm, the NBDs and CTDs might coordinate with the AAD to recruit substrates through direct substrate engagement.…”

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location

“…Similarly, MobB CloDF13 was also expressed in the presence of plasmid pSU1456 and to mimic the in vivo transfer of CloDF13, its location was additionally studied in the presence of plasmids pSU1456 (R388 plasmid that lacks TrwB R388 protein) and pSU4833 (CloDF13 plasmid that contains its mobilization region except for MobB CloDF13 protein). Sample handling was performed as described by Segura et al (2014). The images were acquired in a Leica TCS SP5 confocal fluorescence microscope, with a 60× oil immersion objective.…”

“…Studies about subcellular location of different T4CPs reported up to now have rendered ambiguous results (Kumar and Das, 2002;Gunton et al, 2005;Segura et al, 2014). To gain a deeper knowledge of this matter, specifically regarding the role of the TMD, we have studied the subcellular location of TMD TraJ CD TrwB , MobB CloDF13 , and MobB TMD under different experimental conditions.…”

“…Since the interaction with other conjugative proteins of the T4SS could modify the subcellular location pattern (Segura et al, 2014), the proteins that were active in vivo (i.e., TrwB R388 , TMD TraJ CD TrwB , and MobB CloDF13 ) were also observed in the presence of a T4SS lacking a functional T4CP that could interfere with the studied eGFP variant. On the one hand, TrwB R388 and TMD TraJ CD TrwB were analyzed in the presence of the remaining conjugative proteins of R388 (i.e., in the presence of plasmid pSU1456 that lacks functional TrwB R388 but contains the remaining conjugative proteins).…”

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

“…Each monomer is composed of two main structural domains: the nucleotide-binding domain showing a RecA-like fold, and a small membrane-distal all-alpha domain. The TMD of TrwB plays an important role in TrwB structural integrity and oligomerization (Hormaeche et al 2002;Hormaeche et al 2004;de Paz et al 2010;Vecino et al 2011), subcellular localization 5 (Segura et al 2014), and regulation of ATPase activity (Hormaeche et al 2006;Vecino et al 93 2010). 94 A coupling role for T4CPs is supported by early genetic data (Cabezón et al 1997) and evidence of protein-protein interactions with both the substrate and the T4SS.…”