Enhanced functionalisation of major facilitator superfamily transporters via fusion of C-terminal protein domains is both extensive and varied in bacteria

Abstract

The evolution of gene fusions that result in covalently linked protein domains is widespread in bacteria, where spatially coupling domain functionalities can have functional advantages in vivo. Fusions to integral membrane proteins are less widely studied but could provide routes to enhance membrane function in synthetic biology. We studied the major facilitator superfamily (MFS), as the largest family of transporter proteins in bacteria, to examine the extent and nature of fusions to these proteins. A remarkably diverse variety of fusions are identified and the 8 most abundant examples are described, including additional enzymatic domains and a range of sensory and regulatory domains, many not previously described. Significantly, these fusions are found almost exclusively as C-terminal fusions, revealing that the usually cytoplasmic C-terminal end of MFS protein would the permissive end for engineering synthetic fusions to other cytoplasmic proteins.

Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS)

“…Although the majority of MFS transporters are assumed to be single proteins such as glucose transporters (Figure a), examples of fusions with enzymatic, regulatory, or signaling domains have also emerged. In bacteria, various MFS-fusion proteins have been identified, including some that are not yet characterized. , A lysophospholipid transporter LplT was the first MFS member shown to use a lipid substrate in the transbilayer movement of lysophospholipid 2-acylglycerophosphoethanolamine (2-acyl-GPE). Interestingly, 2-acyl-GPE is thought to be acylated by a fused bifunctional enzyme known as acyltransferase/acyl-ACP synthetase to form phosphatidylethanolamine (Figure b) …”

Physiological Functions of Bacterial “Multidrug” Efflux Pumps

“…In our experience, there is often a general lack of functional importance of the N-and C-terminal 10+ residues in most classes of bacterial efflux proteins, and there is strong bioinformatic support that the C-terminus is the most flexible end for the evolution of natural fusions to partner transport proteins. 533,534 Nevertheless, it is important to verify that the tag does not alter activity. We routinely use plasmid pTTQ18 as vector and E. coli BL21 as host, but there are now legions of vector/host combinations that may be used with membrane proteins.…”

“…coli host (see section ) and modify the gene to add a short sequence of amino acids, a (His) 6–10 “tag” at the N-terminus or C-terminus, that serves both to identify the protein and to aid its purification. In our experience, there is often a general lack of functional importance of the N- and C-terminal 10+ residues in most classes of bacterial efflux proteins, and there is strong bioinformatic support that the C-terminus is the most flexible end for the evolution of natural fusions to partner transport proteins. , Nevertheless, it is important to verify that the tag does not alter activity. We routinely use plasmid pTTQ18 as vector and E.…”

Review on the Antibacterial Mechanism of Plant-Derived Compounds against Multidrug-Resistant Bacteria (MDR)

“…Based on sequence similarities, source of energy, substrate binding, and the number of components, bacterial efflux transporters are classified into five prominent families [ 28 ]; Resistance-nodulation division (RND), which is a specific group for Gram-negative bacteria [ 30 ], adenosine triphosphate (ATP)-binding cassette superfamily (ABC) [ 31 ], multidrug and toxic compound extrusion (MATE) [ 32 ], major facilitator superfamily (MFS) [ 33 ], and small multidrug resistance family (SMR) [ 34 ]. Figure 1 is adopted from Blanco et al, presenting the major efflux transporters in bacteria [ 28 ].…”