Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli

Abstract

The conjugative plasmid pRSD2 carries a raf operon that encodes a peripheral raffinose metabolic pathway in enterobacteria. In addition to the previously known raf genes, we identified another gene, rafY, which in Escherichia coli codes for an outer membrane protein (molecular mass, 53 kDa) similar in function to the known glycoporins LamB (maltoporin) and ScrY (sucrose porin). Sequence comparisons with LamB and ScrY revealed no significant similarities; however, both lamB and scrY mutants are functionally complemented by RafY. Expressed from the tac promoter, RafY significantly increases the uptake rates for maltose, sucrose, and raffinose at low substrate concentrations; in particular it shifts the apparent K m for raffinose transport from 2 mM to 130 M. Moreover, RafY permits diffusion of the tetrasaccharide stachyose and of maltodextrins up to maltoheptaose through the outer membrane of E. coli. A comparison of all three glycoporins in regard to their substrate selectivity revealed that both ScrY and RafY have a broad substrate range which includes ␣-galactosides while LamB seems to be restricted to malto-oligosaccharides. It supports growth only on maltodextrins but not, like the others, on raffinose and stachyose.The first step in carbohydrate metabolism of gram-negative bacteria is the permeation of the substrate through the outer membrane. Molecules up to a mass of about 600 Da can enter the periplasm through the general porins OmpF and OmpC in Escherichia coli; however, tetrasaccharides and larger molecules need specific glycoporins, such as LamB or ScrY (for recent reviews, see references 16 and 31).The maltoporin LamB of E. coli, which also functions as a phage receptor (19), is required for maltodextrin catabolism. In contrast to the general porins, it contains specific sugarbinding sites that facilitate diffusion of oligosaccharides through the pore (4, 14, 15). Moreover, the maltoporin also enhances the uptake of mono-and disaccharides (e.g., glucose and trehalose) at low substrate concentrations. The gene lamB, which is part of the chromosomal mal regulon, is normally induced by maltose, maltodextrins, and trehalose. It seems, however, that the endogenously induced level of the mal regulon is high enough for LamB to play an important general role in the carbohydrate transport of E. coli (8,12).The second glycoporin, ScrY (11,22,23), was found to be involved in the sucrose catabolic pathway encoded by the enterobacterial conjugative plasmid pUR400 (25). Although the sequence similarity between LamB and ScrY is very low, the latter can substitute for the maltoporin in lamB mutants, except for the function as a receptor (23). ScrY also contains sugar-binding sites (26).Here we describe a third glycoporin in enterobacteria, which is also encoded by a conjugative plasmid first isolated from an E. coli strain found in a chicken egg (7). This plasmid, pRSD2, is known to enable E. coli K-12 cells to grow on raffinose by providing a raffinose permease (encoded by rafB), an invertase (encoded by rafD), and ...

The porin RafY encoded by the raffinose plasmid pRSD2 of Escherichia coli forms a general diffusion pore and not a carbohydrate‐specific porin

“…these are about 0.3 M and above. We have to conclude from these results and those of the in vivo experiments [24] that RafY The alternative explanation that the monomeric channels had different conductances and that the channel with the smallest simply forms a wide channel in the outer membrane that allows the passage of high-molecular-mass carbohydrates, in contrast conductance closed first seems to be rather unlikely because the three monomers forming a trimer have the same primary struc-to the 'normal' general diffusion pores OmpF and OmpC, which are not permeable to these carbohydrates. Our result represents ture.…”

“…This means that it is a typical nose, stachyose and maltooligosaccharides, are permeable outer-membrane protein and forms a β-barrel cylinder. In a prethrough the RafY channel in vivo [24]. Our titration experiments vious study it has been noted that there is no identity between demonstrated that RafY does not contain a binding site for car-the primary structures of the carbohydrate-specific porins and bohydrates inside the channel.…”

“…Our titration experiments vious study it has been noted that there is no identity between demonstrated that RafY does not contain a binding site for car-the primary structures of the carbohydrate-specific porins and bohydrates inside the channel. This was concluded from the ex-RafY [24]. We used the program GCG [37] to perform a search perimental observation that the membrane conductance was not for the relation between different outer-membrane proteins, such influenced by the addition of carbohydrates.…”

“…They often form solute-specific stachyose and maltoheptaose, through the outer membrane, pores and contain binding sites for neutral substrates, such as which means that it has a similar wide range of carbohydrate carbohydrates [10,11] and nucleosides [12], and charged sotransport as ScrY, whereas LamB facilitates only the uptake of lutes, such as phosphate [8,13]. Many of these specific porins carbohydrates of the maltooligosaccharide series [24]. It has are part of an uptake system, composed of different proteins.…”

“…constitutive transmembrane protein, called porin, which contains a general diffusion pore with a defined exclusion limit for hydro-Recently, another gene has been cloned and sequenced, which codes for an outer membrane protein and allows E. coli to grow philic solutes [4, 5]. In addition to the constitutive porins the outer membrane may contain porins that are induced under spe-on raffinose, sucrose and maltose [24]. RafY allows the diffusion of a variety of high-molecular-mass carbohydrates, such as cial growth conditions [6Ϫ9].…”