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 gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.Keywords : raffinose transport; carbohydrate specificity ; RafY channel ; enteric bacteria; lipid bilayer membrane.The cell envelope of gram-negative bacteria consists of dif-also contains the gene for a carbohydrate-specific outer-membrane porin [19Ϫ21]. ferent layers. The inner or cytoplasmic membrane contains the proteins for the transport of nutrients and proteins involved in E. coli is normally unable to grow on raffinose as sole carbon source. In some E. coli strains a plasmid has been recogthe synthesis of phospholipids, peptidoglycans and lipopolysaccharides [1]. The periplasmic space between the membranes is nized that allows them to grow on raffinose [22]. The structure of the plasmid has been investigated and several genes have an aqueous compartment isoosmolar to the cytoplasm [2]. The outer membrane is an asymmetric membrane composed on the been found to form an operon on the plasmid [23]. Its genes encode an inner membrane permease (rafB) and two cytoplasmic outside of lipopolysaccharides and on the inside of lipids [3]. It contains only a few major proteins. At least one of them is a enzymes (rafA and rafD), which split raffinose into galactose and sucrose and sucrose into fructose and glucose, respectively. 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]. 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 simil...

The difference in substrate selectivity of the maltodextrin (LamB) and sucrose (ScrY) porins is attributed mainly to differences in loop L3, which is supposed to constrict the lumen of the pores. We show that even a single mutation (D201Y) in loop L3 leads to a narrowing of the substrate range of ScrY to that resembling LamB. In addition, we removed the putative N-terminal coiled-coil structure of ScrY and studied the effect of this deletion on sucrose transport.

The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.