Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species.

Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7%. The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events.     

Structural variation in the O-specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes

The O-polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose-containing homopolymers. Structural analysis, using chemical and high-field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating units OAc 1 2/6 →3)-β-d -Galf-(1 →3)-α- d -Galp-(1→d -Galactan I-OAc →3)-α-d -Galp-(1 →3)-β-d -Galp-(1→d -Galactan II. K. pneumoniae O8 mutant RFK-1 was isolated by resistance to phage KO1-2; strain RFK-1 expressed only d -galactan I-OAc. The ¹H- and ¹³C-NMR resonances from this O-polysaccharide indicate that all of the O-acetyl groups within the K. pneumoniae O8 polysaccharide are carried on d -galactan I and O-acetylation occurs only on the β- d -galactofuranose residues; 60% of the available β- d -galactofuranose residues are non-acetylated. The O-acetylation of the remaining residues is equally distributed between the O-2 and O-6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical to d -galactan I and II expressed by K. pneumoniae O1, accounting for the antigenic cross-reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and the O-acetyl groups present in the K. pneumoniae serotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. The rfb (O-polysaccharide biosynthesis) gene cluster of K. pneumoniae serotype O1 determines the synthesis of d -galactan I. rfb_Kpo1-specific gene probes were used to examine conservation in the rfb gene clusters of other K. pneumoniae serotypes which produce d -galactan I. Six O1 strains were examined and all showed hybridization with rfb_KpO1 probes under conditions of high stringency. Three serotype O2 strains produce d -galactan I and these strains also contained DNA sequences recognized by rfb_KpO1 probes under high stringency. The physical maps of these homologous rfb chromosomal regions showed some polymorphism. Surprisingly, the rfb_KpO8 region from K. pneumoniae serotype O8 was only recognized by rfb_KpO1 probes under low-stringency hybridization conditions, providing evidence for two substantially different clonal groups of rfb genes from K. pneumoniae strains with structurally related O-antigens.     

Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1

A plasmid that included both an 8.9 kb chromosomal DNA insert containing genes from the rfb cluster of Shigella dysenteriae 1 and a smaller insert containing the rfp gene from a S. dysenteriae 1 multicopy plasmid resulted in efficient expression of O antigen in an rfb-deleted strain of Escherichia coli K-12. Eight genes were identified in the rfb fragment: the rfbB-CAD cluster which encodes dTDP-rhamnose synthesis, rfbX which encodes a hydrophobic protein involved in assembly of the O antigen, rfc which encodes the O antigen polymerase, and two sugar transferase genes. The production of an O antigen also required the E. coli K-12 rfe gene, which is known to encode a transferase which adds N-acetyl-glucosamine phosphate to the carrier lipid unde-caprenol phosphate. Thus Rfe protein appears to function as an analogue of the Salmonella RfbP protein to provide the first sugar of the O unit. Functional analysis of the other genes was facilitated by the fact that partial O units of one, two or three sugars were efficiently transferred to the lipopolysaccharide core. This analysis indicated that the plasmid-encoded Rfp protein is the transferase that adds the second sugar of the O unit while the two rfb transferases add the distal sugars to make an O antigen whose structure is (Rha–Rha–Gal–GlcNAc)n. The use of the rfe gene product as the transferase that adds the first sugar of an O unit is a novel mechanism which may be used for the synthesis of other enteric O antigens.     

Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1

The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP-binding site. KpsM and KpsT belong to the ATP-binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using β-lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane-spanning regions, with the N- and C-terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV–SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross-linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker-insertion and site-directed mutagenesis define the N-terminus, the first cytoplasmic loop, and the SV-SVI linker as regions that are important for the function of KpsM in K1 polymer transport.     

Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.     

Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae

Defined chromosomal mutations that lead to assembly failure of the toxin coregulated pilus (TCP) of Vibrio cholerae provide useful insights into the biogenesis of a type-4 pilus. Mutants in rfb affecting LPS O-antigen biosynthesis, and strains depleted of the cytoplasmic membrane-associated ATP-binding protein TcpT, provide contrasting TCP export-defective phenotypes acting at different locations. Mutants in the perosamine biosynthesis pathway of V. cholerae 569B result in an rfb phenotype with an LPS consisting only of core oligosaccharide and lipid A. Such strains are unable to assemble TCP, and TcpA subunits are found in the periplasm and membrane fractions. In both rfb and tcpT mutants, the export defect is specific and complete. TcpT is a member of a large family of cytoplasmic membrane-associated ATP-binding proteins which are essential in type-4 pilin systems and in many non-pilin outer membrane transporters in Gram-negative bacteria. The behaviour of translocation-arrested TcpA in rfb and tcpT mutants is indistinguishable from that within assembled pilus under a range of conditions including flotation in density gradients, chemical cross-linking, and detergent extraction experiments. From the data presently available, it would appear that TcpA requires TcpT-mediated translocation from the cytoplasmic membrane and that TcpT stabilizes the subunit at or immediately beyond this stage, before crossing the outer membrane.     

Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae

D-Galactan I is an O-antigenic polymer with the repeat unit structure [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of D-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of D-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The D-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into D-galactan I biosynthesis by the bifunctional wbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of D-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the D-galactan I polymer requires WbbM for Galp transferase, together with Galf transferase activity provided by WbbO. Comparison of the biosynthetic pathways for D-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.     

Escherichia coli Clone Sonnei (Shigella sonnei) Had a Chromosomal O-Antigen Gene Cluster Prior to Gaining Its Current Plasmid-Borne O-Antigen Genes

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the his and gnd genes on the chromosome of Escherichia coli and related species. Shigella sonnei, essentially a clone of E. coli (E. coli clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of E. coli Sonnei among the 166 known E. coli serotype strains.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide.

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.     

Mapping of chromosomal loci associated with lipopolysaccharide synthesis and serotype specificity in Vibrio cholerae 01 by transposon mutagenesis using Tn5 and Tn2680

Summary Vibrio cholerae strains of the 01 serotype have been classified into three subclasses, Ogawa, Inaba and Hikojima, which are associated with the O-antigen of the lipopolysaccharide (LPS). The DNA encoding the biosynthesis of the O-antigen, the rfb locus, has been cloned and analysed (Manning et al. 1986; Ward et al. 1987). Transposon mutagenesis of the Inaba and Ogawa strains of V. cholerae, using Tn5 or Tn2680 allowed the isolation of a series of independent mutants in each of these serotypes. Some of the insertions were mapped to the rfb region by Southern hybridization using the cloned rfb DNA as a probe, confirming this location to be responsible for both O-antigen production and serotype specificity. The other insertions allowed a second region to be identified which is involved in V. cholerae LPS biosynthesis.     

Isolation of rfb Gene Clusters Directing the Synthesis of O Polysaccharides Consisting of Mannose Homopolymers and Serological Analysis of Lipopolysaccharides

Four serotypes of two genera, Escherichia coli O8 and O9 and Klebsiella O3 and O5, produce the O polysaccharides consisting of mannose homopolymers. Previously we reported the isolation and expression of E. coli O9 rfb in E. coli K-12 strains (Kido et al, J. Bacteriol., 171: 3629–3633, 1989). In this study, R' plasmids carrying his-rfb region of the other three strains were isolated and expressed in E. coli K-12 strain. Serological study of lipopolysaccharides (LPS) synthesized in E. coli K-12 strain was carried out. His-linked rfb genes from E. coli O9 and Klebsiella O3 directed the synthesis of O polysaccharides with the same antigenicity as those of the parental strains in E. coli K-12 strain. On the other hand, rfb genes from E. coli O8 and Klebsiella O5 directed the synthesis of O polysaccharides which were antigenically not identical but partially common to those of the parental strains. A rough strain derived from E. coli O8 synthesized LPS which showed the identical antigenicity as the wild strain when the his-rfb region of E. coli O8 was introduced. The results suggest that some genes located distantly from his are additionally required to complete the synthesis of O polysaccharides of E. coli O8 and Klebsiella O5.     

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPS_C putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPS_C in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPS_C led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (eps^T) in EPS_C containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of eps^T resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPS_C that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.     

Expression, purification and characterization of recombinant phosphomannomutase and GDP-{alpha}-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-{alpha}-D-mannose from D-mannose

The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis)of Salmonella enterica, group B, encoding for the enzymes phosphomannomutase(EC 5.4.2.8 [EC] ) and GDP-     

Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP

Resistance to multiple natural product drugs associated with reduced drug accumulation in human tumor cells may be conferred by either the 170 kDa P-glycoprotein or the 190 kDa multidrug resistance protein, MRP. Both MRP and P-glycoprotein belong to the large and ancient ATP-binding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identity. Unlike P-glycoprotein, MRP actively transports conjugated organic anions such as the cysteinyl leukotriene C₄ and glutathione-conjugated aflatoxin B₁. Transport of unconjugated chemotherapeutic agents appears to require cotransport of glutathione. MRP and several more recently discovered ABC proteins contain an additional NH₂-proximal membrane-spanning domain not found in previously characterized ABC transporters. This domain, whose NH₂-terminus is extracytosolic, is essential for MRP-mediated transport activity. This review summarizes current knowledge of the structural and transport characteristics of MRP which suggest that the physiologic functions of this protein could range from a protective role in chemical toxicity and oxidative stress to mediation of inflammatory responses involving cysteinyl leukotrienes. BioEssays 20:931–940, 1998. © 1998 John Wiley & Sons, Inc.     

Structure and genetics of the O-antigen of Escherichia coli O158

A structure of the O-polysaccharide (O-antigen) of Escherichia coli O158 has been reported (Datta, A. K.; Basu, S.; Roy, N. Carbohydr. Res.1999, 322, 219–227). In this work, we reinvestigated the O158 polysaccharide using sugar analyses, Smith degradation, and ¹H and ¹³C NMR spectroscopy and established the following structure, which is at variance with the structure established earlier:This structure is in agreement with the predicted functions of genes found in the O-antigen gene cluster of E. coli O158.     

ATP-binding cassette transporters in Escherichia coli

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.