Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein

We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, -R, -A and -B mutants of Escherichia coli K-12, which do not form a complete LPS-containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7-deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A-core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild-type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane-impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen.

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1)

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.

Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2"

COMPARISON OF THE GROWTH INHIBITORY AND BACTERIOCIDAL EFFECTS OF TAUROLIDINE AND TAURULTAM ON A CLINICAL ISOLATE OF ESCHERICHIA-COLI

To investigate the mode of action of Taurolin, an antimicrobial preparation, the growth inhibitory and bacteriocidal effects of taurolidine and taurultam solutions on Escherichia coli isolated from a diagnosed urinary tract infection were examined at 37-degrees-C. The inhibitory effects of taurolidine solutions were observed to be greater than those of taurultam solutions at comparative concentrations; however, the presence of sublethal concentrations of formaldehyde (methylene glycol) associated with taurolidine was sufficient to account for this. The bacteriocidal activity of taurolidine (2.0% w/v) was greater than that of taurultam (4.5% w/v). Both compounds produced biphasic death rates with dissimilar initial slopes, suggested to be due to the presence of formaldehyde in taurolidine solutions. These observations indicate that the growth inhibitory and bacteriocidal effects of Taurolin solutions are primarily due to taurultam, however, the presence of sublethal concentrations of formaldehyde is significant in the expression of this activity.

Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase

WaaL is a membrane enzyme implicated in ligating undecaprenyl-diphosphate (Und-PP)-linked O antigen to lipid A-core oligosaccharide. We determined the periplasmic location of a large (EL5) and small (EL4) adjacent loops in the Escherichia coli K-12 WaaL. Structural models of the EL5 from the K-12, R1 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular alpha-helices. One alpha-helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non-functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non-conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates.

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.

The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PPi. We show that, although the GalF protein is expressed in vivo, GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF-/galU+ and galF/galU+ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo. We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110:identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.