Structural characterization of closely related O-antigen lipopolysaccharide (LPS) chain length regulators

The surface O-antigen polymers of Gram-negative bacteria exhibit a modal length distribution that depends on dedicated chain length regulator periplasmic proteins (polysaccharide co-polymerases, PCPs) anchored in the inner membrane by two transmembrane helices. In an attempt to determine whether structural changes underlie the O-antigen modal length specification, we have determined the crystal structures of several closely related PCPs, namely two chimeric PCP-1 family members solved at 1.6 and 2.8 Å and a wild-type PCP-1 from Shigella flexneri solved at 2.8 Å. The chimeric proteins form circular octamers, whereas the wild-type WzzB from S. flexneri was found to be an open trimer. We also present the structure of a Wzz^FepE mutant, which exhibits severe attenuation in its ability to produce very long O-antigen polymers. Our findings suggest that the differences in the modal length distribution depend primarily on the surface-exposed amino acids in specific regions rather than on the differences in the oligomeric state of the PCP protomers.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Structure and heterogeneity of the O-antigen chain of Salmonella Agona lipopolysaccharide

Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.     

Functional analysis of predicted coiled-coil regions in the Escherichia coli K-12 O-antigen polysaccharide chain length determinant Wzz

Wzz is a membrane protein that determines the chain length distribution of the O-antigen lipopolysaccharide by an unknown mechanism. Wzz proteins consist of two transmembrane helices separated by a large periplasmic loop. The periplasmic loop of Escherichia coli K-12 Wzz (244 amino acids from K65 to A308) was purified and found to be a monomer with an extended conformation, as determined by gel filtration chromatography and analytical ultracentrifugation. Circular dichroism showed that the loop has a 60% helical content. The Wzz periplasmic loop also contains three regions with predicted coiled coils. To probe the function of the predicted coiled coils, we constructed amino acid replacement mutants of the E. coli K-12 Wzz protein, which were designed so that the coiled coils could be separate without compromising the helicity of the individual molecules. Mutations in one of the regions, spanning amino acids 108 to 130 (region I), were associated with a partial defect in O-antigen chain length distribution, while mutants with mutations in the region spanning amino acids 209 to 223 (region III) did not have an apparent functional defect. In contrast, mutations in the region spanning amino acids 153 to 173 (region II) eliminated the Wzz function. This phenotype was associated with protein instability, most likely due to conformational changes caused by the amino acid replacements, which was confirmed by limited trypsin proteolysis. Additional mutagenesis based on a three-dimensional model of region I demonstrated that the amino acids implicated in function are all located at the same face of a predicted α-helix, suggesting that a coiled coil actually does not exist in this region. Together, our results suggest that the regions predicted to be coiled coils are important for Wzz function because they maintain the native conformation of the protein, although the existence of coiled coils could not be demonstrated experimentally.     

Specificity of enzymes of biosynthesis of Salmonella anatum O-antigen for polyprenyl derivatives of different chain length and saturation

Polyprenyl phosphates of different structure were prepared and their ability to serve as sugar acceptors in the biosynthesis of O-specific polysaccharides of Salmonella anatum was investigated. It was demonstrated that C30-C80-polyprenyl phosphates with unsaturated alpha-isoprene unit were as active as natural acceptor (undecaprenyl phosphate) in this enzymic system. C15- and C100-polyprenyl phosphates of this series were less effective in O-antigen repeating unit formation. Citronellyl and dolichyl phosphates (derivatives of C10- and C105-polyprenols, respectively, with saturated alpha-isoprene unit) were poor substrates. For polymerization of repeating units, the polyprenol chain-length is of utmost importance: its shortening results in a marked drop in the efficiency of respective compounds as substrates.     

STM2209-STM2208 (opvAB): a phase variation locus of Salmonella enterica involved in control of O-antigen chain length

STM2209 and STM2208 are contiguous loci annotated as putative protein-coding genes in the chromosome of Salmonella enterica. Lack of homologs in related Enterobacteria and low G+C content suggest that S. enterica may have acquired STM2209-STM2208 by horizontal transfer. STM2209 and STM2208 are co-transcribed from a promoter upstream STM2209, and their products are inner (cytoplasmic) membrane proteins. Analysis with the bacterial adenylate cyclase two-hybrid system suggests that STM2209 and STM2208 may interact. Expression of STM2209-STM2208 is subjected to phase variation in wild type Salmonella enterica serovar Typhimurium. Switching frequencies in LB medium are 6.1×10(-5) (OFF→ON) and 3.7×10(-2) (ON→OFF) per cell and generation. Lack of DNA adenine methylation locks STM2209-STM2208 in the ON state, and lack of the LysR-type factor OxyR locks STM2209-STM2208 in the OFF state. OxyR-dependent activation of STM2209-STM2208 expression is independent of the oxidation state of OxyR. Salmonella cultures locked in the ON state show alteration of O-antigen length in the lipopolysaccharide, reduced absorption of bacteriophage P22, impaired resistance to serum, and reduced proliferation in macrophages. Phenotypic heterogeneity generated by STM2209-STM2208 phase variation may thus provide defense against phages. In turn, formation of a subpopulation unable to proliferate in macrophages may restrain Salmonella spread in animal organs, potentially contributing to successful infection.     

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).     

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.     

Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro

We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei which contains C14 acyl and alkyl chains shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.     

Sequencing of laminin B chain cDNAs reveals C-terminal regions of coiled-coil alpha-helix.

cDNAs for laminin B chains have been isolated from a parietal endoderm cDNA library in pUC8 and pUC9. Identification is based on: ability to direct the synthesis in Escherichia coli of polypeptides carrying laminin antigen determinants, in vitro translation of hybrid selected mRNA, and hybridization to high mol. wt. RNA differentially expressed in cells synthesizing large amounts of laminin. The plasmid pPE9 hybrid selects mRNA for the B2 (mol. wt. 185 000) chain and provides 217 residues of C-terminal amino acid sequence. The plasmids pPE386 and 49 both hybrid select mRNAs for the B1a (mol. wt. 205 000) and B1b (mol. wt. 200 000) chains. These two cDNAs are identical over much of their sequence, but pPE386 includes 133 nucleotides of 3' non-coding sequence and a poly(A) tail. Together they provide 495 residues of C-terminal amino acid sequence. Analysis of the predicted sequences reveals a striking heptad repeat, with a high probability that residues a and d are hydrophobic. Such a repeat is typical of the coiled-coil alpha-helices found in proteins such as myosin, tropomyosin and desmin (2-stranded) and fibrinogen (3-stranded).     

Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation

We have determined the primary structure of the myosin heavy chain (MHC) of the striated adductor muscle of the scallop Aequipecten irradians by cloning and sequencing its cDNA. It is the first heavy chain sequence obtained in a directly Ca(2+)-regulated myosin. The 1938-amino acid sequence has an overall structure similar to other MHCs. The subfragment-1 region of the scallop MHC has a 59-62% sequence identity with sarcomeric and a 52-53% identity with nonsarcomeric (smooth and metazoan nonmuscle) MHCs. The heavy chain component of the regulatory domain (Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'Neall-Hennessey, E., Melandri, F. D., and Szent-Gy?rgyi, A. G. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4771-4775) starts at either Leu-755 or Val-760. Ca(2+)-sensitive Trp residues (Wells, C., Warriner, K. E., and Bagshaw, C. R. (1985) Biochem. J. 231, 31-38) are located near the C-terminal end of this segment (residues 818-827). More detailed sequence comparison with other MHCs reveals that the 50-kDa domain and the N-terminal two-thirds of the 20-kDa domain differ substantially between sarcomeric and nonsarcomeric myosins. In contrast, in the light chain binding region of the regulatory domain (residues 784-844) the scallop sequence shows greater homology with regulated myosins (smooth muscle, nonmuscle, and invertebrate striated muscles) than with unregulated ones (vertebrate skeletal and heart muscles). The N-terminal 25-kDa domain also contains several residues which are preserved only in regulated myosins. These results indicate that certain heavy chain sites might be critical for regulation. The rod has features typical of sarcomeric myosins. It is 52-60% and 30-33% homologous with sarcomeric and nonsarcomeric MHCs, respectively. A Ser-rich tailpiece (residues 1918-1938) is apparently nonhelical.     

The structural basis of chain length control in Rv1086

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C₅₀) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.     

Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction.

HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.     

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz

Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.     

Detection and investigation of Campylobacter jejuni by polymerase chain reaction-restriction fragment length polymorphism analysis

A molecular typing approach for Campylobacter jejuni with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni, was generated and studied. Using polymerase chain reaction (PCR)-RFLP with the restriction endonuclease Mbo I, it was demonstrated that C. jejuni could be divided into four types. Genotypic analysis of C. jejuni by PCR-RFLP is a valuable technique for epidemiological typing.