HMW1 Is Required for Cytadhesin P1 Trafficking to the Attachment Organelle in Mycoplasma pneumoniae

Mycoplasma pneumoniae proteins HMW1-HMW3 collectively are essential for cytadherence, but the function or requirement for each has not been defined. Cytadherence mutant M6 lacks HMW1 because of a frameshift in hmw1 and produces a truncated adherence-associated protein P30 because of a deletion at the 3′ end of p30. Genetic manipulation of this mutant was used to evaluate the role of HMW1 in cytadherence. Mutant M6 was transformed with a recombinant transposon containing a wild-type p30 allele. Transformants synthesized both truncated and full-length P30, from the resident and recombinant alleles, respectively. However, these transformants remained hemadsorption negative, suggesting that HMW1 is required for cytadherence. Wild-type M. pneumoniae cells are generally elongated, tapering to form the attachment organelle at one end of the cell. The cytadhesin protein P1 is normally densely clustered on the mycoplasma surface at this differentiated terminal structure. However, both mutant M6 and M6 transformed with recombinant p30 had a striking ovoid morphology with no tapering at the tip structure, making the attachment organelle indistinguishable. Furthermore, protein P1 was randomly distributed on the mycoplasma surface rather than clustered at a polar location. In contrast, mutant M6 transformed with a recombinant transposon expressing the wild-type hmw1 allele exhibited a near-normal morphology and localized P1 to the attachment organelle. Significantly, M6 transformed with an hmw1 gene truncated slightly at the 3′ end failed to restore proper morphology or P1 localization to the attachment organelle, suggesting a functional importance to the C-terminal domain of HMW1.     

Deletion analysis identifies key functional domains of the cytadherence-associated protein HMW2 of Mycoplasma pneumoniae

Mycoplasma pneumoniae attachment to host cells requires biogenesis of a functional attachment organelle, including proper localization of the adhesion protein P1 to this structure. Mutations in the hmw2 gene result in the inability to cytadhere, failure to localize P1 to the attachment organelle, altered cell morphology and accelerated turnover of the cytadherence-associated proteins HMW1, HMW3 and P65. The hmw2 gene encodes HMW2 (190 kDa) and P28 (28 kDa), the latter apparently the product of internal translation initiation near the 3' end of the hmw2 coding region. Transformation of hmw2 mutant I-2 with recombinant wild-type hmw2 restores a wild-type phenotype. In the current study, a severely truncated hmw2 gene with an in frame internal deletion of 80% of the HMW2 coding region that leaves the P28-encoding region intact restored cytadherence to mutant I-2. Transformants produced the expected 38 kDa HMW2 derivative (HMW2Deltamid) at levels comparable to that of HMW2 in wild-type cells; like HMW2, HMW2Deltamid exhibited marked Triton X-100 insolubility. HMW3, P65 and P28 were fully restored, but not HMW1. These transformants were morphologically similar to wild-type M. pneumoniae but failed to localize P1 to the attachment organelle. Finally, a C-terminally truncated HMW2 derivative was partly Triton X-100 soluble and incapable of restoring HMW1, HMW3 and P65 to wild-type levels. These data are consistent with a model in which the C-terminal domain of HMW2 imparts normal localization to the protein, and this localization itself is required for productive interactions with downstream cytadherence-associated proteins. Furthermore, these results emphasize the association of HMW1 with P1 clustering.     

Mycoplasma pneumoniae Cytoskeletal Protein HMW2 and the Architecture of the Terminal Organelle

The terminal organelle of Mycoplasma pneumoniae mediates cytadherence and gliding motility and functions in cell division. The defining feature of this complex membrane-bound cell extension is an electron-dense core of two segmented rods oriented longitudinally and enlarging to form a bulb at the distal end. While the components of the core have not been comprehensively identified, previous evidence suggested that the cytoskeletal protein HMW2 forms parallel bundles oriented lengthwise to yield the major rod of the core. In the present study, we tested predictions emerging from that model by ultrastructural and immunoelectron microscopy analyses of cores from wild-type M. pneumoniae and mutants producing HMW2 derivatives. Antibodies specific for the N or C terminus of HMW2 labeled primarily peripheral to the core along its entire length. Furthermore, truncation of HMW2 did not correlate specifically with core length. However, mutant analysis correlated specific HMW2 domains with core assembly, and examination of core-enriched preparations confirmed that HMW2 was a major component of these fractions. Taken together, these findings yielded a revised model for HMW2 in terminal organelle architecture.Mycoplasma pneumoniae is a cell wall-less pathogen of the human respiratory tract causing community-acquired tracheobronchitis and atypical, or “walking,” pneumonia (38). Colonization of the respiratory mucosa is mediated in large part by the terminal organelle, a polar, tapered extension of the mycoplasma cell having a high density of receptor-binding proteins (4, 22, 28). The terminal organelle also constitutes the motor in gliding motility (5, 11), and its duplication precedes cell division (5, 12, 32).Ultrastructurally, the terminal organelle is defined by a characteristic electron-dense core consisting of a thick rod and a thin rod oriented longitudinally in parallel and capped by a terminal button at the distal end (4, 16, 17, 39, 41). The core and terminal button are elements of the mycoplasma cytoskeleton (triton shell), a complex network of proteins resistant to extraction with Triton X-100 (TX) (1, 7, 24), much like the cytoskeletal fraction of eukaryotic cells (18, 33, 34). The composition of the M. pneumoniae triton shell has been examined by using antibody probes (22) and by mass spectrometry (29), but the identities of proteins specific to the electron-dense core are largely unknown, although cores fail to assemble in the absence of cytoskeletal proteins HMW1 and HMW2, both of which localize to the terminal organelle (3, 31, 36).HMW2 is a large protein (1,818 residues) predicted to have a globular N terminus followed by 10 dimeric or trimeric coiled-coil domains interspersed with leucine zipper motifs (23) (Fig. ​(Fig.1).1). Spontaneously arising mutant I-2 lacks HMW2 due to a frameshift in the corresponding MPN310 open reading frame, which also encodes protein P28 at its 3′ end, in the same reading frame encoding HMW2 (6). Mutants C1 and H9 are similar to mutant I-2 but result from Tn4001 disruption of MPN310 (15, 23) (Fig. ​(Fig.1).1). The loss of HMW2 and the inability of these mutants to assemble a core are accompanied by an abnormal morphology, reduced levels of terminal organelle proteins HMW1, HMW3, P24, P28, P41, and P65, failure to localize the major adhesin P1 to the terminal organelle, and the loss of cytadherence (6, 20, 22, 31). Imprecise transposon excision from mutant C1 yielded excision revertant C1R1, having an in-frame deletion in MPN310 that truncates HMW2 and eliminates P28 (6) (Fig. ​(Fig.1).1). Analysis using immunofluorescence (40) or fluorescent protein fusions (3, 19) localizes HMW2 generally to the terminal organelle. Based on its localization, its requirement for core formation, and its deduced length relative to that of the core, we proposed previously that HMW2 is a major component of the electron-dense core and, with P28, may form bundles oriented longitudinally to yield the large rod of the core (3).Open in a separate windowFIG. 1.Structural features of the indicated wild-type (WT), engineered, and mutant HMW2 proteins. White boxes, predicted dimeric coiled coils; black boxes, predicted trimeric coiled coils; dark gray boxes, leucine zipper motifs; arrows, predicted N terminus of P28; black triangles, cysteine residues. The numbers above each diagram correspond to the coiled-coil regions, while the letters below correspond to the leucine zipper motifs.In the present study, we explored further the role of HMW2 in core formation relative to the current model, by which (i) HMW2 is predicted to orient with its N- and C-terminal domains at the ends of the large rod of the core and (ii) mutants producing shorter HMW2 proteins are expected to have correspondingly shorter cores. We report here the successful localization of HMW2 by immunoelectron microscopy (immuno-EM) and the ultrastructural analysis of electron-dense cores in several HMW2 truncation mutants, allowing us to correlate specific regions of HMW2 with normal core formation. Finally, we evaluated core enrichment following detergent and salt extractions, demonstrating that HMW2 was a major component of a core-enriched fraction (CEF). Alternative models for HMW2 in core architecture are considered, based on our observations.     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

HMW1 is required for stability and localization of HMW2 to the attachment organelle of Mycoplasma pneumoniae

The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.     

Evolutionary and functional relationships among the nontypeable Haemophilus influenzae HMW family of adhesins

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Approximately 75 to 80% of NTHi clinical isolates produce proteins that belong to the HMW family of adhesins, which are believed to facilitate colonization. The prototype HMW adhesins are designated HMW1 and HMW2 and were identified in NTHi strain 12. HMW1 and HMW2 are 71% identical and 80% similar overall, yet display differing cellular binding specificities. In the present study we set out to define more clearly the relationships between HMW1 and HMW2 and other members of the HMW family of adhesins. PCR analysis of 49 epidemiologically distinct isolates revealed that all strains possessing hmw genes as determined by Southern analysis contain two hmw loci in conserved, unlinked physical locations on the chromosome. Functional analysis of the HMW adhesins produced by three unrelated strains demonstrated that each isolate possesses one protein with HMW1-like adherence properties and another with HMW2-like adherence properties. These findings suggest that the hmw1 and hmw2 loci may have arisen via a gene duplication event in an ancestral strain. In addition, they support the hypothesis that the distinct binding specificities of HMW1 and HMW2 emerged early and have persisted over time, suggesting an ongoing selective advantage.     

The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates N-linked glycosylation of the Haemophilus influenzae HMW1 adhesin

The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases.     

Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits

Wheat HMW glutenin subunit genes 1Ax1 and 1Dx5 were introduced, and either expressed or overexpressed, into a commercial wheat cultivar that already expresses five subunits. Six independent transgenic events were obtained and characterized by SDS-PAGE and Southern analysis. The 1Dx5 gene was overexpressed in two events without changes in the other endosperm proteins. Overexpression of 1Dx5 increased the contribution of HMW glutenin subunits to total protein up to 22%. Two events express the 1Ax1 subunit transgene with associated silencing of the 1Ax2* endogenous subunit. In the SDS-PAGE one of them shows a new HMW glutenin band of an apparent M_r lower than that of the 1Dx5 subunit. Southern analysis of the four events confirmed transformation and suggest that the transgenes are present in a low copy number. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic wheat expressing the 1Ax1 subunit transgene and overexpressing the Dx5 gene. Transgenes and expression patterns were stably transmitted to the progenies in all the events except one where in some of the segregating T₂ seeds the silencing of all HMW glutenin subunits was reverted associated with a drastic lost of transgenes from a high to a low copy number. The revertant T₂ seeds expressed the five endogenous subunits plus the 1Ax1 transgene. Received: 16 June 1999 / Accepted: 29 July 1999     

Juxtaposition of the genes encoding Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW3.

The loss and reacquisition of high-Mr (HMW) proteins, HMW1, 2, 3, 4 and 5, by Mycoplasma pneumoniae correlates with cytadherence phase variation. We are cloning and characterizing the genes encoding HMW1-5 to understand the mechanism regulating their coordinate expression. HMW1 was purified by polyacrylamide-gel electrophoresis. Amino acid (aa) sequence data were obtained from enzymatically generated peptide fragments from HMW1. A degenerate 17-mer probe synthesized based upon the aa sequence of one peptide clearly identified a single 4.75-kb BamHI fragment of M. pneumoniae DNA under stringent hybridization conditions. This fragment was cloned into pUC19 to generate pKV16. Restriction mapping of the 4.75-kb BamHI fragment in pKV16 revealed a possible overlap with the 9.4-kb EcoRI fragment containing the gene encoding protein HMW3. Southern blotting and reciprocal hybridization studies confirmed this overlap, establishing the juxtaposition of the genes encoding HMW1 and HMW3. Finally, physical mapping analysis by probing restriction fragments of M. pneumoniae DNA resolved by pulsed-field gel electrophoresis with the cloned genes encoding HMW1 and HMW3 revealed definitively that the hmw locus maps to a 106.8-kb ApaI fragment, rather than a 117.5-kb ApaI fragment, as had been reported previously for hmw3 [Krause and Mawn, J. Bacteriol. 172 (1990) 4790-4797].     

Sequence analysis and characterization of the hmw gene cluster of Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp) cytadherence requires the proper anchoring of cytadhesin proteins in the mycoplasmal membrane at an attachment organelle through their interaction with a cytoskeleton-like network of accessory proteins that includes HMW1 and HMW3. Approximately 8.25 kb of Mp DNA was sequenced, beginning at the 3' end of the hmw3 gene and continuing through hmwl. Comparison of the resulting deduced amino acid (aa) sequence with N terminus and internal peptide aa sequences from purified HMW1 permitted definitive identification of hmwl. HMW1 was characterized with respect to structure, hydrophobicity, possible phosphoacceptor sites and expression of the Mp recombinant protein in Escherichia coli. In addition, HMW1 membrane topography was examined for antibody accessibility on the mycoplasmal surface. hmw3 and hmwl flank four open reading frames (ORFs) spanning approximately 4.3 kb and in the same orientation as the hmw genes. The sequences of their deduced products were evaluated for likely structural features and comparison with protein data banks. Finally, the Mp rpsD analog was identified immediately downstream from hmwl.     

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S^s genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S^sx49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S^sy49077) from the same accession. Consequently, 1S^sx49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S^sx49077 subunit is presented.     

The TpsB translocator HMW1B of haemophilus influenzae forms a large conductance channel

The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane beta-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.     

The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain essential for bacterial two-partner secretion

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and HMW2B outer membrane translocators by an N-terminal region called the secretion domain. Here we present the crystal structure at 1.92 A of the HMW1 pro-piece (HMW1-PP), a region that contains the HMW1 secretion domain and is cleaved and released during HMW1 secretion. Structural analysis of HMW1-PP revealed a right-handed beta-helix fold containing 12 complete parallel coils and one large extra-helical domain. Comparison of HMW1-PP and the Bordetella pertussis FHA secretion domain (Fha30) reveals limited amino acid homology but shared structural features, suggesting that diverse TpsA proteins have a common structural domain required for targeting to cognate TpsB proteins. Further comparison of HMW1-PP and Fha30 structures may provide insights into the keen specificity of TpsA-TpsB interactions.     

Circulating High-Molecular-Weight (HMW) Adiponectin Level Is Related with Breast Cancer Risk Better than Total Adiponectin: A Case-Control Study

The level of total adiponectin, a mixture of different adiponectin forms, has been reported associated with breast cancer risk with inconsistent results. Whether the different forms play different roles in breast cancer risk prediction is unclear. To examine this, we measured total and high molecular weight (HMW) adiponectin in a case-control study (1167 sets). Higher circulating HMW adiponectin was negatively associated with breast cancer risk after adjusting for menopausal status and family history of breast cancer (P=0.024). We analyzed the relationship between adiponectin and breast cancer risk in 6 subgroups. Higher circulating HMW adiponectin was also negatively associated with breast cancer risk (P=0.020, 0.014, 0.035) in the subgroups of postmenopausal women, negative family history of breast cancer, BMI>=24.0. Total adiponectin was positively associated with breast cancer (P=0.028) in the subgroup of BMI<=24.0. Higher HMW/total adiponectin ratio was negatively associated with breast cancer (P=0.019) in the subgroup of postmenopausal women. Interestingly, in the subgroup of women with family history of breast cancer, higher circulating total and HMW adiponectin were positively associated with breast cancer risk (P=0.034, 0.0116). This study showed different forms of circulating adiponectin levels might play different roles in breast cancer risk. A higher circulating HMW adiponectin is associated with a decreased breast cancer risk, especially in postmenopausal, without family history of breast cancer or BMI>=24.0 subgroups, whereas higher circulating HMW adiponectin levels is a risk factor in women with a family history of breast cancer. Further investigation of different forms of adiponectin on breast cancer risk is needed.     

In Vitro Gibberellin A(1) Binding in Zea mays L

The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [³H]gibberellin A₁ (GA₁) to a soluble macromolecular component present in the cytosol was demonstrated at 4°C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the ³H-activity bound to this protein was largely [³H]GA₁ and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA₁. Both biologically active and inactive GAs and non-GAs were able to inhibit GA₁ binding. [³H]GA₁ binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography.     

Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt ( Triticum spelta L.)

A partial promoter region of the high-molecular weight (HMW) glutenin genes was studied in two wheat specimens, a 300 year-old spelt (Triticum spelta L.) and an approximately 250 year-old bread wheat (Triticum aestivum L.) from Switzerland. Sequences were compared to a recent Swiss landrace T. spelta ’Oberkulmer.’ The alleles from the historical bread wheat were most similar to those of modern T. aestivum cultivars, whereas in the historical and the recent spelt specific alleles were detected. Pairwise genetic distances up to 0.03 within 200 bp from the HMW Glu-A1-2, Glu-B1-1 and Glu-B1-2 alleles in spelt to the most-similar alleles from bread wheat suggest a polyphyletic origin. The spelt Glu-B1-1 allele, which was unlike the corresponding alleles in bread wheat, was closer related to an allele found in tetraploid wheat cultivars. The results are discussed in context of the origin of European spelt. Received: 22 July 2000 / Accepted: 27 April 2001     

Structure of the Haemophilus influenzae HMW1B translocator protein: evidence for a twin pore

Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded beta-barrel domain, the approximate size of the beta-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 A resolution. The native HMW1B structure is a dimer of beta-barrels, with each beta-barrel measuring 40 A by 50 A in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.     

Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes

The high-molecular-weight (HMW) subunits of wheat glutenin are the major determinants of the gluten visco-elasticity that allows wheat doughs to be used to make bread, pasta and other food products. In order to increase the proportions of the HMW subunits, and hence improve breadmaking performance, particle bombardment was used to transform tritordeum, a fertile amphiploid between wild barley and pasta wheat, with genes encoding two HMW glutenin subunits (1Ax1 and 1Dx5). Of the 13 independent transgenic lines recovered (a transformation frequency of 1.4%) six express the novel HMW subunits at levels similar to, or higher than, those of the endogenous subunits encoded on chromosome 1B. Small-scale mixograph analysis of T₂ seeds from a line expressing the transgene for 1Dx5 indicated that the addition of novel HMW subunits can result in significant improvements in dough strength and stability, thus demonstrating that transformation can be used to modify the functional properties of tritordeum for improved breadmaking. Received: 15 January 1999 / Accepted: 5 February 1999     

Ecogeographical distribution of HMW glutenin alleles in populations of the wild tetraploid wheat Triticum turgidum var. dicoccoides

Summary Polymorphism of high molecular weight (HMW) glutenin subunits in 466 accessions of the wild tetraploid wheat Triticum turgidum var. dicoccoides in Israel was characterized with regard to the ecogeographical distribution of the HMW glutenin alleles, both between and within 22 populations, and along transects in a single population. While some populations were monomorphic for all the HMW glutenin loci, namely, Glu-A1-1, Glu-A1-2, Glu-B1-1 and Glu-B1-2, others contained up to four alleles per locus. Intrapopulation variability could be predicted by the geographical distribution: marginal populations tended to be more uniform than those at the center of distribution. The various HMW glutenin alleles tended to be clustered, both at a regional level and within a single population along transects of collection. It is suggested that this clustering is due to selection pressures acting both at a regional and at a microenvironmental level. This was confirmed by the significant correlations found between the MW of subunits encoded by Glu-A1-1 and the populations' altitude, average temperature and rainfall. The possible selective values of seed storage proteins are discussed.