Structural characterization of lipid A component of Erwinia carotovora lipopolysaccharide

The chemical structure of the lipid A component of lipopolysaccharide excreted into the liquid medium by the plant pathogenic enterobacterium Erwinia carotovora FERM P-7576 was characterized. It consists of a -1, 6-linked glucosamine disaccharide which carries ester-and amide-bound fatty acids and phosphate similar to the lipid A from other gram-negative bacteria. The lipid A preparation was not uniform in the number and composition of the fatty acids linked to the disaccharide. Four prominent lipids A were involved, they were composed of five to seven residues of fatty acid. Among them the major component was hexa-acyl lipid A, in which the hydroxyl group at position 3 and the amino group of the non-reducing glucosamine unit carry 3-dodecanoyl-oxytetradecanoyl residues. Positions 2 and 3 of the reducing glucosamine unit were substituted by 3-hydroxytetradecanoic acid. In the hepta-acyl lipid A, an additional hexadecanoic acid was linked to the hydroxyl group of the 3-hydroxytetradecanoyl residue at position 2 of the hexa-acyl lipid A. Two penta-acyl lipids A were the homologs of the hexa-acyl lipid A with decreasing acylation. Dodecanoic acid was missing from one, and 3-hydroxytetradecanoic acid from another. 3-Dodecanoyloxytetradecanoyl residue at position 3 differentiates E. carotovora lipid A from that of other gram-negative bacteria.Abbreviations LPS lipopolysaccharide - GlcN glucosamine - KDO 3-deoxy-d-manno-octulosonic acid - FAB-MS fast atom bombardment mass spectrometry - u atomic mass unit     

Geminiviruses: Genome structure and gene function

The plant pathogenic single‐strand DNA‐containing geminiviruses have been the recent focus of intense investigation, owing both to their agronomic importance and to their potential as vectors for the expression of foreign genes in plants. Molecular genetic studies have provided detailed information on the genomic organization of many of these viruses. A greater genetic complexity has been demonstrated among the members of this viral family than had previously been suspected, as well as an apparently rapid rate of evolution of genetic diversity. We now recognize fundamental differences in the genome structure and organization of the whitefly‐ and leafhopper‐transmitted viruses, as well as among those geminiviruses infecting dicotyledonous or monocotyledonous hosts. This knowledge has provided new insights into the evolution of these viruses. The viral genes involved in replication and in systemic movement in the plant have been defined, and viral origins for single‐strand (ss) and double‐strand (ds) DNA replication have been mapped to small nucleotide regions. With the structural features of the viral genomes now well defined, current efforts are focused on elucidating the molecular aspects of viral gene regulation and interactions with host‐cell components that lead to the production of disease. Recent progress in determining the mechanism of replication and systemic movement and the contributions of these to symptom and disease development are discussed in the context of the potential for genetically engineering disease‐resistant plants.     

Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels.

An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root. The algorithm encoded in STAMP (STructural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases. In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij' provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij' values and thus provide a reproducible resource for studies of residue conservation within structural motifs.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.     

Application of a directed conformational search for generating 3-D coordinates for protein structures from alpha-carbon coordinates.

A directed conformational search algorithm using the program CONGEN (ref. 3), which samples backbone conformers, is described. The search technique uses information from the partially built structures to direct the search process and is tested on the problem of generating a full set of backbone Cartesian coordinates given only alpha-carbon coordinates. The method has been tested on six proteins of known structure, varying in size and classification, and was able to generate the original backbone coordinates with RMSs ranging from 0.30-0.87A for the alpha-carbons and 0.5-0.99A RMSs for the backbone atoms. Cis peptide linkages were also correctly identified. The procedure was also applied to two proteins available with only alpha-carbon coordinates in the Brookhaven Protein Data Bank; thioredoxin (SRX) and triacylglycerol acylhydrolase (TGL). All-atom models are proposed for the backbone of both these proteins. In addition, the technique was applied to randomized coordinates of flavodoxin to assess the effects of irregularities in the data on the final RMS. This study represents the first time a deterministic conformational search was used on such a large scale.     

Developmental changes in barrier web structure under different levels of predation risk inNephila clavipes (Araneae: Tetragnathidae)

Individuals of the orb-weaving spider Nephila clavipesbuild complex webs with a region used for prey capture, the orb, and tangle webs opposite either face, the barrier webs. Barrier webs have been hypothesized to serve a variety of functions, including predator defense, and the primary function of the barrier web should be reflected in the relative size of the barrier to the orb under varying conditions of foraging success and predation risk. To investigate the effects of predation pressure and foraging success on barrier web structure, I conducted a comparative study in three disjunct populations that differed in predation risk and foraging success. Although both the orb web and the barrier webs are silk, there was no indication of a foraging-defense trade-off. Barrier web structure did not change during seasonal shifts in orb web size related to changes in preycapture rate, and barrier web silk density and orb radius were positively correlated. The hypothesis that the construction of barrier webs is in part a response to predation pressure was supported. Barrier webs do deflect attacks by some predators, and barrier webs built by small spiders, suffering frequent predation attempts, had a higher silk density than barrier webs built by larger individuals. Additionally, barrier web complexity decreased at a later age in areas with higher predation risk.     

Succession of sessile organisms on experimental plates immersed in Nabeta Bay,Izu Peninsula,Japan V. An integrated consideration on the definition and prediction of succession

Successional changes of sessile organisms over about 3 years on concrete plates immersed at depths of 1.0, 2.5, 4.0 and 5.5 m in Nabeta Bay, Shimoda, Pacific coast of Japan, were investigated from their species composition, percentage cover, and the structure, metabolism, biomass and maturity of the community for the purpose of verifying the hypothesis of succession by Margalef (1968) and Odum (1969). The process of succession was divided into five stages from a cluster analysis based on Horn's (1966) similarity in community structure. The five stages were characterized from dominant, species and 18 community attributes in terms of structure, metabolism, biomass and maturity. With the advance of stages, two large-sized, long-lived species, the kelpEisenia bicyclis and the oysterCrassostrea nippona, dominated; 18 community attributes showed a directional succession ending in a constant community structure, low community metabolism, accumulation of community biomass except chlorophylla and an enhanced degree of community maturity. This directionality in succession of community attributes was valid for the prediction of Margalef and Odum, despite a tendency for estimated daily energy budget to be more heterotrophic in such an open system. From these results, it was suggested that the definition of succession by Odum (1969) should be interpreted as the regularity in shift of dominant species, not individual species, and as the control of system by feedback mechanism developing with succession rather than community-controlled mechanism.     

Size dependency of gametophytes decay inAthyrium brevifrons Nakai during spring desiccation

Gametophyte populations inAthyrium brevifrons were analysed with respect to population size and surviving area (%) of individual thalli in a transplant garden at Sapporo during 5–26 April 1983, to study the safe-microsite for gametophyte establishment in nature. Spores dispersed in August 1982 germinated and grew into thalli of various widths (<10 mm); 10.3% of the thalli matured by early October 1982. Maturation was attained by gametophytes of width 4–7 mm. The number of gametophytes gradually decreased with increasing width. By April 1983, 20.5% of total gametophytes were mature with a mode of 5–6 mm in width. The relative number of gametophytes with surviving area of 2–20% increased and that of 85–100% decreased in accordance with collection days delayed until after snow-melt. Surviving area (%) on gametophyte of all widths decreased with decreasing soil moisture contents. In particular, immature gametophytes of 2–4 mm width showed a significant correlation (P<0.01) between soil moisture content and relative number of gametophytes with 0–20% surviving area and mean surviving area (%) of every width of thalli. The spring desiccation might be a factor that reduces or limits gametophyte populations in nature.