Attachment and Biofilm Formation of <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> on a Hydrophobic Surface at the Air Interface

Properties of attachment of Mycobacterium marinum to hydrophobic surfaces and subsequent␣biofilm formation were investigated. Binding of M. marinum to polypropylene occured under aerobic and anaerobic/microaerophilic conditions. However, aerobic conditions were necessary for biofilms to persist. Highly non-polar organic solvents were found to efficiently remove attached bacteria from the polypropylene surface, indicating strong hydrophobic interactions between the M. marinum cell wall and the surface. Increased capsular material, occurring during stationary phase, correlated with a decrease in attachment of cells to polypropylene. A protein of approximately 40 kDa appears to be present in increased amounts during the stationary phase. The protein has been identified by LC MS/MS analysis as alanine dehydrogenase.     

Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum

Lipoteichoic acid is a major cell wall virulence factor of gram-positive bacteria. LTAs from various bacteria have differential immunostimulatory potentials due to heterogeneity in their structures. Although recent studies have demonstrated that LTA isolated from Lactobacillus plantarum (pLTA) has anti-inflammatory properties and is less inflammatory than LTAs from pathogenic bacteria, little is known about the structure of pLTA. In this study, high-field NMR spectra of the pLTA were compared with those of LTA from pathogenic bacterium, Staphylococcus aureus (aLTA). The 2D NMR results demonstrated that pLTA possesses α-linked hexose sugar substituents on the poly-glycerophosphate backbone instead of N-acetylglucosamine substituents, and unsaturated fatty acids in its glycolipids. The sugar substituents were revealed as an approximately 29:1 molar ratio of the glucose to galactose by HPAEC-PAD analysis. MALDI-TOF/TOF MS analyses identified the presence of unsaturated fatty acids in the glycolipid moieties of pLTA. In addition, the glycolipid structure was found to be composed of trihexosyl-diacyl- and/or trihexosyl-triacyl-glycerol ceramide units by means of unique fragment ions of the glycolipids. These results enabled us to elucidate the pLTA structure, which is distinctively different from canonical LTA structure, and suggest that the unique immunological property of pLTA might be caused by the pLTA structure.     

A structural model for the adduct between cytochrome c and cytochrome c oxidase

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein–protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc ₁ to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc ₁–cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space. Protein Data Bank Accession numbers The coordinates of the five best structural models for each of the four clusters have been deposited in the Protein Data Bank (PDB ID 1ZYY).     

Structural Basis of a Physical Blockage Mechanism for the Interaction of Response Regulator PmrA with Connector Protein PmrD from Klebsiella pneumoniae

In bacteria, the two-component system is the most prevalent for sensing and transducing environmental signals into the cell. The PmrA-PmrB two-component system, responsible for sensing external stimuli of high Fe³⁺ and mild acidic conditions, can control the genes involved in lipopolysaccharide modification and polymyxin resistance in pathogens. In Klebsiella pneumoniae, the small basic connector protein PmrD protects phospho-PmrA and prolongs the expression of PmrA-activated genes. We previously determined the phospho-PmrA recognition mode of PmrD. However, how PmrA interacts with PmrD and prevents its dephosphorylation remains unknown. To address this question, we solved the x-ray crystal structure of the N-terminal receiver domain of BeF₃⁻-activated PmrA (PmrA_N) at 1.70 Å. With this structure, we applied the data-driven docking method based on NMR chemical shift perturbation to generate the complex model of PmrD-PmrA_N, which was further validated by site-directed spin labeling experiments. In the complex model, PmrD may act as a blockade to prevent phosphatase from contacting with the phosphorylation site on PmrA.     

Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c)

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. ¹H and ¹³C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap2Ac-(1→3)[α-d-Glcp-(1→2)-α-d-Glcp-(1→4)]-β-d-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Annular and non-annular binding sites on the (Ca + Mg)-ATPase

Quenching of the fluorescence of the (Ca²⁺ + Mg²⁺)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

O-antigen structure and gene clusters of Escherichia coli O51 and Salmonellaenterica O57; another instance of identical O-antigens in the two species

The O-polysaccharides were released by mild acid hydrolysis from the lipopolysaccharides of Escherichia coli O51 and Salmonella enterica O57 and found to possess the same structure, which was established by sugar analysis and 1D and 2D NMR spectroscopy: The O-antigen gene clusters of E. coli O51 and S. enterica O57 were sequenced and found to contain the same genes with a high-level similarity. All genes expected for the synthesis of the O-antigen were identified based on their similarity to genes from available databases.     

An NMR microscopic study of grape (Vitis vinifera L.)

Summary Mature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by¹H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T₂-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T₁-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T₁-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T₂-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.Abbrevations BF bright field - FDA fluorescein diacetate - FI field inhomogeneity - FOV field of view - NMR nuclear magnetic resonance - RF radiofrequency - T₁ spin-lattice relaxation time - T₂ spin-spin relaxation time - TE echo time - TMS tetramethylsilane - TR repeat time     

A theoretical model of Aquifex pyrophilus flagellin: implications for its thermostability

Aquifex pyrophilus is a flagellated hyperthermophilic eubacterial species that grows optimally at 85°C. The thermostable A. pyrophilus flagellar filament is primarily composed of a single protein called flagellin (FlaA). The N- and C-terminal sequence regions of FlaA are important for self-assembly and share high sequence similarity with mesophilic bacterial flagellins. We have developed a predictive 3D-structure of FlaA, using the published structure of mesophilic Salmonella typhimurium flagellin (FliC) as a template and analyzed it with respect to possible determinants of thermostability. A sequence comparison of FlaA and FliC revealed a +7.0% increase in FlaA hydrophobic residues, a +0.6% increase in charged residues and a corresponding decrease of −6.0% in polar residues. The FlaA N- and C-termini also have higher proportions of hydrophobic and charged residues at the expense of polar residues and higher non-polar surface areas. Thus, a predominant stabilizing factor in FlaA appears to be increased hydrophobicity, which often confers greater rigidity to proteins. Fewer intramolecular ion pairs were observed in FlaA than FliC, although an increase in the positive charge potential of the FlaA D0 and D1 domains was also observed; increased intermolecular salt bridges may also contribute to the thermal stability of the oligomeric flagellar fiber. Figure a Model 3D structure of thermostable A. pyrophilus FlaA flagellin. b 3D structure of mesophilic S. typhimurium FliC flagellin. α-helices are shown in red, β-sheets are shown in yellow, turns are shown in blue and coils are shown in green. Both the structures show domains D0, D1, D2 and D3. c, d Stereo view of surface charge distribution of FlaA. e, f Stereo view of surface charge distribution of FliC. The legend shows the scale for coloring of the charge spectrum. Blue represents positive charge, red represents negative charge and white represents neutral     

Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix α C corresponding to a substitution of Met¹¹⁵ by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.     

High-resolution solution structure of Bacillus subtilis IIAglc

The high-resolution solution structure of the phosphocarrier protein IIA^glc from Bacillus subtilis is determined using 3D and 4D heteronuclear NMR methods. B. subtilis IIA^glc contains 162 amino acid residues and is one of the larger proteins for which high-resolution solution structure has been determined by NMR methods. The structures have been calculated from a total of 2,232 conformational constraints. Comparison with the X-ray crystal structure indicates that the overall fold is the same in solution and in crystalline environments, although some local structural differences are observed. These occur largely in turns and loops, and mostly correspond to regions with high-temperature factors in the crystal structure. The N-terminus of IIA^glc is disordered in solution. The active site is located in a concave region of the protein surface. The histidine, which accepts the phosphoryl group (His 83), interacts with a neighboring histidine (His 68) and is surrounded by hydrophobic residues. Proteins 31:258–270, 1998. © 1998 Wiley-Liss, Inc.     

棉花DUR3基因的鉴定及进化分析

植物DUR3同源蛋白属于钠离子/溶质共运蛋白家族的尿素高亲和力运输蛋白，在植物体对外源尿素的主动吸收及内源尿素的再分配过程中具有重要作用。为明确棉花DUR3基因的结构和进化情况，基于生物信息学的方法，从全基因组水平鉴定陆地棉和雷蒙德氏棉的DUR3基因，并对基因结构、跨膜结构域、基序分布、进化关系等进行分析。结果表明：(1)从陆地棉A亚组和D亚组染色体各鉴定出1个DUR3基因，从雷蒙德氏棉基因组鉴定出1个DUR3基因。这3个棉花DUR3同源蛋白同其他植物DUR3同源蛋白一样，具有15个跨膜结构域，具有3个位置一致、高度保守的基序。(2)基因结构分析表明，双子叶植物DUR3基因的外显子个数明显多于单子叶植物，这3个棉花DUR3基因的外显子个数亦是如此。(3)根据物种间种属亲缘关系，对不同物种DUR3氨基酸序列构建的进化树显示，棉花的同双子叶植物的聚在一起。(4)DUR3直系同源基因和旁系同源基因的K_a/K_s比值普遍均大于1,说明这些基因在进化过程中主要受到正向选择的作用。该研究结果为深入研究棉花DUR3同源蛋白提供了理论基础。     

Identification of a chloroform-soluble membrane miniprotein in Escherichia coli and its homolog in Salmonella typhimurium

Two homologous 29 amino acid-long highly hydrophobic membrane miniproteins were identified in the Bligh–Dyer lipid extracts of Escherichia coli and Salmonella typhimurium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The amino acid sequences of the proteins were determined by collision-induced dissociation tandem mass spectrometry, in conjunction with a translating BLAST (tBLASTn) search, i.e., comparing the MS/MS-determined protein query sequence against the six-frame translations of the nucleotide sequences of the E. coli and S. typhimurium genomes. Further MS characterization revealed that both proteins retain the N-terminal initiating formyl-methionines. The methodologies described here may be amendable for detecting and characterizing small hydrophobic proteins in other organisms that are difficult to annotate or analyze by conventional methods.     

Building native protein conformation from NMR backbone chemical shifts using Monte Carlo fragment assembly

We have been analyzing the extent to which protein secondary structure determines protein tertiary structure in simple protein folds. An earlier paper demonstrated that three-dimensional structure can be obtained successfully using only highly approximate backbone torsion angles for every residue. Here, the initial information is further diluted by introducing a realistic degree of experimental uncertainty into this process. In particular, we tackle the practical problem of determining three-dimensional structure solely from backbone chemical shifts, which can be measured directly by NMR and are known to be correlated with a protein's backbone torsion angles. Extending our previous algorithm to incorporate these experimentally determined data, clusters of structures compatible with the experimentally determined chemical shifts were generated by fragment assembly Monte Carlo. The cluster that corresponds to the native conformation was then identified based on four energy terms: steric clash, solvent-squeezing, hydrogen-bonding, and hydrophobic contact. Currently, the method has been applied successfully to five small proteins with simple topology. Although still under development, this approach offers promise for high-throughput NMR structure determination.     

Mechanism of isoproturon resistance in <Emphasis Type="Italic">Phalaris minor:</Emphasis> in silico design,synthesis and testing of some novel herbicides for regaining sensitivity

Isoproturon, 3-p-cumenyl-1 dimethylurea was the only herbicide controlling Phalaris minor, a major weed growing in wheat fields till the early 1980s. Since it has acquired resistance against isoproturon, like other substituted urea herbicides, where the identified target site for isoproturon is in the photosynthetic apparatus at D1 protein of Photosystem-II (PS-II). Nucleotide sequence of susceptible and resistant psbA gene of P. minor has been reported to have four point mutations. During the present work D1 protein of both susceptible and resistant biotypes of P Minor has been modeled. Transmembrane segments of amino acids were predicted by comparing with the nearest homolog of bacterial D1 protein. Volume and area of active site of both susceptible and resistant biotypes has been simulated. Isoproturon was docked at the active site of both, susceptible and resistant D1 proteins. Modeling and simulation of resistance D1 protein indicates that the resistance is due to alteration in secondary structure near the binding site, resulting in loss in cavity area, volume and change in binding position, loss of hydrogen bonds, hydrophobic interaction and complete loss of hydrophobic sites. To regain sensitivity in resistant biotype new derivatives of isoproturon molecules have been proposed, synthesized and tested. Among the 17 derivatives we found that the N-methyl triazole substituted isoproturon is a potential substitute for isoproturon.     

Structure of the N-terminal region of Haemophilus influenzae H10017: implications for function

Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. HI0017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of HI0017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel -sheet and two short -helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of HI0017 has an amino acid sequence that closely resembles pyruvate formate-lyase – an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of HI0017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate.     

Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm

A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine -class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.Figure: A three-dimensional (3D) structure of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. This modeled 3D structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668).     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.