Characterization of Colony Morphology Variants Isolated from Pseudomonas aeruginosa Biofilms

ABSTRACT In this study, we report the isolation of small, rough, strongly cohesive colony morphology variants from aging Pseudomonas aeruginosa PAO1 biofilms. Similar to many of the P. aeruginosa colony morphology variants previously described in the literature, these variants autoaggregate in liquid culture and hyperadhere to solid surfaces. They also exhibit increased hydrophobicity and reduced motility compared to the wild-type parent strain. Despite the similarities in appearance of our colony morphology variant isolates on solid medium, the isolates showed a range of responses in various phenotypic assays. These variants form biofilms with significant three-dimensional structure and more biomass than the wild-type parent. To further explore the nature of the variants, their transcriptional profiles were evaluated. The variants generally showed increased expression of the psl and pel loci, which have been previously implicated in the adherence of P. aeruginosa to solid surfaces. When a mutation in the psl locus was introduced into a colony morphology variant, the colony morphology was only partially affected, but hyperadherence and autoaggregation were lost. Finally, similar colony morphology variants were found in isolates from cystic fibrosis patients. These variants displayed many of the same characteristics as the laboratory variants, suggesting a link between laboratory and cystic fibrosis biofilms.

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Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

Journal of Bacteriology ◽

10.1128/jb.181.14.4397-4403.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4397-4403 ◽

Cited By ~ 22

Author(s):

Casper Jørgensen ◽

Gert Dandanell

Keyword(s):

Amino Acids ◽

Purine Nucleoside Phosphorylase ◽

Mutational Analysis ◽

Regulatory Protein ◽

Three Dimensional ◽

Dimensional Structure ◽

Wild Type ◽

Isolation And Characterization ◽

In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

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Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614229 ◽

1998 ◽

Vol 79 (01) ◽

pp. 104-109 ◽

Cited By ~ 6

Author(s):

Osamu Takamiya

Keyword(s):

Monoclonal Antibodies ◽

Tissue Factor ◽

Human Factor ◽

Solid Phase ◽

Three Dimensional ◽

Coagulation Factors ◽

Factor Vii ◽

Dimensional Structure ◽

Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

Genetics ◽

10.1093/genetics/139.1.267 ◽

1995 ◽

Vol 139 (1) ◽

pp. 267-286 ◽

Cited By ~ 2

Author(s):

J D Fackenthal ◽

J A Hutchens ◽

F R Turner ◽

E C Raff

Keyword(s):

Amino Acid ◽

Three Dimensional ◽

Variable Region ◽

Internal Variable ◽

Microtubule Assembly ◽

Dimensional Structure ◽

Wild Type ◽

Beta Tubulin ◽

Assembly Kinetics ◽

Beta 2

Abstract We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

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Ribosomal protein S17: Characterization of the three-dimensional structure by proton and nitrogen-15 NMR

Biochemistry ◽

10.1021/bi00210a033 ◽

1993 ◽

Vol 32 (47) ◽

pp. 12812-12820 ◽

Cited By ~ 43

Author(s):

Barbara L. Golden ◽

David W. Hoffman ◽

V. Ramakrishnan ◽

Stephen W. White

Keyword(s):

Ribosomal Protein ◽

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure

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Nutritional Effects on Host Response to Lung Infections with Mucoid Pseudomonas aeruginosa in Mice

Infection and Immunity ◽

10.1128/iai.72.3.1479-1486.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1479-1486 ◽

Cited By ~ 15

Author(s):

Anna M. van Heeckeren ◽

Mark Schluchter ◽

Lintong Xue ◽

Juan Alvarez ◽

Steven Freedman ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Docosahexaenoic Acid ◽

Knockout Mice ◽

Host Response ◽

Inflammatory Responses ◽

Liquid Diet ◽

Wild Type ◽

Agarose Beads ◽

Lung Infections

ABSTRACT In cystic fibrosis, a recessive genetic disease caused by defects in the cystic fibrosis conductance regulator (CFTR), the main cause of death is lung infection and inflammation. Nutritional deficits have been proposed to contribute to the excessive host inflammatory response in both humans and Cftr-knockout mice. Cftr-knockout mice and gut-corrected Cftr-knockout mice expressing human CFTR primarily in the gut were challenged with Pseudomonas aeruginosa-laden agarose beads; they responded similarly with respect to bronchoalveolar lavage cell counts and levels of the acute-phase cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6. Wild-type mice fed the liquid diet used to prevent intestinal obstruction in Cftr-knockout mice had inflammatory responses to P. aeruginosa-laden agarose beads similar to those of wild-type mice fed an enriched solid diet, so dietary effects are unlikely to account for differences between wild-type mice and mice with cystic fibrosis. Finally, since cystic fibrosis patients and Cftr-knockout mice have an imbalance in fatty acids (significantly lower-than-normal levels of docosahexaenoic acid), the effects of specific supplementation with docosahexaenoic acid of wild-type and Cftr-knockout mice on their inflammatory responses to P. aeruginosa-laden agarose beads were tested. There were no significant differences (P = 0.35) in cumulative survival rates between Cftr-knockout mice and wild-type mice provided with either the liquid diet Peptamen or Peptamen containing docosahexaenoic acid. In conclusion, diet and docosahexaenoic acid imbalances alone are unlikely to explain the differences in the host response to lung infections with mucoid P. aeruginosa between mice with cystic fibrosis and their wild-type counterparts.

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Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

Biochemical Journal ◽

10.1042/bj3560217 ◽

2001 ◽

Vol 356 (1) ◽

pp. 217-222 ◽

Cited By ~ 5

Author(s):

Ricardo FRANCO ◽

Alice S. PEREIRA ◽

Pedro TAVARES ◽

Arianna MANGRAVITA ◽

Michael J. BARBER ◽

...

Keyword(s):

Absorption Spectra ◽

Catalytic Mechanism ◽

Protoporphyrin Ix ◽

Three Dimensional ◽

Iron Chelation ◽

Enzymic Activity ◽

Dimensional Structure ◽

Wild Type ◽

Wild Type Enzyme ◽

Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

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Structural characterization of quaternary selenites of tungsten(VI), A 2W3SeO12 (A = NH4, Cs, Rb, K or Tl)

Acta Crystallographica Section E Crystallographic Communications ◽

10.1107/s2056989021002735 ◽

2021 ◽

Vol 77 (4) ◽

pp. 406-411

Author(s):

Vineela Balisetty ◽

Kanamaluru Vidyasagar

Keyword(s):

Three Dimensional ◽

Spectroscopic Studies ◽

Solid State Reactions ◽

Dimensional Structure ◽

X Ray Diffraction ◽

Compound K ◽

Three Dimensional Structure ◽

Thermal And Spectroscopic Studies ◽

Anionic Framework

The quaternary A 2W3SeO12 (A = NH4, Cs, Rb, K or Tl) selenites have been prepared in the form of single crystals by hydrothermal and novel solid-state reactions. They were characterized by X-ray diffraction, thermal and spectroscopic studies. All of them have a hexagonal tungsten oxide (HTO) related [W3SeO12]2− anionic framework with pyramidally coordinated Se4+ ions. The known A 2W3SeO12 (A = NH4, Cs or Rb) compounds are isostructural with the Cs2W3TeO12 compound and have a non-centrosymmetric layered structure containing intra-layer Se—O bonds. The new compound K2W3SeO12(α) is isostructural with the K2W3TeO12 compound and has a centrosymmetric three-dimensional structure containing interlayer Se—O bonds. It is inferred that the new Tl2W3SeO12 compound has the same three-dimensional structure as K2W3SeO12(α).

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Solvent Accessibility of Residues Undergoing Pathogenic Variations in Humans: From Protein Structures to Protein Sequences

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.626363 ◽

2021 ◽

Vol 7 ◽

Author(s):

Castrense Savojardo ◽

Matteo Manfredi ◽

Pier Luigi Martelli ◽

Rita Casadio

Keyword(s):

Solvent Accessibility ◽

Protein Structures ◽

Three Dimensional ◽

Protein Sequences ◽

Large Data ◽

Human Protein ◽

Dimensional Structure ◽

Wild Type ◽

Solvent Exposure ◽

Data Set

Solvent accessibility (SASA) is a key feature of proteins for determining their folding and stability. SASA is computed from protein structures with different algorithms, and from protein sequences with machine-learning based approaches trained on solved structures. Here we ask the question as to which extent solvent exposure of residues can be associated to the pathogenicity of the variation. By this, SASA of the wild-type residue acquires a role in the context of functional annotation of protein single-residue variations (SRVs). By mapping variations on a curated database of human protein structures, we found that residues targeted by disease related SRVs are less accessible to solvent than residues involved in polymorphisms. The disease association is not evenly distributed among the different residue types: SRVs targeting glycine, tryptophan, tyrosine, and cysteine are more frequently disease associated than others. For all residues, the proportion of disease related SRVs largely increases when the wild-type residue is buried and decreases when it is exposed. The extent of the increase depends on the residue type. With the aid of an in house developed predictor, based on a deep learning procedure and performing at the state-of-the-art, we are able to confirm the above tendency by analyzing a large data set of residues subjected to variations and occurring in some 12,494 human protein sequences still lacking three-dimensional structure (derived from HUMSAVAR). Our data support the notion that surface accessible area is a distinguished property of residues that undergo variation and that pathogenicity is more frequently associated to the buried property than to the exposed one.

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Structural characterization of a Thorarchaeota profilin indicates eukaryotic-like features but with an extended N-terminus

10.1101/2021.08.06.455371 ◽

2021 ◽

Author(s):

Celestine N Chi ◽

Ravi Teja Inturi ◽

Sandra Martinez Lara ◽

Mahmoud Darweesh

Keyword(s):

Common Ancestor ◽

Three Dimensional ◽

Eukaryotic Cell ◽

Dimensional Structure ◽

Resonance Spectroscopy ◽

Last Eukaryotic Common Ancestor ◽

Rigid Core ◽

N Terminus ◽

Metagenomics Analysis

The emergence of the first eukaryotic cell was preceded by evolutionary events which are still highly debatable. Recently, comprehensive metagenomics analysis has uncovered that the Asgard super-phylum is the closest yet known archaea host of eukaryotes. However, it remains to be established if a large number of eukaryotic signature proteins predicated to be encoded by the Asgard super-phylum are functional at least, in the context of a eukaryotic cell. Here, we determined the three-dimensional structure of profilin from Thorarchaeota by nuclear magnetic resonance spectroscopy and show that this profilin has a rigid core with a flexible N-terminus which was previously implicated in polyproline binding. In addition, we also show that thorProfilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirm the notion that Asgardean encoded proteins possess eukaryotic-like characteristics and strengthen likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor

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Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

Scientific Reports ◽

10.1038/srep11666 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 14

Author(s):

A.E. Naas ◽

A.K. MacKenzie ◽

B. Dalhus ◽

V.G.H. Eijsink ◽

P.B. Pope

Keyword(s):

Active Site ◽

Three Dimensional ◽

Structural Features ◽

Structure And Function ◽

Dimensional Structure ◽

Active Site Cleft ◽

Polysaccharide Utilization Locus ◽

And Function ◽

Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

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