Isolation and Characterization ofEscherichia coli tolC Mutants Defective in Secreting Enzymatically Active Alpha-Hemolysin

ABSTRACT This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.

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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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Hemoglobin Biosynthesis in Vitreoscilla stercoraria DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene

Applied and Environmental Microbiology ◽

10.1128/aem.64.6.2220-2228.1998 ◽

1998 ◽

Vol 64 (6) ◽

pp. 2220-2228 ◽

Cited By ~ 14

Author(s):

Meenal Joshi ◽

Shekhar Mande ◽

Kanak L. Dikshit

Keyword(s):

Globin Gene ◽

Three Dimensional ◽

Fold Increase ◽

Binding Pocket ◽

Growth Conditions ◽

Dimensional Structure ◽

Strictly Aerobic ◽

Coding Region ◽

Oxygen Control ◽

Constitutive Production

ABSTRACT In the strictly aerobic, gram-negative bacteriumVitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure ofVitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla,V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation ofV. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within theV. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscillastrain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species ofVitreoscilla. The relatively slower autooxidation rate ofV. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercorariaDW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.

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The htrA Gene Plays an Important role During Yersinia Pseudotuberculosis Growth at High Temperature

10.21203/rs.3.rs-50599/v1 ◽

2020 ◽

Author(s):

Elena Escobar Garduño ◽

Lucia Soto Urzua ◽

Rogelio Rodriguez Sotres ◽

Luis Javier Martinez Morales

Keyword(s):

Growth Rate ◽

Mutant Strain ◽

Growth Rates ◽

Yersinia Pseudotuberculosis ◽

Wild Strain ◽

Three Dimensional ◽

Data Bank ◽

Immunoblot Analysis ◽

Dimensional Structure ◽

Bacteria Growth

Abstract htrA is a gene coding for the stress inducible HtrA protein, identified as a temperature stress response protein in several Gram positive and Gram negative bacteria. Growth rates at several temperatures (30ºC, 37ºC and 42ºC) were compared for Yersinia pseudotuberculosis YPIII wild strain and the isogenic mutant 1YPIII (htrA::Km), which was obtained by insertion of a kanamycin resistance cassette into the htrA gene.Y. pseudotuberculosis 1YPIII growth rates did not differ from the Y. pseudotuberculosis wild strain growth rates when cultivated at 30°C, which is consistent with a non-essential role for the HtrA protein at this temperature. However, 1YPIII mutant strain growth rate decreased by 18.73% at 37°C, and by 60.14% at 42°C, as compared to the Y. pseudotuberculosis YPIII wild strain growth rate. HtrA complementation in the strain 1YPIII/pAHTRA46 suppressed the differences in growth rates. Immunoblot analysis confirmed the absence of the HtrA protein in the 1YPIII mutant strain at any of the growth temperatures under analysis. In silico predictions were obtained for the three-dimensional structure of amino acid sequence belonging to HtrA from Y. pseudotuberculosis YPIII, Yersinia pestis CO92, using the protein data bank structure 1KY9:B from Escherichia coli, as template. The model's quality was found to be acceptable. Southern blot analysis shows a single htrA gene signal. These data indicate that the unique htrA gene in Y. pseudotuberculosis YPIII is required for the adaptive response of this species to high temperatures and although it is not a pathogenicity factor, it can be targeted by antibiotics.

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Temperature Induced Protein Unfolding and Folding of RNase a Studied by Time-Resolved Infrared Spectroscopy

Laser Chemistry ◽

10.1155/1999/28202 ◽

1999 ◽

Vol 19 (1-4) ◽

pp. 233-235 ◽

Cited By ~ 10

Author(s):

H. Georg ◽

C. W. Wharton ◽

F. Siebert

Keyword(s):

Time Course ◽

Protein Unfolding ◽

Structural Changes ◽

Three Dimensional ◽

Rnase A ◽

Dimensional Structure ◽

Time Range ◽

Lag Phase ◽

Structural Level ◽

Time Resolved Infrared Spectroscopy

When a protein finds its native three-dimensional structure from the unstructured amino-acid chain various processes spanning a large time range are relevant. To understand the mechanism of protein folding one needs to cover the entire folding/ refolding (U↔N) reaction on a structural level. In the case of RNase A, the main structural changes occur in the ms time range, that can be monitored with rapid-scan- FTIR spectroscopy combined with rapid mixing techniques. To induce unfolding we inject aqueous protein solution into a hot IR cuvette and record the time course of the spectral changes. A lag phase is found when the unfolding conditions are relatively weak, suggesting an unfolding intermediate.

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Isolation and Characterization of Tissue Resident CD29-Positive Progenitor Cells in Livestock to Generate a Three-Dimensional Meat Bud

Cells ◽

10.3390/cells10092499 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2499

Author(s):

Yuna Naraoka ◽

Yo Mabuchi ◽

Yosuke Yoneyama ◽

Eriko Grace Suto ◽

Daisuke Hisamatsu ◽

...

Keyword(s):

Complex Structure ◽

Three Dimensional ◽

Collagen Gel ◽

Dimensional Structure ◽

Meat Production ◽

Proliferating Cells ◽

Isolation And Characterization ◽

Bovine Tissue ◽

The Matrix

The current process of meat production using livestock has significant effects on the global environment, including high emissions of greenhouse gases. In recent years, cultured meat has attracted attention as a way to acquire animal proteins. However, the lack of markers that isolate proliferating cells from bovine tissues and the complex structure of the meat make it difficult to culture meat in a dish. In this study, we screened 246 cell-surface antibodies by fluorescence-activated cell sorting for their capacity to form colonies and their suitability to construct spheroid “meat buds”. CD29+ cells (Ha2/5 clone) have a high potency to form colonies and efficiently proliferate on fibronectin-coated dishes. Furthermore, the meat buds created from CD29+ cells could differentiate into muscle and adipose cells in a three-dimensional structure. The meat buds embedded in the collagen gel proliferated in the matrix and formed large aggregates. Approximately 10 trillion cells can theoretically be obtained from 100 g of bovine tissue by culturing and amplifying them using these methods. The CD29+ cell characteristics of bovine tissue provide insights into the production of meat alternatives in vitro.

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Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro

Biochemical Journal ◽

10.1042/bj3610409 ◽

2002 ◽

Vol 361 (2) ◽

pp. 409-416 ◽

Cited By ~ 8

Author(s):

Eric DI LUCCIO ◽

Alessandra MATAVEL ◽

Sandrine OPI ◽

Imed REGAYA ◽

Guillaume SANDOZ ◽

...

Keyword(s):

Time Course ◽

Three Dimensional ◽

Secondary Structures ◽

Dimensional Structure ◽

Electrophysiological Recordings ◽

Initial Stage ◽

On Line ◽

Α Helix ◽

Β Sheet

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the α-helix within 5h, followed by the formation of the β-sheet within 22h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the α-helix and two- or three-disulphide-bridged sMTX for the β-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K+ currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.

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The mosquito protein AEG12 displays both cytolytic and antiviral properties via a common lipid transfer mechanism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019251118 ◽

2021 ◽

Vol 118 (11) ◽

pp. e2019251118

Author(s):

Alexander C. Y. Foo ◽

Peter M. Thompson ◽

Shih-Heng Chen ◽

Ramesh Jadi ◽

Brianna Lupo ◽

...

Keyword(s):

Lipid Bilayers ◽

Acyl Chain ◽

Three Dimensional ◽

Cytolytic Activity ◽

Dimensional Structure ◽

Lipid Transfer ◽

Adeno Associated Virus ◽

Nonenveloped Virus ◽

Flavivirus Infection ◽

Blood Meals

The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.

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Cell Differentiation during Sexual Development of the Fungus Sordaria macrospora Requires ATP Citrate Lyase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.450 ◽

1999 ◽

Vol 19 (1) ◽

pp. 450-460 ◽

Cited By ~ 71

Author(s):

Minou Nowrousian ◽

Sandra Masloff ◽

Stefanie Pöggeler ◽

Ulrich Kück

Keyword(s):

Amino Acid ◽

Mutant Strain ◽

Sexual Development ◽

Fruiting Body ◽

Time Course ◽

Sexual Cycle ◽

Cosmid Library ◽

Acetyl Coa ◽

Sordaria Macrospora ◽

Isolation And Characterization

ABSTRACT During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrosporadevelopmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of theacl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.

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A Second Lysine-Specific Serine Protease from Lysobacter sp. Strain IB-9374

Journal of Bacteriology ◽

10.1128/jb.186.15.5093-5100.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 5093-5100 ◽

Cited By ~ 14

Author(s):

Shigeru Chohnan ◽

Kentaro Shiraki ◽

Kiyonobu Yokota ◽

Makoto Ohshima ◽

Natsuki Kuroiwa ◽

...

Keyword(s):

Amino Acids ◽

Tertiary Structure ◽

Guanidine Hydrochloride ◽

Sodium Dodecyl ◽

Three Dimensional ◽

Catalytic Triad ◽

Dimensional Structure ◽

Gene Encoding ◽

Lysyl Endopeptidase ◽

Mature Enzyme

ABSTRACT A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA. The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.

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Systematic mutational analysis of the yeast ACT1 gene.

Genetics ◽

10.1093/genetics/132.2.337 ◽

1992 ◽

Vol 132 (2) ◽

pp. 337-350 ◽

Cited By ~ 36

Author(s):

K F Wertman ◽

D G Drubin ◽

D Botstein

Keyword(s):

Mutational Analysis ◽

Three Dimensional ◽

Dimensional Structure ◽

High Yield ◽

Temperature Sensitive ◽

Rabbit Muscle ◽

Isolation And Characterization ◽

Charged Residues ◽

Actin Structure ◽

Mutant Phenotypes

Abstract We report the isolation and characterization of a synoptic set of site-directed mutations distributed throughout the single actin gene of Saccharomyces cerevisiae. Mutations were systematically targeted to the surface of the protein by identifying clusters of 2 or more charged residues in the primary sequence; every charged residue in a cluster was replaced with alanine. Mutations were recovered in high yield (34 of 36 constructed) as heterozygous diploids. Mutant phenotypes were examined in haploid segregants: 11 were recessive lethal, 16 conditional-lethal (including temperature-sensitive and salt-sensitive) and 7 had no discernible phenotype. Genetic analysis suggested that the two mutations constructed but not recovered in yeast may have a dominant defective phenotype. Location of the mutant residues on the three-dimensional structure of the rabbit muscle actin monomer confirmed that most (81%) of the charged residues we altered lie at or near the surface of the protein, confirming a key assumption of the method. Many of the new act1 alleles have properties readily interpreted in light of the actin structure and should prove useful in both genetic and biochemical studies of actin function.

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