scholarly journals Natural Selection in the Chicken Host Identifies 3-Deoxy-d-manno- Octulosonic Acid Kinase Residues Essential for Phosphorylation of Pasteurella multocida Lipopolysaccharide

2010 ◽  
Vol 78 (9) ◽  
pp. 3669-3677 ◽  
Author(s):  
Marina Harper ◽  
Andrew D. Cox ◽  
Frank St. Michael ◽  
Mark Ford ◽  
Ian W. Wilkie ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Pasteurella Multocida ◽  
Inner Core ◽  
Single Amino Acid ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Fowl Cholera ◽  
Mutant Strains ◽  
High Doses

ABSTRACT Pasteurella multocida is the causative agent of a number of diseases in animals, including fowl cholera. P. multocida strains simultaneously express two lipopolysaccharide (LPS) glycoforms (glycoforms A and B) that differ only in their inner core structure. Glycoform A contains a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated by the Kdo kinase, KdkA, whereas glycoform B contains two unphosphorylated Kdo residues. We have previously shown that P. multocida mutants lacking the heptosyltransferase, HptA, produce full-length glycoform B LPS and a large amount of truncated glycoform A LPS, as they cannot add heptose to the glycoform A inner core. These hptA mutants were attenuated in chickens because the truncated LPS made them vulnerable to host defense mechanisms, including antimicrobial peptides. However, here we show that birds inoculated with high doses of the hptA mutant developed fowl cholera and the P. multocida isolates recovered from diseased birds no longer expressed truncated LPS. Sequencing analysis revealed that the in vivo-derived isolates had mutations in kdkA, thereby suppressing the production of glycoform A LPS. Interestingly, a number of the spontaneous KdkA mutant strains produced KdkA with a single amino acid substitution (A112V, R123P, H168Y, or D193N). LPS structural analysis showed that complementation of a P. multocida kdkA mutant with wild-type kdkA restored expression of glycoform A to wild-type levels, whereas complementation with any of the mutated kdkA genes did not. We conclude that in P. multocida KdkA, the amino acids A112, R123, H168, and D193 are critical for Kdo kinase function and therefore for glycoform A LPS assembly.

scholarly journals N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways

2021 ◽  
Vol 22 (14) ◽  
pp. 7565
Author(s):  
Kyungho Woo ◽  
Dong Ho Kim ◽  
Man Hwan Oh ◽  
Ho Sung Park ◽  
Chul Hee Choi
Keyword(s):  
Bone Marrow ◽  
Quorum Sensing ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Host Responses ◽  
Wild Type ◽  
Mutant Strains

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

2021 ◽  
Vol 85 (2) ◽  
pp. 297-306
Author(s):  
Zui Fujimoto ◽  
Le Thi Thu Hong ◽  
Naomi Kishine ◽  
Nobuhiro Suzuki ◽  
Keitarou Kimura
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
X Ray Crystallography ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5342-5348 ◽  
Author(s):  
Ute Meisel ◽  
Joachim-Volker Höltje ◽  
Waldemar Vollmer
Keyword(s):  
Escherichia Coli ◽  
Single Amino Acid ◽  
Penicillin Binding Protein ◽  
Amino Acid Exchange ◽  
Lytic Transglycosylases ◽  
Mutant Strains ◽  
Ph Conditions ◽  
Expression Plasmids ◽  
Acid Exchange

ABSTRACT Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (α, β, and γ) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals The Entner-Doudoroff Pathway Has Little Effect on Helicobacter pylori Colonization of Mice

2003 ◽  
Vol 71 (5) ◽  
pp. 2920-2923 ◽  
Author(s):  
Amy E. Wanken ◽  
Tyrrell Conway ◽  
Kathryn A. Eaton
Keyword(s):  
Helicobacter Pylori ◽  
Wild Type ◽  
Mutant Strains ◽  
H Pylori ◽  
Type Strains

ABSTRACT Helicobacter pylori mutants deficient in 6-phosphogluconate dehydratase (6PGD) were constructed. Colonization densities were lower and minimum infectious doses were higher for mutant strains than for wild-type strains. In spite of better colonization, however, wild-type strains did not displace the mutant in cocolonization experiments. Loss of 6PGD diminishes the fitness of H. pylori in vivo, but the pathway is nonessential for colonization.

scholarly journals phgABC, a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo

2004 ◽  
Vol 72 (8) ◽  
pp. 4579-4588 ◽  
Author(s):  
Jeremy S. Brown ◽  
Sarah M. Gilliland ◽  
Shilpa Basavanna ◽  
David W. Holden
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mutant Strain ◽  
Compatible Solutes ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Mutant Strains ◽  
Increased Sensitivity ◽  
Related Proteins ◽  
And Oxidative Stress

ABSTRACT To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg−1. While investigating Streptococcus pneumoniae genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed phgABC. PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria. phgA and phgC mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the phgA mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the phgA mutant strain was unaffected by osmotic stress. The phgA and phgC mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the phgA mutant strain was not restored in gp91phox−/− mice and the phgA and phgC mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.

scholarly journals Modulation of the Effects of Lung Immune Response on Bone Marrow by Oral Antigen Exposure

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
P. Xavier-Elsas ◽  
C. L. C. A. Silva ◽  
L. Pinto ◽  
T. Queto ◽  
B. M. Vieira ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Lymphocytes ◽  
Ex Vivo ◽  
Allergen Challenge ◽  
Allergic Airway Inflammation ◽  
Oral Exposure ◽  
Wild Type ◽  
Homologous Antigen ◽  
Mutant Strains

Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.

scholarly journals Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

2007 ◽  
Vol 75 (8) ◽  
pp. 3885-3893 ◽  
Author(s):  
Marina Harper ◽  
John D. Boyce ◽  
Andrew D. Cox ◽  
Frank St. Michael ◽  
Ian W. Wilkie ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Inner Core ◽  
Phase Variation ◽  
Outer Core ◽  
Core Oligosaccharide ◽  
Bacterial Mutants ◽  
Major Antigen ◽  
First Time ◽  
Mucosal Pathogens

ABSTRACT Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-d-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

Metribuzin-Resistant Mutants of Chlamydomonas reinhardii

1984 ◽  
Vol 39 (5) ◽  
pp. 437-439 ◽  
Author(s):  
N. Pucheu ◽  
W. Oettmeier ◽  
U. Heisterkamp ◽  
K. Masson ◽  
G.F. Wildner
Keyword(s):  
Electron Transport ◽  
Photosynthetic Electron Transport ◽  
Wild Type ◽  
Binding Studies ◽  
Molecular Weight Range ◽  
Chlamydomonas Reinhardii ◽  
Thylakoid Membrane Proteins ◽  
Mutant Strains ◽  
Resistant Mutants

Herbicide resistance in Chlamydomonas reinhardii cells was induced by mutagenesis with 5-fluorodeoxyuridine and ethylmethanesulfonate. Four mutant strains were isolated and analyzed for resistance against DCMU-type or phenolic inhibitors of photosynthetic electron transport. The mutants were different in both the extent and the pattern of their resistance: the R/S value, i.e. the ratio of I50 values of the inhibition of photosynthetic electron transport in isolated resistant and susceptible thylakoids, varied for metribuzin from 10 000 to 36. The mutant MZ-1 was resistant against metribuzin, atrazine and DCMU, whereas the mutant MZ-2 showed resistance mainly against metribuzin and atrazine. The mutant MZ-3 was similar to MZ-1, but showed a lesser extent of resistance against DCMU. The mutant MZ-4 showed resistance against metribuzin, but not against atrazine. These results demonstrate that the resistance against one herbicide of the DCMU-type (metribuzin) must not be accompanied by similar resistance against te other inhibitors. Binding studies with radioactively labeled herbicides, [14C]metribuzin, [14C]atrazine and [3H]DCMU, and isolated thylakoids supported these observations. Phosphorylation of thylakoid membrane proteins was studied with wild-type cells and resistant mutants under in vivo conditions in the light. The 32P-labeled main proteins bands were in the molecular weight range of 10-14 kDa, 26-29 kDa, 32-35 kDa and 46-48 kDa. The pattern and the extent of incorporation of 32P were similar for the mutants and the wild-type cells.

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