Characterization of the Chromosomalaac(6′)-Iz Gene of Stenotrophomonas maltophilia

1999 ◽  
Vol 43 (10) ◽  
pp. 2366-2371 ◽  
Author(s):  
Thierry Lambert ◽  
Marie-Cécile Ploy ◽  
François Denis ◽  
Patrice Courvalin
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Type I ◽  
Aminoglycoside Resistance ◽  
Reading Frame ◽  
A Value ◽  
Calculated Mass ◽  
Total Dna ◽  
Good Agreement

ABSTRACT The aac(6′)-Iz gene of Stenotrophomonas maltophilia BM2690 encoding an aminoglycoside 6′-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 462 bp corresponding to a protein with a calculated mass of 16,506 Da, a value in good agreement with that of ca. 16,000 found by in vitro coupled transcription-translation. Analysis of the deduced amino acid sequence indicated that the protein was a member of the major subfamily of aminoglycoside 6′-N-acetyltransferases. The enzyme conferred resistance to amikacin but not to gentamicin, indicating that it was an AAC(6′) of type I. The open reading frame upstream from the aac(6′)-Izgene was homologous to the fprA gene of Myxococcus xanthus (61% identity), which encodes a putative pyridoxine (pyridoxamine) 5′-phosphate oxidase. Pulsed-field gel electrophoresis of total DNA from BM2690 and S. maltophilia ATTC 13637 digested with XbaI, DraI, and SpeI followed by hybridization with rRNA and aac(6′)-Iz-specific probes indicated that the gene was located in the chromosome. Theaac(6′)-Iz gene was detected by DNA-DNA hybridization in all 80 strains of S. maltophilia tested. The MICs of gentamicin against these strains of S. maltophilia were lower than those of amikacin, netilmicin, and tobramycin, indicating that production of AAC(6′)-Iz contributes to aminoglycoside resistance in S. maltophilia.

scholarly journals The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea

2002 ◽  
Vol 68 (10) ◽  
pp. 4812-4819 ◽  
Author(s):  
Silvia T. Cardona ◽  
Francisco P. Chávez ◽  
Carlos A. Jerez
Keyword(s):  
Sulfolobus Solfataricus ◽  
Open Reading Frame ◽  
Inorganic Polyphosphate ◽  
Reading Frame ◽  
Highly Active ◽  
Calculated Mass ◽  
Polyphosphate Metabolism ◽  
Long Chain

ABSTRACT Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

scholarly journals The Emergence of Human Coronavirus EMC: How Scared Should We Be?

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Renee W. Y. Chan ◽  
Leo L. M. Poon
Keyword(s):  
Airway Epithelial Cells ◽  
Type I ◽  
Airway Epithelial ◽  
Human Coronavirus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Airway Epithelium ◽  
Mammalian Cell Lines ◽  
Physiological Relevance

ABSTRACT A novel betacoronavirus, human coronavirus (HCoV-EMC), has recently been detected in humans with severe respiratory disease. Further characterization of HCoV-EMC suggests that this virus is different from severe acute respiratory syndrome coronavirus (SARS-CoV) because it is able to replicate in multiple mammalian cell lines and it does not use angiotensin-converting enzyme 2 as a receptor to achieve infection. Additional research is urgently needed to better understand the pathogenicity and tissue tropism of this virus in humans. In their recent study published in mBio, Kindler et al. shed some light on these important topics (E. Kindler, H. R. Jónsdóttir, M. Muth, O. J. Hamming, R. Hartmann, R. Rodriguez, R. Geffers, R. A. Fouchier, C. Drosten, M. A. Müller, R. Dijkman, and V. Thiel, mBio 4[1]:e00611-12, 2013). These authors report the use of differentiated pseudostratified human primary airway epithelial cells, an in vitro model with high physiological relevance to the human airway epithelium, to characterize the cellular tropism of HCoV-EMC. More importantly, the authors demonstrate the potential use of type I and type III interferons (IFNs) to control viral infection.

scholarly journals Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
Author(s):  
Sarah L. Barker ◽  
Linda Lee ◽  
B. Daniel Pierce ◽  
Lymarie Maldonado-Báez ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

scholarly journals Characterization of IS18, an Element Capable of Activating the Silent aac(6′)-Ij Gene ofAcinetobacter sp. 13 Strain BM2716 by Transposition

1998 ◽  
Vol 42 (10) ◽  
pp. 2759-2761 ◽  
Author(s):  
Eric Rudant ◽  
Patrice Courvalin ◽  
Thierry Lambert
Keyword(s):  
Resistance Gene ◽  
Insertion Sequence ◽  
Resistant Mutant ◽  
Open Reading Frame ◽  
Inverted Repeats ◽  
Aminoglycoside Resistance ◽  
Reading Frame ◽  
Terminal Inverted Repeats ◽  
Hybrid Promoter

ABSTRACT Insertion sequence IS18 was detected by analysis of the spontaneous aminoglycoside resistant mutant Acinetobactersp. 13 strain BM2716-1. Insertion of the element upstream from the silent acetyltransferase gene aac(6′)-Ij created a hybrid promoter that putatively accounts for the expression of the aminoglycoside resistance gene. The 1,074-bp IS18 element contained partially matched (20 out of 26 bases) terminal inverted repeats, one of which overlapped the 3′ end of a 935-bp open reading frame potentially encoding a protein related to the transposases of the IS30 family. IS18 was found in 6 out of 29 strains of Acinetobacter sp. 13 but not in 10 strains each of A. baumannii and A. haemolyticus.

scholarly journals Molecular Characterization of Two-Component Systems of Helicobacter pylori

2000 ◽  
Vol 182 (8) ◽  
pp. 2068-2076 ◽  
Author(s):  
Dagmar Beier ◽  
Rainer Frank
Keyword(s):  
Helicobacter Pylori ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Response Regulators ◽  
Reading Frame ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogenHelicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3′ end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

scholarly journals Aph(3′)-IIc, an Aminoglycoside Resistance Determinant from Stenotrophomonas maltophilia

2006 ◽  
Vol 51 (1) ◽  
pp. 359-360 ◽  
Author(s):  
Aki Okazaki ◽  
Matthew B. Avison
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolate ◽  
Stenotrophomonas Maltophilia ◽  
Resistance Determinant ◽  
Aminoglycoside Resistance ◽  
Aminoglycoside Phosphotransferase

ABSTRACT We report the characterization of an intrinsic, chromosomally carried aph(3′)-IIc gene from Stenotrophomonas maltophilia clinical isolate K279a, encoding an aminoglycoside phosphotransferase enzyme that significantly increases MICs of kanamycin, neomycin, butirosin, and paromomycin when expressed in Escherichia coli. Disruption of aph(3′)-IIc in K279a results in decreased MICs of these drugs.

scholarly journals Mammalian Casein Kinase 1α and Its Leishmanial Ortholog Regulate Stability of IFNAR1 and Type I Interferon Signaling

2009 ◽  
Vol 29 (24) ◽  
pp. 6401-6412 ◽  
Author(s):  
Jianghuai Liu ◽  
Lucas P. Carvalho ◽  
Sabyasachi Bhattacharya ◽  
Christopher J. Carbone ◽  
K. G. Suresh Kumar ◽  
...  
Keyword(s):  
Er Stress ◽  
Mammalian Cells ◽  
Leishmania Major ◽  
Type I Interferon ◽  
Casein Kinase ◽  
Type I ◽  
Bona Fide ◽  
Activity Dependent

ABSTRACT Phosphorylation of the degron of the IFNAR1 chain of the type I interferon (IFN) receptor triggers ubiquitination and degradation of this receptor and, therefore, plays a crucial role in negative regulation of IFN-α/β signaling. Besides the IFN-stimulated and Jak activity-dependent pathways, a basal ligand-independent phosphorylation of IFNAR1 has been described and implicated in downregulating IFNAR1 in response to virus-induced endoplasmic reticulum (ER) stress. Here we report purification and characterization of casein kinase 1α (CK1α) as a bona fide major IFNAR1 kinase that confers basal turnover of IFNAR1 and cooperates with ER stress stimuli to mediate phosphorylation-dependent degradation of IFNAR1. Activity of CK1α was required for phosphorylation and downregulation of IFNAR1 in response to ER stress and viral infection. While many forms of CK1 were capable of phosphorylating IFNAR1 in vitro, human CK1α and L-CK1 produced by the protozoan Leishmania major were also capable of increasing IFNAR1 degron phosphorylation in cells. Expression of leishmania CK1 in mammalian cells stimulated the phosphorylation-dependent downregulation of IFNAR1 and attenuated its signaling. Infection of mammalian cells with L. major modestly decreased IFNAR1 levels and attenuated cellular responses to IFN-α in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling.

scholarly journals Establishment and Characterization of a Novel Fibroblastic Cell Line (SCI13D) Derived from the Broncho-Alveolar Lavage of a Patient with Fibrotic Hypersensitivity Pneumonitis

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1193
Author(s):  
Paolo Giannoni ◽  
Marco Grosso ◽  
Giuseppina Fugazza ◽  
Mario Nizzari ◽  
Maria Cristina Capra ◽  
...  
Keyword(s):  
Cell Line ◽  
Hypersensitivity Pneumonitis ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Usual Interstitial Pneumonia ◽  
Type I ◽  
Fibroblastic Cell ◽  
Primary Fibroblasts

Hypersensitivity pneumonitis (HP) is a diffuse interstitial lung disease (ILD) caused by the inhalation of a variety of antigens in susceptible individuals. Patients with fibrotic HP (fHP) may show histopathological and radiological manifestations similar to patients with idiopathic pulmonary fibrosis (usual interstitial pneumonia-like pattern of fibrosis) that are associated with a worse prognosis. We describe here the establishment and characterization of a fibroblastic cell line derived from the broncho-alveolar lavage (BAL) of a patient with fHP, a 53 year old man who presented at our Pneumology Unit with cough and dyspnea. The fHP diagnosis was based on international criteria and multidisciplinary discussion. Primary fibroblasts were expanded in vitro until passage 36. These fibroblasts displayed morpho/phenotypical features of myofibroblasts, showing high positivity for α-smooth muscle actin, type I collagen, and fibronectin as determined by quantitative RT-PCR and cyto-fluorographic analysis. Cytogenetic analyses further evidenced trisomy of chromosome 10, which interestingly harbors the FGF2R gene. To our knowledge, this is the first fibroblastic cell line derived from an fHP patient and might, therefore, represent a suitable tool to model the disease in vitro. We preliminarily assessed here the activity of pirfenidone, further demonstrating a consistent inhibition of cells growth by this antifibrotic drug.

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