Molecular and Functional Characterization of MobK Protein—A Novel-Type Relaxase Involved in Mobilization for Conjugational Transfer of Klebsiella pneumoniae Plasmid pIGRK

Conjugation, besides transformation and transduction, is one of the main mechanisms of horizontal transmission of genetic information among bacteria. Conjugational transfer, due to its essential role in shaping bacterial genomes and spreading of antibiotics resistance genes, has been widely studied for more than 70 years. However, new and intriguing facts concerning the molecular basis of this process are still being revealed. Most recently, a novel family of conjugative relaxases (Mob proteins) was distinguished. The characteristic feature of these proteins is that they are not related to any of Mobs described so far. Instead of this, they share significant similarity to tyrosine recombinases. In this study MobK—a tyrosine recombinase-like Mob protein, encoded by pIGRK cryptic plasmid from the Klebsiella pneumoniae clinical strain, was characterized. This study revealed that MobK is a site-specific nuclease and its relaxase activity is dependent on both a conserved catalytic tyrosine residue (Y179) that is characteristic of tyrosine recombinases and the presence of Mg2+ divalent cations. The pIGRK minimal origin of transfer sequence (oriT) was also characterized. This is one of the first reports presenting tyrosine recombinase-like conjugative relaxase protein. It also demonstrates that MobK is a convenient model for studying this new protein family.

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Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4

Genes ◽

10.3390/genes11101207 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1207

Author(s):

Sandra Daniel ◽

Kelly Goldlust ◽

Valentin Quebre ◽

Minjia Shen ◽

Christian Lesterlin ◽

...

Keyword(s):

Escherichia Coli ◽

Plasmid Stability ◽

Horizontal Transmission ◽

Viable Cell ◽

Time Lapse ◽

Conjugative Plasmid ◽

Triple Mutant ◽

Conjugational Transfer ◽

Transposon Insertion ◽

Free Cells

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors—active partitioning, toxin-antitoxin and conjugational transfer—are all involved in the prevalence of pESBL in the E. coli population.

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Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization

Biochemistry and Cell Biology ◽

10.1139/o03-084 ◽

2004 ◽

Vol 82 (2) ◽

pp. 295-303 ◽

Cited By ~ 14

Author(s):

Emi Mishiro ◽

Ming-Yih Liu ◽

Yoichi Sakakibara ◽

Masahito Suiko ◽

Ming-Cheh Liu

Keyword(s):

Molecular Cloning ◽

Divalent Cations ◽

Functional Characterization ◽

The Other ◽

Inhibitory Effects ◽

Amino Acid Sequence Level ◽

Ph Optimum ◽

Rapid Amplification ◽

High Degree

By employing the reverse transcriptase – polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.Key words: zebra fish, tyrosylprotein sulfotransferase, molecular cloning.

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Imipenem Resistance in a Salmonella Clinical Strain Due to Plasmid-Mediated Class A Carbapenemase KPC-2

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.4.1297-1300.2003 ◽

2003 ◽

Vol 47 (4) ◽

pp. 1297-1300 ◽

Cited By ~ 126

Author(s):

Vivi Miriagou ◽

Leonidas S. Tzouvelekis ◽

Shannon Rossiter ◽

Eva Tzelepi ◽

Frederick J. Angulo ◽

...

Keyword(s):

Sequence Analysis ◽

Klebsiella Pneumoniae ◽

Salmonella Enterica ◽

Conjugative Plasmid ◽

Clinical Strain ◽

Class A ◽

Potential Association ◽

Imipenem Resistance ◽

Flanking Regions

ABSTRACT A Salmonella enterica serotype Cubana isolate exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. β-Lactam resistance was mediated by a conjugative plasmid that encoded KPC-2, a class A carbapenemase previously found in a Klebsiella pneumoniae isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of bla KPC-2 with mobile structures.

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OXA-24 Carbapenemase Gene Flanked by XerC/XerD-Like Recombination Sites in Different Plasmids from Different Acinetobacter Species Isolated during a Nosocomial Outbreak

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01674-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2724-2727 ◽

Cited By ~ 73

Author(s):

María Merino ◽

Joshi Acosta ◽

Margarita Poza ◽

Francisca Sanz ◽

Alejandro Beceiro ◽

...

Keyword(s):

Horizontal Transmission ◽

Carbapenem Resistance ◽

Acinetobacter Calcoaceticus ◽

Multidrug Resistant ◽

Clinical Strain ◽

Acinetobacter Species ◽

Gene Comparison ◽

Carbapenemase Gene ◽

Large Outbreak ◽

Culture Sample

ABSTRACT A clinical strain of Acinetobacter calcoaceticus resistant to carbapenems was isolated from a blood culture sample from an inpatient in a hospital in Madrid (Spain) during a large outbreak of infection (affecting more than 300 inpatients), caused by a multidrug-resistant Acinetobacter baumannii clone. The carbapenem resistance in both the A. calcoaceticus and A. baumannii clones was due to a bla OXA-24 gene harbored in different plasmids. The plasmids were fully sequenced, revealing the presence of site-specific recombination binding sites putatively involved in mobilization of the bla OXA-24 gene. Comparison of plasmids contained in the two strains revealed possible horizontal transmission of resistance genes between the Acinetobacter species.

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Structural determination of the polysaccharide isolated from biofilms produced by a clinical strain of Klebsiella pneumoniae

Carbohydrate Research ◽

10.1016/j.carres.2016.05.001 ◽

2016 ◽

Vol 430 ◽

pp. 29-35 ◽

Cited By ~ 11

Author(s):

Paola Cescutti ◽

Gianluigi De Benedetto ◽

Roberto Rizzo

Keyword(s):

Klebsiella Pneumoniae ◽

Clinical Strain ◽

Structural Determination

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Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates

Frontiers in Microbiology ◽

10.3389/fmicb.2015.01354 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 10

Author(s):

Ella Kaplan ◽

Noa Sela ◽

Adi Doron-Faigenboim ◽

Shiri Navon-Venezia ◽

Edouard Jurkevitch ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Municipal Wastewater ◽

Functional Characterization

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Molecular and Biochemical Characterization of SHV-56, a Novel Inhibitor-Resistant β-Lactamase from Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00387-08 ◽

2008 ◽

Vol 52 (10) ◽

pp. 3792-3794 ◽

Cited By ~ 17

Author(s):

Véronique Dubois ◽

Laurent Poirel ◽

François Demarthe ◽

Corinne Arpin ◽

Laure Coulange ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biochemical Characterization ◽

Clinical Strain ◽

Chromosomal Gene ◽

Inhibitor Resistance ◽

Critical Site ◽

Single Substitution

ABSTRACT A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla SHV-56, encoding a new inhibitor-resistant β-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.

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Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies

International Journal of Molecular Sciences ◽

10.3390/ijms23010116 ◽

2021 ◽

Vol 23 (1) ◽

pp. 116

Author(s):

Olamide Jeje ◽

Reabetswe Maake ◽

Ruan van Deventer ◽

Veruschka Esau ◽

Emmanuel Amarachi Iwuchukwu ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Conformational Changes ◽

Metal Ion ◽

Therapeutic Potential ◽

Divalent Cations ◽

Homology Modelling ◽

Substrate Binding ◽

Structure Stability ◽

Atp Binding ◽

The Impact

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

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Draft Genome Sequence of a Polymyxin-Resistant Klebsiella pneumoniae Clinical Strain Carrying mcr-8.1 and blaNDM-5

Microbiology Resource Announcements ◽

10.1128/mra.01224-20 ◽

2021 ◽

Vol 10 (12) ◽

Author(s):

Na Pei ◽

Zijuan Jian ◽

Yirui Liu ◽

Tianzhu Liang ◽

Wenen Liu ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Global Health ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Clinical Strain ◽

Content Type ◽

Major Threat ◽

Carbapenem Resistant

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to global health. Here, we report the draft genome sequence of a Klebsiella pneumoniae clinical strain carrying mcr-8.1 and blaNDM-5.

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