Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04185-7 ◽

2021 ◽

Author(s):

Fatma Ben Abid ◽

Clement K. M. Tsui ◽

Yohei Doi ◽

Anand Deshmukh ◽

Christi L. McElheny ◽

...

Keyword(s):

Common Species ◽

Clinical Samples ◽

Whole Genome ◽

E Coli ◽

Carbapenem Resistant ◽

Genes Encoding ◽

Encoding Genes ◽

Sequence Types ◽

Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

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Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s00775-021-01854-y ◽

2021 ◽

Author(s):

I. B. Trindade ◽

G. Hernandez ◽

E. Lebègue ◽

F. Barrière ◽

T. Cordeiro ◽

...

Keyword(s):

Functional Characterization ◽

Paramagnetic Nmr ◽

E Coli ◽

The Past ◽

Internal Cavities ◽

Structural Differences ◽

Bond Strengths ◽

Redox Bohr Effect ◽

Micromolar Range

AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

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Sa1806 Molecular and Functional Characterization of Fimh in Crohn's Disease Associated E. Coli

Gastroenterology ◽

10.1016/s0016-5085(13)61117-8 ◽

2013 ◽

Vol 144 (5) ◽

pp. S-310

Author(s):

Brendan Chandler ◽

Belgin Dogan ◽

Ellen J. Scherl ◽

Kenneth W. Simpson

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Functional Characterization ◽

E Coli

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Molecular and Functional Characterization of Genes Encoding Anti-Thyroglobulin and Anti-Tsh Receptor Antibodies

Thyroid Autoimmunity ◽

10.1007/978-1-4613-0945-1_20 ◽

1987 ◽

pp. 175-180

Author(s):

Marc Monestier ◽

Constantin A. Bona

Keyword(s):

Functional Characterization ◽

Tsh Receptor ◽

Genes Encoding ◽

Receptor Antibodies ◽

Tsh Receptor Antibodies

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Expression, secretion and functional characterization of three laccases in E. coli

Synthetic and Systems Biotechnology ◽

10.1016/j.synbio.2021.12.002 ◽

2022 ◽

Vol 7 (1) ◽

pp. 474-480

Author(s):

Yating Mo ◽

Hou Ip Lao ◽

Sau Wa Au ◽

Ieng Chon Li ◽

Jeremy Hu ◽

...

Keyword(s):

Functional Characterization ◽

E Coli

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Identification of Genes Encoding Heat Shock Protein 40 Family and the Functional Characterization of Two Hsp40s, MHF16 and MHF21, in Magnaporthe oryzae

The Plant Pathology Journal ◽

10.5423/ppj.2008.24.2.131 ◽

2008 ◽

Vol 24 (2) ◽

pp. 131-142 ◽

Cited By ~ 3

Author(s):

Mi-Hwa Yi ◽

Yong-Hwan Lee

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Magnaporthe Oryzae ◽

Functional Characterization ◽

Genes Encoding

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Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

10.1101/303594 ◽

2018 ◽

Author(s):

Krithika Rajagopalan ◽

Jonathan Dworkin

Keyword(s):

Biochemical Characterization ◽

Regulatory Protein ◽

Biochemical Properties ◽

Bacterial Genomes ◽

Phosphatase Inhibitors ◽

E Coli ◽

Specificity And Sensitivity ◽

Genes Encoding ◽

Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

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The characterization of ESBL genes in Escherichia coli and Klebsiella pneumoniae causing nosocomial infections in Vietnam

The Journal of Infection in Developing Countries ◽

10.3855/jidc.2938 ◽

2013 ◽

Vol 7 (12) ◽

pp. 922-928 ◽

Cited By ~ 15

Author(s):

Nguyen Hoang Thu Trang ◽

Tran Vu Thieu Nga ◽

James I Campbell ◽

Nguyen Trong Hiep ◽

Jeremy Farrar ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Nosocomial Infections ◽

Enteric Bacteria ◽

E Coli ◽

Plasmid Analysis ◽

Resistance Plasmids ◽

Genes Encoding ◽

Third Generation Cephalosporins

Background: Extended-spectrum β-lactamases (ESBLs) are enzymes capable of hydrolyzing oxyimino-β-lactams and inducing resistance to third generation cephalosporins. The genes encoding ESBLs are widespread and generally located on highly transmissible resistance plasmids. We aimed to investigate the complement of ESBL genes in E. coli and Klebsiella pneumoniae causing nosocomial infections in hospitals in Ho Chi Minh City, Vietnam. Methodology: Thirty-two non-duplicate isolates of E. coli and Klebsiella pneumoniae causing nosocomial infections, isolated between March and June 2010, were subjected to antimicrobial susceptibility testing. All isolates were PCR-amplified to detect the blaSHV, blaTEM and blaCTX-M ESBL genes and subjected to plasmid analysis. Results: We found that co-resistance to multiple antimicrobials was highly prevalent, and we report the predominance of the blaCTX-M-15 and blaCTX-M-27 genes, located on highly transmissible plasmids ranging from 50 to 170 kb in size. Conclusions: Our study represents a snap shot of ESBL-producing enteric bacteria causing nosocomial infections in this setting. We suggest that antimicrobial resistance in nosocomial E. coli and Klebsiella pneumoniae is rampant in Vietnam and ESBL organisms are widespread. In view of these data and the dramatic levels of antimicrobial resistance reported in Vietnam we advocate an urgent review of antimicrobial use in the Vietnamese healthcare system.

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