Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika MeSH
- beta-laktamasy genetika MeSH
- Enterobacter MeSH
- infekce bakteriemi rodu Klebsiella * MeSH
- Klebsiella pneumoniae genetika MeSH
- mikrobiální testy citlivosti MeSH
- plazmidy genetika MeSH
- Providencia MeSH
- vrány * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Spojené státy americké MeSH
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
- MeSH
- DNA vazebné proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- endopeptidasy biosyntéza chemie genetika izolace a purifikace MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- membránové proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- molekulární biologie metody MeSH
- proteiny z Escherichia coli biosyntéza chemie genetika izolace a purifikace MeSH
- Providencia enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the 'water retention site', suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.
- MeSH
- chloromethylketony aminokyselin chemická syntéza farmakologie MeSH
- DNA vazebné proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- endopeptidasy chemie genetika metabolismus MeSH
- Escherichia coli chemie enzymologie genetika MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- membránové proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- molekulární modely * MeSH
- mutace MeSH
- proteiny z Escherichia coli antagonisté a inhibitory chemie genetika metabolismus MeSH
- Providencia chemie MeSH
- rekombinantní proteiny MeSH
- simulace molekulární dynamiky * MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- alosterická regulace * MeSH
- buněčná membrána enzymologie MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- membránové proteiny metabolismus MeSH
- Providencia enzymologie MeSH
- serinové proteasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
- MeSH
- fosfatasy fyziologie MeSH
- infekce spojené se zdravotní péčí MeSH
- kultivační média MeSH
- lidé MeSH
- potravinářská mikrobiologie MeSH
- potraviny MeSH
- Proteus izolace a purifikace metabolismus růst a vývoj MeSH
- Providencia izolace a purifikace metabolismus růst a vývoj MeSH
- urogenitální systém mikrobiologie MeSH
- Check Tag
- lidé MeSH
