ABSTRACT
Carbapenemase Inactivation Method (CIM) is a test to detect presence of the carbapenemase in Gram-negative bacteria. Determination of the carbapenemase production by inactivation of meropenem requires that a zone of control E. coli inhibition be measured approximately 6-24 h after plating. We have modified the CIM test by developing a rapid method which instead measures the growth of E. coli indicator strain ATCC 25922 using real-time PCR, referred to as a nucleic acid testing CIM (natCIM). Our natCIM, therefore reduces the detecting time from 6 to 24 h to approximately 4 h.
ABSTRACT
The hyperthermophilic archaeon, Pyrococcus furiosus, expresses a small, alpha-crystallin-like protein in response to exposure to extreme temperatures, above 103 degrees C. The P. furiosus small heat shock protein (Pfu-sHSP) forms large oligomeric complexes. Based on the available crystal structures of the Methanocaldococcus jannaschii and wheat sHSPs, the protruding carboxy terminal domain is probably involved in subunit interactions. We constructed Pfu-sHSP mutants to analyze chaperone function and to study multi-subunit assembly. The results confirmed that the carboxy terminus of Pfu-sHSP is involved in inter-dimer interactions, whereas the amino terminal deletion mutant still exhibited the wild-type assembly characteristics. The ability to form oligomeric complexes via the carboxy terminal domain was shown to be necessary for thermotolerance of Escherichia coli overexpressing Pfu-sHSP. The amino terminal domain was not required for inter-species thermotolerance.
Subject(s)
Archaeal Proteins/chemistry , Heat-Shock Proteins/chemistry , Pyrococcus furiosus/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Base Sequence , DNA, Archaeal/genetics , Dimerization , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hot Temperature , Models, Molecular , Protein Structure, Quaternary , Protein Subunits , Pyrococcus furiosus/genetics , Pyrococcus furiosus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , alpha-Crystallins/chemistry , alpha-Crystallins/genetics , alpha-Crystallins/metabolismABSTRACT
The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105 degrees C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105 degrees C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56 degrees C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50 degrees C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.
Subject(s)
Heat-Shock Proteins/metabolism , Pyrococcus furiosus/metabolism , Animals , Cattle , Cloning, Molecular , Escherichia coli , Glutamate Dehydrogenase/metabolism , Heat-Shock Proteins/genetics , Hot Temperature , Molecular Sequence DataABSTRACT
To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif.
