RESUMEN
Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.
Asunto(s)
Proteínas de Unión Periplasmáticas , Sideróforos , Sideróforos/metabolismo , Proteínas de Unión Periplasmáticas/química , Geobacillus stearothermophilus/metabolismo , Compuestos Férricos/metabolismo , Hierro/metabolismoRESUMEN
The aim of this study is to develop the real-time PCR assays to detect and qualify C. coli from stool specimen. The primers and probe were designed based on the specific sequence of ceuE gene in C. coli using Primer 5.0 and Vector NTI Suite 6.0 e. The PCR assay was optimized with the reference C. coli strains. The standard curve based on the dilutions of genomic DNA showed a linear relationship between log CFU/mL and threshold cycles. The detectable limitation was 5.62 CFU/mL by using purified DNA from bacteria culture. The reproducibility of this assay was assessed by calculating the variation of the threshold cycle value and the slope from test repeats for the same strains and different strains. Our results indicate that the developed assay has high sensitivity and specificity for identification of C. coli.
RESUMEN
Campylobacter coli is the most common Campylobacter species found in pig (95%), but the ability of this bacterium to survive in swine manure as well as the potential for causing human illness are poorly understood. We present here laboratory-scale experiments to investigate the effect of temperature on the survival of C. coli in spiked swine manure samples at temperatures from 4 to 52°C. The survival of C. coli during storage for 30 days was studied by three different methods: bacterial culture (plate counting), DNA qPCR, and mRNA RT-qPCR. The results indicate that C. coli could survive in swine manure up to 24 days at 4°C. At higher temperatures, this bacterium survived only 7 days (15°C) or 6 days (22°C) of storage. The survival of C. coli was extremely short (few hours) in samples incubated at 42 and 52°C. The results from the RT-qPCR method were consistent with the data from the bacterial culture method, indicating that it detected only viable C. coli cells, thus eliminating false-positive resulting from DNA from dead C. coli cells.