Analysis of the presence of prtR proteinase gene in natural isolates of Lactobacillus rhamnosus.
ABSTRACT The region of the prtR gene coding for the active site of PrtR proteinase was detected in natural isolates of lactobacilli, previously determined as Lactobacillus rhamnosus. This region was present in all L. rhamnosus strains with proteolytic activity. The PCR primers used were constructed on the basis of the sequence of the catalytic domain of the prtR proteinase gene. These primers generated in colony-PCR procedure specific 611 1-bp product with DNA from natural isolates of L. rhamnosus. No PCR amplifications using these primers were obtained for closely related bacteria of genus Lactobacillus, regardless of their proteolytic activity. In addition, these primers could be used singly or in multiplex PCR together with the Lactobacillus genus-specific primers. Compared with the other proteinases within the genus Lactobacillus (PrtP, PrtB and PrtH) which retained the activity in cell-free proteinase extracts, PrtR proteinase showed proteolytic activity only under in vivo conditions (whole cells of the producing strains).
Full-textDOI: · Available from: Ljubisa Topisirovic, Nov 08, 2014
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ABSTRACT: The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.Bioorganic & medicinal chemistry letters 08/2011; 21(20):6067-70. DOI:10.1016/j.bmcl.2011.08.057 · 2.33 Impact Factor
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ABSTRACT: The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40 °C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.Applied Microbiology and Biotechnology 12/2014; 99(10). DOI:10.1007/s00253-014-6258-6 · 3.81 Impact Factor