Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently introduced to many diagnostic microbiological laboratories. Besides the identification of bacteria and fungi, that technique provides a potentially useful tool for the detection of antimicrobial resistance, especially of that conferred by β-lactamases. Here, we describe an assay allowing a detection of meropenem hydrolysis in clinical isolates of Enterobacteriaceae, Pseudomonas spp., and Acinetobacter baumannii using MALDI-TOF MS. This method is able to confirm carbapenemases within 3 h. The results are important for proper and fast intervention to limit the spread of carbapenemase-producing bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.
- MeSH
- Acinetobacter baumannii účinky léků enzymologie genetika izolace a purifikace MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- beta-laktamasy genetika metabolismus MeSH
- beta-laktamová rezistence genetika MeSH
- biotest * MeSH
- exprese genu MeSH
- hydrolýza MeSH
- infekce bakteriemi rodu Acinetobacter diagnóza farmakoterapie mikrobiologie MeSH
- lidé MeSH
- pseudomonádové infekce diagnóza farmakoterapie mikrobiologie MeSH
- Pseudomonas účinky léků enzymologie genetika izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- thienamyciny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Repetitive extragenic palindromic elements (REPs) constitute a group of bacterial genomic repeats known for their high abundance and several roles in host cells´ physiology. We analyzed the phylogenetic distribution of particular REP classes in genomic sequences of sixty-three bacterial strains belonging to the Pseudomonas fluorescens species complex and ten strains of Stenotrophomonas sp., in order to assess intraspecific REP diversity and to gain insight into long-term REP evolution. RESULTS: Based on proximity to RAYT (REP-associated tyrosine transposase) genes, twenty-two and thirteen unique REP classes were determined in fluorescent pseudomonads and stenotrophomonads, respectively. In stenotrophomonads, REP elements were typically found in tens or a few hundred copies per genome. REPs of fluorescent pseudomonads were generally more numerous, occurring in hundreds or even over a thousand perfect copies of particular REP class per genome. REP sequences showed highly heterogeneous distribution. The abundances of REP classes roughly followed host strains´ phylogeny, differing markedly among individual clades. High abundances of particular REP classes appeared to depend on the presence of the cognate RAYT gene, and deviations from this state could be attributed to recent or ancient mutations of rayt-flanking REPs, or RAYT loss. RAYTs of both studied bacterial groups are monophyletic, and their cognate REPs show species-specific characteristics, suggesting shared evolutionary history of REPs, RAYTs and their hosts. CONCLUSIONS: The results of our large-scale analysis show that REP elements constitute intriguingly dynamic components of genomes of fluorescent pseudomonads and stenotrophomonads, and indicate that REP diversification and proliferation are ongoing processes. High numbers of REPs have apparently been retained during the entire evolutionary time since the establishment of these two bacterial lineages, probably because of their beneficial effect on host long-term fitness. REP elements in these bacteria represent a suitable platform to study the interplay between repeated elements, their mobilizers and host bacterial cells.
Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099(T)), Pseudomonas aureofaciens (NCIM2026(T)), and Pseudomonas aeruginosa (MTCC2582(T)). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH(4))(2)SO(4) precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn(2+), Cu(2+), and Ni(2+) ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- endopeptidasy genetika metabolismus MeSH
- Fabaceae mikrobiologie MeSH
- fylogeneze MeSH
- molekulární sekvence - údaje MeSH
- Pseudomonas klasifikace enzymologie genetika izolace a purifikace MeSH
- půdní mikrobiologie MeSH
- rhizosféra MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Indie MeSH
DNA replication of plasmids in Gram-negative bacteria has been an object of study at CIB-CSIC for nearly 30 years. We have been focused on the enterobacterial antibiotic resistance factor R1 (1981-1992) and the pPS10 replicon from the phytopathogen Pseudomonas savastanoi (since 1984). Our group has used multidisciplinary (genetic, biochemical and biophysical-structural) approaches to unravel the molecular mechanism for the activation of RepA. Rep-type plasmidic proteins are either transcriptional repressors or replication initiators/inhibitors, depending on their association state (dimers vs. monomers) and targeting of alternative (operator or iteron) DNA sites. We discovered that allosteric DNA-binding remodels the structure of RepA N-terminal domain (WH1), transforming alpha-helical portions into beta-strands. This precisely tunes the distances between the DNA reading heads in WH1 and the C-terminal domain (WH2), to match the target operator or iteron sequences. We have recently moved into engineering such structural transformation in RepA-WH1 to build-up synthetic protein devices that allow for customized ligand (DNA)-promoted amyloidogenesis. Our basic studies on plasmid DNA replication are relevant for settling the bases of a minimalist bacterial model to tackle transmissible amyloid proteinopathies and are a valuable tool for bottom-up synthetic biology.
- MeSH
- amyloid biosyntéza genetika MeSH
- amyloidóza enzymologie genetika MeSH
- bakteriální léková rezistence genetika imunologie účinky léků MeSH
- bakteriální proteiny biosyntéza genetika účinky léků MeSH
- financování organizované MeSH
- gramnegativní bakterie genetika růst a vývoj MeSH
- klinické laboratorní techniky využití MeSH
- lidé MeSH
- plazmidy biosyntéza genetika chemie MeSH
- Pseudomonas enzymologie genetika růst a vývoj MeSH
- replikace DNA genetika účinky léků MeSH
- rostliny MeSH
- statistika jako téma MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Sphingolipid ceramide N-deacylase (SCDase, EC 3.5.1.69) is a hydrolytic enzyme isolated from Pseudomonas sp. TK 4. In addition to its primary deacylation function, this enzyme is able to reacylate lyso-sphingolipids under specific conditions. We immobilised this enzyme on magnetic macroporous cellulose and used it to semisynthesise C17:0 glucosylceramide and C17:0 sulphatide, which are required internal standards for quantification of the corresponding glycosphingolipids (GSL) by tandem mass spectrometry. A high rate of conversion was achieved for both lipids (80% for C17:0 sulphatide and 90% for C17:0 glucosylceramide). In contrast to synthesis with a soluble form of the enzyme, use of immobilised SCDase significantly reduced the contamination of the sphingolipid products with other isoforms, so further purification was not necessary. Our method can be effectively used for the simple preparation of specifically labelled sphingolipids of high isoform purity for application in mass spectrometry.
- MeSH
- amidohydrolasy chemie MeSH
- bakteriální proteiny chemie MeSH
- enzymy imobilizované chemie MeSH
- glukosylceramidy chemická syntéza chemie MeSH
- glykosfingolipidy analýza MeSH
- hmotnostní spektrometrie metody normy MeSH
- hydrolýza MeSH
- Pseudomonas enzymologie MeSH
- referenční standardy MeSH
- stereoizomerie MeSH
- sulfoglykosfingolipidy chemická syntéza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30-84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G(+) bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS (-) mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.
- MeSH
- antibióza MeSH
- bakteriální proteiny genetika metabolismus MeSH
- houby fyziologie MeSH
- karboxylesterhydrolasy genetika metabolismus MeSH
- laktony metabolismus MeSH
- mutace MeSH
- polygalakturonasa genetika metabolismus MeSH
- Pseudomonas enzymologie fyziologie genetika MeSH
- půdní mikrobiologie MeSH
- regulace genové exprese u bakterií MeSH
- transkripční faktory genetika metabolismus MeSH
- vývojová regulace genové exprese MeSH
