N-acyl homoserine lactones (N-HLs) are signaling molecules used by Gram-negative bacteria in a phenomenon called quorum sensing. Bacteria will detect N-HLs as a way of monitoring their population which, upon reaching a critical level, will express a specific phenotype. An example is the expression of bioluminescence by Vibrio fischeri. Most studies have not considered the chirality of these molecules nor have they used highly sensitive detection methods. Here, the production of d,l-N-HLs are monitored for V. fischeri, Burkholderia cepacia, Pseudomonas fluorescens, and P. putida, using highly sensitive tandem mass spectrometry analysis. Novel N-HLs are reported for both V. fischeri and B. cepacia, including a plethora of previously unknown d-N-HLs, including the first d-N-HLs containing oxo and hydroxy functionalities. Anomalously, N-HLs were not detected in any cultures of P. fluorescens and P. putida, which are species that previously were reported to produce N-HLs. However, it is apparent that differences in the reported occurrence and levels of N-HLs can result from (a) different strains of bacteria, (b) different growth media and environmental conditions, and (c) sometimes false-positive results from detection methodologies. Time studies of V. fischeri suggest the possibility that separate synthetic and elimination pathways exist between d- and l-N-HLs. Possible biological processes that could be the source of d-N-HL production are considered.

• Klíčová slova
• bioluminescence, chemical communication, chirality, homoserine lactones, phenotype expression,
• MeSH
• Aliivibrio fischeri * chemie   metabolismus   MeSH
• Burkholderia cepacia * metabolismus   MeSH
• chromatografie kapalinová MeSH
• gama-butyrolakton metabolismus   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• quorum sensing MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gama-butyrolakton MeSH

Recent years have witnessed an increased prevalence of intrinsic and acquired beta-lactamase-producing bacteria, severely limiting human and veterinary medicine therapeutic options. The present study aimed to design specific oligonucleotides for rapid PCR detection of the cephalosporinase-encoding gene blaEC (BlaEC family class C beta-lactamase). A total of three primers were designed to detect 2281 variants of the blaEC gene and two sets of primer pairs were also tested against DNA from 11 strains. The study indicates that the proposed primers should be able to detect 100% of all described blaEC genes in different bacterial strains and monitor their spread. After comparing the amino acid sequences, a phylogenetic tree was created based on the presence of conserved amino acids and homologous motifs. More than 24 760 mutations in BlaEC enzymes have been identified. The mutations involving 371 amino acid positions and these hotspots can change the structure and activity of the monitored enzymes. We predicted several BlaEC enzymes with a broadened substrate activity against higher-generation cephalosporins.

• Klíčová slova
• AmpC beta-lactamase, PCR, antibiotic resistance, mutation, primer,
• Publikační typ
• časopisecké články MeSH

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed.

• Klíčová slova
• Rhodococcus erythropolis, in vitro transcription, RNA-seq, sigma factor, transcriptional regulatory network,
• MeSH
• bakteriální proteiny * metabolismus   MeSH
• genetická transkripce MeSH
• genové regulační sítě MeSH
• regulace genové exprese u bakterií * MeSH
• Rhodococcus MeSH
• sigma faktor metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• sigma faktor MeSH

Fungal metabolic carbon acquisition and its subsequent partitioning between biomass production and respiration, i.e. the carbon-use efficiency (CUE), are central parameters in biogeochemical modeling. However, current available techniques for estimating these parameters are all associated with practical and theoretical shortcomings, making assessments unreliable. Gene expression analyses hold the prospect of phenotype prediction by indirect means, providing new opportunities to obtain information about metabolic priorities. We cultured four different fungal isolates (Chalara longipes, Laccaria bicolor, Serpula lacrymans and Trichoderma harzianum) in liquid media with contrasting nitrogen availability and measured growth rates and respiration to calculate CUE. By relating gene expression markers to measured carbon fluxes, we identified genes coding for 1,3-β-glucan synthase and 2-oxoglutarate dehydrogenase as suitable markers for growth and respiration, respectively, capturing both intraspecific variation as well as within-strain variation dependent on growth medium. A transcript index based on these markers correlated significantly with differences in CUE between the fungal isolates. Our study paves the way for the use of these markers to assess differences in growth, respiration and CUE in natural fungal communities, using metatranscriptomic or the RT-qPCR approach.

• Klíčová slova
• carbon-use efficiency, fungi, gene markers, growth, metatranscriptomics, respiration,
• MeSH
• Ascomycota genetika   metabolismus   MeSH
• Basidiomycota genetika   MeSH
• biologické markery * analýza   MeSH
• fungální proteiny * genetika   metabolismus   MeSH
• houby * genetika   metabolismus   MeSH
• Hypocreales genetika   metabolismus   MeSH
• Laccaria genetika   metabolismus   MeSH
• transkriptom * MeSH
• Trichoderma genetika   metabolismus   MeSH
• uhlík * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery * MeSH
• fungální proteiny * MeSH
• uhlík * MeSH

Numerous serotypes which belong to the genus Enterovirus (EV) show variability in their virulence and clinical manifestations. They are also known to undergo changes caused by mutations and recombination during their circulation in the environment and the population. Various EV serotypes are prevalent in groundwater, wastewater and surface waters. Our previous studies showed that oral infection induces pancreatitis depending on specific conditions, such as gravidity, in an outbred murine model. Our aim in the present study was to further explore the pancreatic histopathology in an outbred mouse model following oral infection with clinical isolates from a patient who had aseptic meningitis and an isolate from a treated-sewage sample recovered from the residential area of the patient. The isolates were identified as coxsackievirus B4 (CVB4) in tissue culture. The CVB4 sewage-isolate induced pancreatitis after oral infection. In contrast, pancreatitis was absent following infection with the clinical isolates. Comparison of polyprotein sequences showed that the treated-sewage strains differed from the patient's isolates by 9 and 11 amino acids. We conclude that the isolates of clinical and environmental origin differed in their pathogenic properties and showed genetic variation.

• Klíčová slova
• coxsackievirus B4, genetic variability, isolates, pancreatitis, pathogenesis,
• MeSH
• coxsackie virózy * virologie   MeSH
• enterovirus B lidský * patogenita   fyziologie   MeSH
• lidé MeSH
• myši MeSH
• odpadní vody * virologie   MeSH
• pankreatitida * chemicky indukované   virologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• odpadní vody * MeSH

Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.

• Klíčová slova
• β-lactamase, PCR, antibiotic resistance, bacteria, primer,
• MeSH
• Bacteria enzymologie   genetika   izolace a purifikace   MeSH
• bakteriologické techniky * MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• beta-laktamová rezistence genetika   MeSH
• DNA bakterií genetika   MeSH
• DNA primery MeSH
• lidé MeSH
• polymerázová řetězová reakce * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-laktamasy MeSH
• DNA bakterií MeSH
• DNA primery MeSH

Inclusions in evaporitic minerals sometimes contain remnants of microorganisms or biomarkers, which can be considered as traces of life. Raman spectroscopy with resonance enhancement is one of the best analytical methods to search for such biomarkers in places of interest for astrobiology, including the surface and near subsurface of planet Mars. Portable Raman spectrometers are used as training tools for detection of biomarkers. Investigations of the limits and challenges of detecting biomolecules in crystals using Raman spectroscopy is important because natural occurrences often involve mineral assemblages as well as their fluid and solid inclusions. A portable Raman spectrometer with 532 nm excitation was used for detection of carotenoid biomarkers: salinixanthin of Salinibacter ruber (Bacteroidetes) and α-bacterioruberin of Halorubrum sodomense (Halobacteria) in laboratory-grown artificial inclusions in compound crystals of several chlorides and sulfates, simulating entrapment of microorganisms in evaporitic minerals. Crystals of halite (NaCl), sylvite (KCl), arcanite (K2SO4) and tschermigite ((NH4)Al(SO4)2·12H2O) were grown from synthetic solutions that contained microorganisms. A second crystalline layer of NaCl or K2SO4 was grown subsequently so that primary crystals containing microorganisms are considered as solid inclusions. A portable Raman spectrometer with resonance enabling excitation detected signals of both carotenoid pigments. Correct positions of diagnostic Raman bands corresponding to the specific carotenoids were recorded.

• Klíčová slova
• carotenoids, halophilic prokaryotes, inclusions, miniature Raman spectrometer, salts,
• MeSH
• Bacteria chemie   MeSH
• chloridy chemie   MeSH
• exobiologie * MeSH
• karotenoidy analýza   MeSH
• krystalizace MeSH
• Mars * MeSH
• prokaryotické buňky chemie   MeSH
• Ramanova spektroskopie přístrojové vybavení   MeSH
• sírany chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloridy MeSH
• karotenoidy MeSH
• sírany MeSH

A specific technique of nuclear magnetic resonance (NMR) spectroscopy, filter-exchange spectroscopy (FEXSY), was employed to investigate water transport through the plasma membrane in intact yeast cells. This technique allows water transport to be monitored directly, thus avoiding the necessity to subject the cells to any rapid change in the external conditions, e.g. osmotic shock. We established a sample preparation protocol, a data analysis procedure and verified the applicability of FEXSY experiments. We recorded the exchange rates in the temperature range 10-40°C for Saccharomyces cerevisiae. The resulting activation energy of 29 kJ mol-1 supports the hypothesis that water exchange is facilitated by water channels-aquaporins. Furthermore, we measured for the first time water exchange rates in three other phylogenetically unrelated yeast species (Schizosaccharomyces pombe, Candida albicans and Zygosaccharomyces rouxii) and observed remarkably different water exchange rates between these species. Findings of our work contribute to a better understanding of as fundamental a cell process as the control of water transport through the plasma membrane.

• Klíčová slova
• Saccharomyces cerevisiae, activation energy, membrane permeability, nuclear magnetic resonance spectroscopy, water transport, yeast,
• MeSH
• akvaporiny metabolismus   MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• Candida albicans metabolismus   MeSH
• kinetika MeSH
• magnetická rezonanční spektroskopie MeSH
• Schizosaccharomyces metabolismus   MeSH
• teplota MeSH
• termodynamika MeSH
• voda metabolismus   MeSH
• Zygosaccharomyces metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akvaporiny MeSH
• voda MeSH

Infection with Toxoplasma gondii has usually been connected with consumption of improperly treated meat. However, contaminated water and products of plant origin have emerged as new sources of infection in the last few years. Here, 292 vegetable samples-carrot, cucumber and lettuce-obtained from nine farms in the Czech Republic were examined using triplex real time PCR targeting two specific T. gondii sequences. Irrigation water and water used for washing of vegetables were also included. Overall, a positivity rate of 9.6% was found in vegetables. The concentration varied between 1.31 × 100 and 9.00 × 102 oocysts/g of sample. A significant difference was found between the positivity of vegetables collected directly from fields and that of vegetables collected from farm storage rooms (4.4-8.6% vs 10-24.1%, respectively). All samples of irrigation water and water used to rinse vegetables were negative. Genotyping based on restriction fragment length polymorphism (RFLP) analysis using seven markers revealed the exclusive presence of genotype II.

• Klíčová slova
• Toxoplasma gondii, farm, food safety, genotyping, qPCR, vegetable,
• MeSH
• bezpečnost potravin MeSH
• farmy * MeSH
• genotyp MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• potravinářská parazitologie * MeSH
• Toxoplasma * klasifikace   genetika   izolace a purifikace   MeSH
• zelenina parazitologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

We investigated the effect of Kluyveromyces lactis ERG6 gene deletion on plasma membrane function and showed increased susceptibility of mutant cells to salt stress, cationic drugs and weak organic acids. Contrary to Saccharomyces cerevisiae, Klerg6 mutant cells exhibited increased tolerance to tunicamycin. The content of cell wall polysacharides did not significantly vary between wild-type and mutant cells. Although the expression of the NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGPD1) in the Klerg6 mutant cells was only half of that in the parental strain, it was induced in the presence of calcofluor white. Also, cells exposed to this drug accumulated glycerol. The absence of KlErg6p led to plasma membrane hyperpolarization but had no statistically significant influence on the plasma membrane fluidity. We propose that the phenotype of Klerg6 mutant cells to a large extent was a result of the reduced activity of specific plasma membrane proteins that require proper lipid composition for full activity.

• MeSH
• delece genu MeSH
• fungální proteiny genetika   metabolismus   MeSH
• fyziologická adaptace * MeSH
• kationické antimikrobiální peptidy metabolismus   MeSH
• Kluyveromyces účinky léků   enzymologie   genetika   fyziologie   MeSH
• kyseliny karboxylové toxicita   MeSH
• methyltransferasy genetika   metabolismus   MeSH
• osmotický tlak MeSH
• regulace genové exprese u hub * MeSH
• tolerance léku MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fungální proteiny MeSH
• kationické antimikrobiální peptidy MeSH
• kyseliny karboxylové MeSH
• methyltransferasy MeSH