Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes.

• MeSH
• Borrelia * genetika   klasifikace   MeSH
• lidé MeSH
• myši MeSH
• návratná horečka * mikrobiologie   MeSH
• testy genetické komplementace MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective. METHODS: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function. RESULTS: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B6 (ϱ = 0.93). CONCLUSIONS: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.

• MeSH
• cystathionin-beta-synthasa genetika   metabolismus   MeSH
• fenotyp MeSH
• genetické testování metody   MeSH
• genotyp MeSH
• homocystinurie genetika   MeSH
• lidé MeSH
• missense mutace * MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae MeSH
• testy genetické komplementace metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Candida krusei is a pathogenic yeast species that is phylogenetically outside both of the well-studied yeast groups, whole genome duplication and CUG. Like all other yeast species, it needs to accumulate high amounts of potassium cations, which are needed for proliferation and many other cell functions. A search in the sequenced genomes of nine C. krusei strains revealed the existence of two highly conserved genes encoding putative potassium uptake systems. Both of them belong to the TRK family, whose members have been found in all the sequenced genomes of species from the Saccharomycetales subclade. Analysis and comparison of the two C. krusei Trk sequences revealed all the typical features of yeast Trk proteins but also an unusual extension of the CkTrk2 hydrophilic N-terminus. The expression of both putative CkTRK genes in Saccharomyces cerevisiae lacking its own potassium importers showed that only CkTrk1 is able to complement the absence of S. cerevisiae's own transporters and provide cells with a sufficient amount of potassium. Interestingly, a portion of the CkTrk1 molecules were localized to the vacuolar membrane. The presence of CkTrk2 had no evident phenotype, due to the fact that this protein was not correctly targeted to the S. cerevisiae plasma membrane. Thus, CkTrk2 is the first studied yeast Trk protein to date that was not properly recognized and targeted to the plasma membrane upon heterologous expression in S. cerevisiae.

• MeSH
• Candida klasifikace   genetika   růst a vývoj   metabolismus   MeSH
• draslík metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genom fungální genetika   MeSH
• iontový transport MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae klasifikace   genetika   růst a vývoj   metabolismus   MeSH
• Saccharomycetales klasifikace   genetika   MeSH
• testy genetické komplementace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dekkera bruxellensis is important for lambic beer fermentation but is considered a spoilage yeast in wine fermentation. We compared two D. bruxellensis strains isolated from wine and found that they differ in some basic properties, including osmotolerance. The genomes of both strains contain two highly similar copies of genes encoding putative glycerol-proton symporters from the STL family that are important for yeast osmotolerance. Cloning of the two DbSTL genes and their expression in suitable osmosensitive Saccharomyces cerevisiae mutants revealed that both identified genes encode functional glycerol uptake systems, but only DbStl2 has the capacity to improve the osmotolerance of S. cerevisiae cells.

• MeSH
• Dekkera genetika   izolace a purifikace   metabolismus   fyziologie   MeSH
• druhová specificita MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genom bakteriální genetika   MeSH
• glycerol metabolismus   MeSH
• osmoregulace genetika   MeSH
• protony MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• symportéry genetika   metabolismus   MeSH
• testy genetické komplementace MeSH
• víno mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální geny genetika   MeSH
• DNA bakterií genetika   MeSH
• Escherichia coli účinky léků   genetika   izolace a purifikace   MeSH
• genotyp MeSH
• kur domácí MeSH
• membránové transportní proteiny genetika   MeSH
• mikrobiální testy citlivosti veterinární   MeSH
• mnohočetná bakteriální léková rezistence účinky léků   genetika   MeSH
• mutace MeSH
• nemoci drůbeže mikrobiologie   MeSH
• proteiny z Escherichia coli genetika   MeSH
• regulace genové exprese u bakterií MeSH
• represorové proteiny genetika   MeSH
• rezistence na chloramfenikol účinky léků   genetika   MeSH
• testy genetické komplementace veterinární   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Metal accumulation in seeds is a prerequisite for germination and establishment of plants but also for micronutrient delivery to humans. To investigate metal transport processes and their interactions in seeds, we focused on METAL TOLERANCE PROTEIN8 (MTP8), a tonoplast transporter of the manganese (Mn) subclade of cation diffusion facilitators, which in Arabidopsis (Arabidopsis thaliana) is expressed in embryos of seeds. The x-ray fluorescence imaging showed that expression of MTP8 was responsible for Mn localization in subepidermal cells on the abaxial side of the cotyledons and in cortical cells of the hypocotyl. Accordingly, under low Mn availability, MTP8 increased seed stores of Mn, required for efficient seed germination. In mutant embryos lacking expression ofVACUOLAR IRON TRANSPORTER1(VIT1), MTP8 built up iron (Fe) hotspots inMTP8-expressing cells types, suggesting that MTP8 transports Fe in addition to Mn. Inmtp8 vit1double mutant seeds, Mn and Fe were distributed in all cell types of the embryo. An Fe transport function of MTP8 was confirmed by its ability to complement Fe hypersensitivity of a yeast mutant defective in vacuolar Fe transport. Imbibingmtp8-1mutant seeds in the presence of Mn or subjecting seeds to wet-dry cycles showed that MTP8 conferred Mn tolerance. During germination, MTP8 promoted reallocation of Fe from the vasculature. These results indicate that cell type-specific accumulation of Mn and Fe in seeds depends on MTP8 and that this transporter plays an important role in the generation of seed metal stores as well as for metal homeostasis and germination efficiency under challenging environmental conditions.

• MeSH
• Arabidopsis embryologie   genetika   metabolismus   MeSH
• biologické modely MeSH
• genový knockout MeSH
• homeostáza * MeSH
• klíčení * genetika   MeSH
• mangan metabolismus   MeSH
• mutace genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny přenášející kationty metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• semena rostlinná embryologie   genetika   MeSH
• spektrometrie rentgenová emisní MeSH
• testy genetické komplementace MeSH
• vývojová regulace genové exprese MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Yeasts usually have one or two high-affinity potassium transporters. Two complete and one interrupted gene encoding three types of putative potassium uptake system exist in Candida albicans SC5314. As high intracellular potassium is essential for many yeast cell functions, the existence of three transporters with differing transport mechanisms (Trk uniporter, Hak cation-proton symporter, Acu ATPase) may help pathogenic C. albicans cells to acquire the necessary potassium in various organs and tissues of the host. When expressed in Saccharomyces cerevisiae lacking their own potassium uptake systems, all three putative transporters were able to provide cells with the ability to grow with low amounts of potassium over a broad range of external pH. Only CaTrk1 was properly recognized and secreted to the plasma membrane. Nevertheless, even the small number of CaHak1 and mainly CaAcu1 molecules which reached the plasma membrane resulted in an improved growth of cells in low potassium concentrations, suggesting a high affinity and capacity of the transporters. A single-point mutation restored the complete CaACU1 gene, and the resulting protein not only provided cells with the necessary potassium but also improved their tolerance to toxic lithium. In contrast to its known homologues, CaAcu1 did not seem to transport sodium.

• MeSH
• Candida albicans genetika   metabolismus   MeSH
• draslík metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média chemie   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• proteiny přenášející kationty nedostatek   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   metabolismus   MeSH
• testy genetické komplementace MeSH
• Publikační typ
• časopisecké články MeSH

Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. Increased stress tolerance, including the thermotolerance of some Cronobacter strains, can promote their survival in production facilities and thus raise the possibility of contamination of dried infant formula which has been identified as a potential source of infection. Some Cronobacter strains contain a genomic island, which might be responsible for increased thermotolerance. By analysis of Cronobacter sequenced genomes this determinant was found to be present in only 49/73 Cronobacter sakazakii strains and in 9/14 Cronobacter malonaticus strains. The island was also found in 16/17 clinical isolates originating from two hospitals. Two configurations of the locus were detected; the first one with the size of 18 kbp containing the thrB-Q genes and a shorter version (6 kbp) harbouring only the thrBCD and thrOP genes. Strains containing the thermotolerance island survived significantly better at 58 °C comparing to a C. sakazakii isogenic mutant lacking the island and strains with the longer version of the island were 2-10 times more tolerant than those with the shortened sequence. The function of the genomic island was further confirmed by its cloning into a low-copy vector and transforming it into the isogenic mutant. Different levels of rpoS, encoding for stress-response sigma factor, expression were also associated with variability in strain thermotolerance.

• MeSH
• bakteriální geny MeSH
• biologická adaptace genetika   MeSH
• Cronobacter klasifikace   genetika   metabolismus   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• genom bakteriální * MeSH
• genomové ostrovy * MeSH
• infekce spojené se zdravotní péčí MeSH
• klonování DNA MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• plazmidy genetika   MeSH
• pořadí genů MeSH
• reakce na tepelný šok genetika   MeSH
• teplota * MeSH
• testy genetické komplementace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres.

• MeSH
• buněčné jádro genetika   metabolismus   MeSH
• Candida genetika   metabolismus   MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• genom fungální * MeSH
• genom mitochondriální * MeSH
• homeostáza telomer MeSH
• klonování DNA MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• regulace genové exprese u hub * MeSH
• rekombinace genetická MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• telomery chemie   metabolismus   MeSH
• testy genetické komplementace MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei.

• MeSH
• ABC transportéry antagonisté a inhibitory   genetika   metabolismus   MeSH
• akonitáthydratasa metabolismus   MeSH
• biologické modely MeSH
• cytosol metabolismus   MeSH
• fumarasa metabolismus   MeSH
• fylogeneze MeSH
• genový knockdown MeSH
• hem metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• proteiny obsahující železo a síru metabolismus   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům antagonisté a inhibitory   genetika   metabolismus   MeSH
• protozoální geny MeSH
• protozoální proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• testy genetické komplementace MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH