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Geny kodující acetyhydroxy syntázu u Streptomyces cinnamonensis a jejich regulační oblast:Mutace v katalytické podjednotce zjią»ující necitlivost k inhibici konečného produktu
Kyselková, Martina ; Kopecký, Jan ; Šigutová, Lucie ; Pospíšil, Stanislav ; Felsberg, Jürgen ; Spížek, Jaroslav ; Janata, Jiří
Reaction of acetohydroxy acid synthase (AHAS) is the key step in branched-chain amino acids biosynthesis. AHAS is feed-back inhibited by valin that binds its regulatory subunit. In Streptomyces cinnamonensis, production of a secondary metabolite, polyether antibiotic monensin A, is associated with valin biosynthesis via 2-oxoisovalerate. Thus, mutants with higher AHAS levels or AHAS insensitive to valin show increased monensin A production. Gene ilvB coding for the AHAS catalytic subunit of the S. cinnamonensis parental strain and four mutants resistant to valin analogues was cloned and sequenced. Nucleotide sequence of ilvB from the strain BVR-18 differed from the wild type in a single substitution C574A resulting in an amino acid change E139A. In the ACB-NLR-2 strain, a triplet AGC in the position 805-807 was deleted resulting in the deletion of Q217. A homology model of the catalytic subunit, created by a virtue of the known three-dimensional structure of the yeast AHAS catalytic subunits dimer, revealed that the mutation found in the BVR-18 was located in a loop at the subunit-subunit interface in a close proximity of the TPP binding site in the active centre. The Q217 deletion in the ACB-NLR-2 occurred in a helix distant from the catalytic site, and seemed to shorten it. AHAS assays in a crude cell extract from the S. cinnamonensis BVR-18 and ACB-NLR-2 strains showed not only the insensitivity to valin but even higher activities of both enzymes in the presence of 10 mM valine. Catalytic and regulatory subunits from the parental and mutant strains were separately expressed in E. coli and combined in vitro to reconstitute the holoenzyme. Properties of such in vitro prepared enzymes were tested. The ACBR-2 and NLR-3 strains with increased AHAS levels have no mutation in the AHAS catalytic subunit. The ilvB regulatory region of those two strains and of the parental strain was cloned and sequenced using the method of chromosome walking. A putative leader peptide and a transcription termination sequence were identified within this region indicating that ilvB expression is controlled by attenuation. However, neither the ACBR-2, nor the NLR-3 have any mutation in the ilvB regulatory region suggesting that some other regulatory mechanism may participate in ilvB expression.
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Analýza genového clusteru anthramycinových a lincosamidových antibiotik
Jelínková, Markéta ; Koběrská, Markéta ; Čermák, Lukáš ; Kopecký, Jan ; Janata, Jiří ; Spížek, Jaroslav
Lincomycin, produced by Streptomyces lincolnensis, is important, clinically used antibiotic. Its gene cluster consists of twenty-seven putative open reading frames with biosynthetic or regulatory functions and three resistance genes. Comparison of the chemical structure of the lincomycin and celesticetin indicates, that in the biosynthesis of celesticetin the whole propylproline branch is absent and proline is substrate of the penultimate step of biosynthesis, the condensation reaction. Second part of biosynthetic pathway including conversion of L-tyrosine to L-proline should be involved in the biosynthesis of functionally different anthramycin antibiotics. Thus it appears, that genetic information on lincomycin and anthramycin biosynthesis must also share common elements (genes), both biosynthetic and regulatory. The organization of transcription units was found. The analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed sooner. A set of probes based on the sequence of lincomycin biosynthetic cluster was used to search for analogous genes in celesticetin producer Streptomyces caelestis and three producers of anthramycin antibiotics: Streptomyces refuineus, Streptomyces albus and Streptosporangium sibiricum. Hybridization experiments proved presence of several analogues of genes coding for sugar moiety biosynthesis in S. caelestis and analogues of some genes coding for anthramycin antibiotics in different anthramycin producers. A rough restriction map of a part of celesticetin gene cluster presumably responsible for sugar moiety biosynthesis shows the gene arrangement to be very similar to that of lincomycin biosynthetic cluster.
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Mechanismus rezistence makrolidů a linkosamidů v koaguláze negativních stafylokoků v ČR a nové typy na linkosamid resistentních S. haemolyticus
Novotná, Gabriela ; Janata, Jiří ; Spížek, Jaroslav
Objectives: The aim of this study was to investigate an incidence of different resistance mechanisms to macrolides and lincosamides in methicillin-resistant coagulase-negative staphylococci in the Czech Republic. Methods: Phenotypes were determined by triple-disc diffusion method. The presence of the genes ermA, ermC (resistance by target site alteration) msrA (efflux resistance) and linA (resistance by antibiotic inactivation) was tested by Southern blot analysis. A bioassay for the detection of lincomycin inactivation mechanism was performed in lincosamide-resistant strains, where none of the resistance genes was detected. In these strains also PFGE analysis was done. Results: In 99 clinical isolates in vitro resistant to one of erythromycin, lincomycin or clindamycin, triple-disc diffusion method reveals seven different phenotypes corresponding with resistance mechanisms. The resistance was mainly due to the presence of msrA gene, which was detected in 53 strains. Genes ermC and ermA were detected in 42 strains and the linA was detected in 28 strains. In 15 lincosamides-resistant strains no resistance gene was detected. Lincosamides were not inactivated in those strains indicating a new type of resistance different from inactivation. Conclusion: The dissemination of resistance types differs strongly from the published data. While in other countries cross-resistance to macrolides and lincosamides conferred by ermA and ermC predominates, in the Czech Republic the gene msrA is the most frequent genetic determinant conferring resistance to macrolides only. It follows that one third of macrolide-resistant staphylococci remains lincosamide-sensitive. A homogenous group of lincosamide-resistant S. haemolyticus with unknown resistance mechanism was newly defined.
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Antibiotics-modes of action, resistance mechanisms and the search for new compounds
Spížek, Jaroslav
According to their mechanism of action antibiotics can be divided into 4 groups. 1. Antibiotics inhibiting bacterial cell wall biosynthesis. 2. Antibiotics inhibiting DNA synthesis. 3. Antibiotics inhibiting RNA synthesis and 4. Antibiotics inhibiting protein synthesis. Basic mechanisms of antibiotic resistance include cell impermeability, modification of target site, modification or degradation of the antibiotic and increased efflux of the antibiotic. The following can be used in the search for new compounds: 1. Microorganisms isolated from extreme life conditions 2. Environmental DNA (eDNA) in which genes coding for secondary metabolites are looked for. 3. Streptomyces genomics yielding information about genes coding biosynthesis of new compounds. 4. Combinatorial biosynthesis for the production of hybrid antibiotics.
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Ivan Málek and Czechoslovak microbiology
Spížek, Jaroslav
Ivan Málek was one of the founders of the Czechoslovak microbiology. His main fields of interest included medical and industrial microbiology. He introduced the term "physiological state of the cell" and was the leader of the Czechoslovak school of continuous cultivation of microorganisms.
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