We studied the impact of a sublethal concentration of erythromycin on the fitness and proteome of a continuously cultivated population of Escherichia coli. The development of resistance to erythromycin in the population was followed over time by the gradient plate method and minimum inhibitory concentration (MIC) measurements. We measured the growth rate, standardized efficiency of synthesis of radiolabeled proteins, and translation accuracy of the system. The proteome changes were followed over time in two parallel experiments that differed in the presence or absence of erythromycin. A comparison of the proteomes at each time point (43, 68, and 103 h) revealed a group of unique proteins differing in expression. From all 35 proteins differing throughout the cultivation, only three were common to more than one time point. In the final population, a significant proportion of upregulated proteins was localized to the outer or inner cytoplasmic membranes or to the periplasmic space. In a population growing for more than 100 generations in the presence of antibiotic, erythromycin-resistant bacterial clones with improved fitness in comparison to early resistant culture predominated. This phenomenon was accompanied by distinct changes in protein expression during a stepwise, population-based development of erythromycin resistance.

• Klíčová slova
• Continuous cultivation system, Escherichia coli, erythromycin, fitness, proteome, resistance,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• časové faktory MeSH
• erythromycin farmakologie   MeSH
• Escherichia coli účinky léků   genetika   metabolismus   MeSH
• genetická zdatnost účinky léků   MeSH
• kultivační média MeSH
• mikrobiální testy citlivosti MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• erythromycin MeSH
• kultivační média MeSH
• proteiny z Escherichia coli MeSH
• proteom MeSH

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

• MeSH
• bakteriální geny MeSH
• bakteriální proteiny analýza   genetika   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• luciferasy bakteriální analýza   genetika   MeSH
• methyltransferasy genetika   MeSH
• mutageneze cílená MeSH
• nesmyslný kodon MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) MeSH
• proteosyntéza * MeSH
• reportérové geny * MeSH
• Streptomyces lividans účinky léků   genetika   fyziologie   MeSH
• streptomycin farmakologie   MeSH
• supresorové geny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• inhibitory syntézy proteinů MeSH
• luciferasy bakteriální MeSH
• methyltransferasy MeSH
• nesmyslný kodon MeSH
• rRNA (adenosine-O-2'-)methyltransferase MeSH Prohlížeč
• streptomycin MeSH