RESUMO
Gene E-L, a chimeric lysis construct from bacteriophages phi X174 and MS2 lysis proteins E and L, respectively, was subjected to internal deletions to create a series of new E-L clones with altered lysis or killing properties. The lytic activities of the parental genes E. L. E-L and the internal truncated forms of E-L were investigated in this study to characterize the different lysis mechanisms, based on differences in the architecture of the different membrane spanning domains. Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that two different lysis mechanisms can be distinguished depending on penetrating of the proteins either the inner membrane or the inner and outer membranes of Escherichia coli. Several candidates, which share efficient lysis properties, have biotechnological applications in terms of cell disruption.
Assuntos
Bacteriólise/fisiologia , Escherichia coli/virologia , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Virais/fisiologia , Sequência de Bases , Escherichia coli/ultraestrutura , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Fatores de Tempo , Transformação Genética , Proteínas Virais/genética , beta-Galactosidase/análise , beta-Lactamases/análiseRESUMO
A concept study devised for the development of a biological containment system has been conducted. We show that the lysis genes of different phage origin function in a variety of bacteria. They may therefore be suited for conditional suicide cassettes. Moreover, we tested whether the Escherichia coli rrnB P1 promoter could function as an environmentally responsive element sensing poor growth conditions expected after an accidental release of E. coli production strains from a bioreactor. Mimicking poor nutrient conditions by production of the alarmone guanosine tetraphosphate (ppGpp) with a plasmid encoded ppGpp synthetase I, the rrnB P1 promoter activity was completely turned off. These experiments suggested that the rrnB P1 promoter may be used as an efficient biosensor for altered growth conditions. A concept for a conditional suicide system employing the rrnB P1 promoter and phage-derived lysis genes as key components is discussed.
Assuntos
Colífagos/genética , Contenção de Riscos Biológicos/métodos , Escherichia coli/genética , Regiões Promotoras Genéticas , Ribossomos/metabolismo , Óperon de RNAr/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Genes Virais , Dados de Sequência Molecular , RNA ViralRESUMO
The lambda kil gene has been shown to be responsible for premature lysis effected by addition of chloramphenicol between 15 and 20 min after thermal induction of a lambda prophage. Here, we localized the kil reading frame. The kil gene, represented by lambda orf47, overlaps genes cIII and gam. Expression of the plasmid-borne kil gene resulted in growth arrest, a reduction of colony-forming units and filament formation. However, kil-mediated cell lysis could not be triggered by chloramphenicol when the plasmid borne kil gene was expressed, suggesting that kil-induced cell lysis requires the phage context.
