The Escherichia coli RNA polymerase alpha subunit: structure and function.

Publisher Summary This chapter discusses recent discoveries in this field, with special emphasis on molecular systems operating to sense the current status of the host physiology and to switch bacteriophage development into potentially the most effective pathway. The development of bacteriophage λ must be precisely controlled in response to environmental conditions and the physiological state of the host cell. Such a control, performed at the genetic level, ensures choosing an optimal developmental strategy for successful propagation of the virus. This chapter have distinguished five genetic switches in bacteriophage λ development (Ag43 phase variation, the ‘‘lysis versus lysogenization’’ decision, prophage induction, a change from early to late DNA replication mode, and induction of the host cell lysis) whose molecular mechanisms can be proposed. These processes include repression and activation of transcription initiation, antitermination of transcription, general and site-specific recombination, a role for chaperone proteins in macromolecular assembly and DNA replication, and even the control of development.

Genetic switches during bacteriophage lambda development.

For Escherichia coli, there have been more and more examples illustrating that the alpha subunit of RNA polymerase is directly involved in the activation of gene transcription by interaction with activator proteins. Because of the vital function of the alpha subunit in cell growth, only a limited number of mutations in its structural gene, rpoA, have been isolated. We obtained a number of these mutants and examined the effects of these mutations on the acid induction of adi and cad gene expression. Several mutations caused a small reduction in adi promoter activity at inducing pH. One mutation, rpoA341, essentially eliminated adi promoter activity, while it had little effect on the cad promoter. During the course of a separate study, we isolated a plasmid that enhanced adi expression. Further characterization of this plasmid showed that it contained cysB, the structural gene for the positive regulator for most cys operon genes. Introduction of a cysB mutation into an adi::lac fusion strain and beta-galactosidase assay studies of the resultant adi::lac cysB mutant established that a wild-type cysB gene was required for efficient acid induction of adi expression. These results suggest that a possible interaction between CysB and the alpha subunit of RNA polymerase is involved in activation of adi transcription.

Effects of rpoA and cysB mutations on acid induction of biodegradative arginine decarboxylase in Escherichia coli.

Architecture of fis-activated transcription complexes at the Escherichia coli rrnB P1 and rrnE P1 promoters

Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage. Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the λimm434 prophage. Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains.

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Grzegorz Wȩgrzyn

Papers

Involvement of the Escherichia coli RNA polymerase α subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins