The bacteriophage Mu com gene appears to specify a translation factor required for mom gene expression

Abstract

Expression of the bacteriophage Mu mom gene is subject to a variety of regulatory controls. Both the host Dam DNA-adenine methylase and the phage Mu C protein are required for mom gene transcription. In addition, the Mu com gene product is required for production of the mom protein. Because the com and mom genes overlap on the same mRNA transcript (with com being located proximal to the 5 ' end), it is likely that Com function is exerted after transcription initiation. To study the role of Com, two segments of Mu were cloned in both orientations (+) and -) into the HindIII site of the galactokinase expression vector, pKG1800; the HindIII site is located between the galK structural gene and its promoter. In (+) plasmids, the Mu DNA inserts were transcribed from the gal promoter in the same orientation as in the phage genome; (-) plasmids had the Mu DNA inserted in the reverse orientation. Each Mu insert contained the same segment of the mom gene from the 3' terminus, but differed in the extent of com gene included at the 5' terminus; one contained a truncated com gene and the other a complete com gene, as well as upstream Mu regulatory sequences. The results are summarized as follows: (1)both (-) plasmids produced only about 10% as much galactokinase activity following fucose induction as the parental vector, pKGlSOO; (2) plasmid pGTVH (+), with an intact com gene produced about 30% as much galactokinase as pKGlSOO; (3) plasmid pMTVH(+), with a truncated com gene, produced only about 10% as much enzyme as pKG1800; (4)pMTVH (+) could be trans-activated to produce galactokinase (to about 50% the level of pKG1800) when a Mu com + prophage was induced at the time of galK induction. In contrast, com − prophage could not trans-activate plasmid pMTVH(+). Neither (-) plasmid was transactivated by induced Mu com + prophage; (5) in a rho-15 mutant, all the plasmids directed galactokinase synthesis independently of the Com function; (6) when the plasmid pMTVH (+) insert was excised and cloned into the SmaI site, about 18 nucleotides downstream from HindIII (this insertion inactivates a Rho-dependent terminator located in this region), the new construct, pMTVS (+), produced about 1 3 as much galactokinase as the parental pKG1800 vector. We conclude from these data that the Com protein is not an anti-termination factor per se; rather, it appears to be required for translation of the mom gene. In the plasmids used here, this translation is necessary to suppress transcription polarity.