A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli

Abstract

The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-l mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-l mutant and no UV-induced mutation.We showed earlier (1) that a plasmid containing the mucA gene from pKM101 caused UV mutagenesis in Haemophilus influenzae, which is normally not operative in this species (4, 7). We now report on the biological effects in H. influenzae and Escherichia coli of a plasmid, pMucAMucB, we have constructed that carries both mucA and mucB genes from pKM101 in our cloning vector pDM2 (5).Starting material for construction of the plasmid was the plasmid pGW1700 (10), kindly provided by David Sobell of Graham Walker's laboratory (Massachusetts Institute of Technology, Cambridge), which contains the two muc genes from pKM101 but is considerably smaller (15). The genes were subcloned into a derivative of pBR322 (10). A 2.4-kilobase-pair fragment was cut out of pGW1700 with the restriction endonucleases XmnI and Scal, and PstI linkers were attached, making it possible to clone the fragment in the vector pDM2, which has a unique PstI site (5), and also to remove the fragment from pDM2. The fragment with PstI linkers was first inserted into PstI-cut pBR322 in order to amplify the PstI-ended fragment for successful cloning in H. influenzae via pDM2. Restriction endonuclease mapping of the plasmid showed the presence of both muc genes, as judged by comparison with previous mapping of these genes (9). H. influenzae and E. coli were transformed by the plasmids as previously described (11,17). Crude plasmid preparations (8) were used to transfer pMucAMucB between H. influenzae species. Figure 1 shows survival curves of AB1157 umu-36 (GW3198), kindly supplied by Graham Walker and previously described (3), containing plasmids pDM2 and pBR322 with and without the two muc genes. The inserts increased the survival to the same extent, whether in pDM2 or pBR322. Thus, it was clear that the lethal effect of UV irradiation of E. coli was similarly influenced by the muc genes in the two different plasmids. The survival was not affected by the plasmids pDM2 and pBR322. The data are in accord with the increase in survival seen in E. coli strain TK610 (AB1157 umuC36 and also containing a mutation affecting excision of pyrimidine dimers) with the plasmid pSK100 carrying both the umuD and umuC genes (16). Figure 2 shows survival curves of wild-type strain Rd with the cloning vector pDM2 and with the vector containing the muc genes. The strains were irradiated together to eliminate possible differences in dosimetry and were distinguished on plates by appropriate antibiotics in the agar. The difference in UV sensi...

Heterospecific expression of misrepair-enhancing activity of mucAB in Escherichia coli and Bacillus subtilis

“…Moreover, the Rec protein activity of B. subtilis is similar to that of the E. coli RecA protein (7,22,23). Expression of introduced mucAB in Haemophilus influenzae (1,49), and to a lesser extent in yeast (43) and mouse cells (27), has been shown.…”