F de la Cruz

GENETIC EVIDENCE OF A COUPLING ROLE FOR THE TRAG PROTEIN FAMILY IN BACTERIAL CONJUGATION

Abstract The ability of conjugative plasmids from six different incompatibility groups to mobilize a set of mobilizable plasmids was examined. The mobilization frequencies of plasmids RSF1010, ColE1, ColE3, and CloDF13 varied over seven orders of magnitude, depending on the helper conjugative plasmid used. Mobilization of CloDF13 was unique in that it did not require TrwB, TraG or TraD (all members of the TraG family) for mobilization by R388, RP4 or F, respectively. CloDF13 itself codes for an essential mobilization protein (MobB) which is also a TraG homolog, only requiring a source of the genes for pilus formation. Besides, CloDF13 was mobilized efficiently by all conjugative plasmids, suggesting that TraG homologs are the primary determinants of the mobilization efficiency of a plasmid, interacting differentialy with the various relaxosomes. Previous results indicated that TraG and TrwB were interchangeable for mobilization of RSF1010 and ColE1 by PILW (the pilus system of IncW plasmids) but TraG could not complement conjugation of trwB mutants, suggesting that additional interactions were taking place between TrwB and oriT(R388) that were not essential for mobilization. To further test this hypothesis, we analyzed the mobilization frequencies of ColE1 and RSF1010 by the P, W, and F pili in the presence of alternative TraG homologs. The results obtained indicated that the frequency of mobilization was determined both by the particular TraG-like protein used and by the pilus system. Thus, TraG-like proteins are not generally interchangeable for mobilization. Therefore we suggest that the factors that determine the frequencies of transfer of different MOB regions are the differential interactions of TrwB with pilus and relaxosome.

Purification of α-hemolysin from an overproducing E. coli strain

SummaryThe genetic determinant of the α-hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions.α-hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified α-hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the α-hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.

Plasmids containing one inverted repeat of Tn21 can fuse with other plasmids in the presence of Tn21 transposase

SummaryIn the presence of the Tn21 transposase, plasmids that contain a single Tn21 inverted repeat sequence fuse efficiently with other plasmids. This reaction occurs in recA strains, is independent of the transposon-encoded resolution system, and results in insertions into different sites in the recipient plasmid. All fusion products studied contained at least one complete copy of the donor plasmid; most also contained some duplication of it as well. The data are consistent with processive models of transposition.

Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum.

The pSUM vectors, a family of Mycobacterium/Escherichia coli shuttle cloning vectors derived from the pSU series of vectors are described. They all contain the pACYC184 origin of replication enabling them to replicate in E. coli, the mycobacterial pAL5000 origin of replication and a kanamycin-resistance (KmR) gene which allows selection in both E. coli and mycobacteria. They carry a multiple cloning site (MCS) to facilitate cloning and the lacZ alpha reporter gene for screening inserts in E. coli. A library of total DNA from a strain of Mycobacterium fortuitum was constructed with one of these vectors, pSUM36, and a mycobacterial gentamicin-resistance (GmR) gene was cloned by selecting for GmR in Mycobacterium smegmatis.

Site-specific recombination and shuffling of resistance genes in transposon Tn21

Many multidrug-resistant transposons found in natural isolates of Gram-negative bacteria are close relatives of Tn21. Thus, the Tn21 subgroup of the Tn3 family of transposable elements is the most successful homogeneous group in acquiring resistance to newly introduced antibiotics. This paper summarizes the mode of acquisition of resistance genes by these elements.