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A 1,3 kb DNA fragment of the IncM plasmid R446, if cloned in a multicopy plasmid, inhibits in trans the expression of conjugation pili by IncM plasmid-harbouring host bacteria as indicated by their insensitivity to the IncM pilus-dependent bacteriophage phi M (Iml, insensitivity to phi M-mediated lysis). Determination of the nucleotide sequence of this DNA fragment, the introduction of deletions an the analysis of transposon insertions reveal two determinants, imlA and imlB, responsible for the Iml phenotype. A stretch of 80 bp of DNA containing imlA and about 450 bp of adjacent DNA comprising imlB, together, bring about inhibition of the typical expression of conjugation pili at 30 degrees C. The introduction of a transposable promoter probe and the construction of respective lacZ fusions indicate transcription of complementary strands in vivo overlapping in the region comprising imlA and imlB. Moreover, the expression of reporter genes discloses temperature-dependent transcription of the imlA-imlB-region in one direction. A particular subfragment of the 1.3 kb IncM plasmid-derived DNA does not inhibit conjugation pilus expression at 30 degrees C but stimulates in trans the formation of pili at 42 degrees C to give rise to untypical sensitivity to phi M at 42 degrees C in addition to 30 degrees C. Other subfragments reveal vital interferences with IncM plasmid-harbouring host cells. The putative nature of the cloned determinants interfering with the normal expression of IncM plasmid DNA is discussed.

Probes for the detection of streptothricin resistance genes have been derived from recombinant plasmids. These include the streptothricin resistance gene probe sat 1/2 derived from Tn 1826 and specific for both the sat-1 determinant of Tn 1825 and the sat-2 determinant of Tn 1826, and the probe sat D derived from and specific for the sat-1 determinant of transposon Tn 1825. A third streptothricin resistance gene probe, sat 3, represents the streptothricin resistance determinant sat-3 of the IncQ R plasmid pIE639. Hybridization studies did not reveal any sequence homology between sat-3 and the transposon-localized sat-1 and sat-2 determinants. Moreover, non of the different sat-determinants isolated from plasmids of gram negative bacteria hybridized with the analogous resistance determinant of Streptomyces noursei, which had been cloned and named nat by Krügel et al. (Gene, 1988, 62, 209-214). The sat 1/2 probe in combination with the sat D probe proved to be suitable for the identification and the differentiation of sat-1 and sat-2 determinants in different genetic environments. Streptothricin resistance genes related to those present on transposons Tn 1825 and Tn 1826 have been detected by hybridization with the probe sat 1/2 on plasmids isolated a long time ago before the application of streptothricins. The sat-3 determinant appears to be exclusively associated with the IncQ plasmid pIE639.

14 conjugative plasmids originated from different bacterial hosts and geographical sources were identified as members of the incompatibility group IncX with R6K as the reference plasmid. In spite of their close genetic relatedness (transfer and incompatibility properties), their molecular sizes ranged between 39 and 77 kb and DNA fingerprinting with endonucleases such as EcoRI, EcoRV, BamHI, BglI, BglII, PstI and SalI revealed a great variety of fragment patterns.

New IncQ R plasmids, pIE639 and pIE723, are characterized and compared to the prototype IncQ plasmid RSF1010. Additional resistance determinants not common on other R plasmids are located on small stretches of DNA interspacing essential regions at different positions in an otherwise unchanged core of IncQ plasmid DNA. The contribution of IncQ plasmids to resistance evolution in bacteria is discussed.

Streptothricin resistance determinants have been cloned from the transposons Tn1825 and Tn1826 to the vector plasmid pUC8. The recombinant plasmids were characterized with respect to their physical structure. The resistance properties of their hosts were characterized with respect to the minimal inhibitory concentration of streptothricin and the activity of the streptothricin acetyltransferase. The proteins encoded by the cloned resistance determinants were analysed in a maxicell system. The results of our investigations suggest that the resistance to streptothricin is mediated by the activity of a streptothricin acetyltransferase with both, Tn1825 and Tn1826. However, the resistance determinant of Tn1825 is more complex than Tn1826 and additional functions involved in realizing the resistance phenotype must be recognized.