[Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans]. / Tn5037-Tn21-podobnyi rtutnyi transpozon, obnaruzhennyi u Thiobacillus ferrooxidans.

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.

[Molecular genetic analysis of the Tn5041 transposition system]. / Molekuliarno-geneticheskii analiz sistemy transpozitsii Tn5041.

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).

[Incorporation of Tn5053 and Tn402 into various plasmids]. / Izuchenie vstraivaniia Tn5053 i Tn402 v razlichnye plazmidy.

Transposons Tn5053 and Tn402 that belong to the novel family of Tn elements are characterized by high selectivity when choosing a target. These transposons integrated with a high frequency into only two of seven large plasmids of various incompatibility groups: RP1 and R446b. The res region of the RP1 plasmid par locus and the res region of the transposon Tn701, included into R446b plasmid, served as targets for both transposons. When Tn701 or par locus integrated into plasmids previously unsuitable for Tn5053 and Tn402 transposition, these plasmids became good targets for both transposons. On the contrary, when the res region of RP1 was damaged impaired, this good target became unsuitable. The insertion sites of Tn5053 and Tn402 were concentrated in the res region of Tn1721 and RP1, but, in some cases, they were at a distance of 100-2000 bp from it.

[Inversion activity in the resolution of transposons Tn5053 and Tn402 possessing a nonstandardly organized res-region]. / Inverversionnaia aktivnost' sistemy rezoliutsii transpozonov Tn5053 i Tn402, obladaiushchikh nestandartno organizovanno res oblast'iu.

The classical bacterial site-specific systems of DNA resolution are characterized, despite their considerable divergence, by a regularly arranged res region represented as a triad of res sites. DNA resolution (deletion) is carried out more efficiently than DNA inversion, primarily due to this structure. In this report, the ratio of deletion to inversion activities is evaluated for the resolution systems of Tn5053 and Tn402, which belong to a novel family and possess an irregularly arranged res region. By this criterion, the system studied is shown to be similar to the classical ones. These data suggest that in the course of evolution of the resolvase systems, the same degree of recombination specialization could be attained by different arrangement of the res region.

[Rpo pathway as a possible recombination mechanism of the interaction between DNA of transducing phages and the Escherichia coli genome]. / Rpo-put' kak vozmozhnyi rekombinatsionnyi mekhanizm vo vzaimodeistviiakh DNK transdutsiruiushchikh fagov s genomom Escherichia coli.

The conditions affecting recombination of DNAs of transducing lambdoid rifd phages with the chromosome of Escherichia coli K-12 recA in the region of homology were studied. In support to the previously obtained data, the Int system was shown to take no part in the process. The homologous character of recombination interactions and their dependence on efficiency of transcription were demonstrated. It is therefore suggested that recombination takes the Rpo pathway. Similar peculiarities were revealed in the processes of interaction between DNAs of rifd phages as well as of lambdoid phages carrying trp genes and the host genome. A hypothesis is put forward that the Rpo pathway operates, depending on the density of DNA supercoiling.

[Mechanism of non-tandem integration of prophage phi 80 into the chromosome of the wild-type Escherichia coli]. / O mekhanizme netandemnoi integratsii profaga phi 80 v khromosomu Escherichia coli dikogo typa.

We studied the ability of lambda, phi 80 and their hybrid lambda att80 to lysogenize homoimmune monolysogens and examined the prophage locations on the chromosome of the resulting polylysogens. We observed an effective integration of phi 80 and lambda att80, in contrast to lambda, into the host chromosome, exclusively, at the attachment sites that were not occupied by the resident prophage (nontandem). Besides, the lambda att80 (int+) prophage was observed to ensure effective nontandem integration of a homoimmune int mutant DNA. Hence, we inferred that the expression of the int gene in the phi 80 prophage is constitutive, cI-independent and results in nontandem integration of the homoimmune prophage. The validity of this inference has been supported experimentally: (i) the only lysogen that was found to contain a phi 80 tandem was highly unstable (spontaneous segregation of monolysogens occurred 6-7 times more frequently than with the lambda tandem); (ii) an int inactivating mutation stabilized the phi 80 tandem; as a result, the int mutant has the frequency of tandem integration as high as that of lambda, while no nontandem integration was observed. A hypothesis is proposed which accounts for the instability of the phi 80 tandems and explains the relation between this phenomenon and the prophage ability to integrate into secondary attachment sites in the presence of the primary (normal) one.

[Formation and genetic structure of polylysogens for lambda and phi 80 and their lambda att80 hybrid infecting wild-type Escherichia coli]. / Obrazovanie i geneticheskaia struktura polilizogenov po lambda, fi 80 i ikh gibridu lambda att80 pri zarazhenii Escherichia coli dikogo tipa.

The frequency of polylysogeny and the genetic structure of polylysogens were studied for phages lambda, phi 80 and lambda att80. For none of these phages does frequency of polylysogeny vary by more than a factor of 2 within a wide range of multiplicities of infection (from 10(-3) up to 10) but the relative location of the prophages on the host chromosome is different. In the case of lambda, polylysogens are formed with a high frequency (0.20-0.41) and the prophages are inserted in tandem into the primary (normal) att site. In the case of phi 80 and lambda att80, polylysogens occur about 10 times less frequently and usually have one prophage inserted into the primary attachment site and another (sometimes, also a third) in one of the secondary ones. Wild-type Escherichia coli was shown to possess at least four secondary att80 sites, two of which (close to the his and tolC loci) are preferred. The frequency of secondary integration of phi 80 and lambda att80 does not differ significantly in the wild-type host and in cells deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at MOI 10). Certain properties of the phi 80 lysogens make it more difficult to decode their genetic structure.

[Integration of the related prophages lambda, phi 80 and their hybrid lambda att80 into the secondary chromosomal att sites of wild-type Escherichia coli]. / Vkliuchenie rodstvennykh profagov lambda, fi 80 i ikh gibrida lambda att80 vo vtorichnye att-saity khromosomy Escherichia coli dikogo tipa.

The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The lambda phage DNA failed to get inserted at the secondary attachment site(s) of the gal operon (frequency less than 2.6 X 10(-8)) in the presence of the primary (normal) one. By contrast, phi 80 and the lambda att80 hybrid integrated into wild-type Escherichia coli at least, at two secondary att sites of the btuB locus, the latter phage being also capable of integration in the vicinity of purE and purC (frequency 2 X 10(-3) to 10(-4)). Integration of phi 80 and lambda att80 into btuB occurred with about the same frequency as in cells deleted for normal insertion site (0.7 divided by 4.0 X 10(-6)). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found in the primary att site. We also failed to observe integration of phi 80 and lambda att80 with formation of secondary monolysogens into other foci (frequency less than 0.0035, if multiplicity of infection was 10(-3) or 10). It is presumed that phi 80 and lambda att80 prophages get only integrated at secondary att sites in case the primary site is occupied.

[Effect of coumermycin A1 and nalidixic acid on lambda phage integration and transducing lambdoid phages containing RNA-polymerase genes]. / Deistvie kumermitsina A1 i nalidiksovoi kisloty na integratsiiu faga lambda i transdutsiruiushchikh liambdoidnykh fagov, soderzhashchikh liambodiodnykh fagov, soderzhashchikh geny RNK-polimerazy.

A specific inhibitor of DNA gyrase, coumermycin A1, inhibits in vivo the interaction of bacteriophage lambda DNA and DNA of rifd transducing phages (lambda rifd47 and lambda att80 rifd35) carrying genes rpoB and rpoC of RNA polymerase. Besides, coumermycin A1 inhibits recA-dependent recombination between the bacterial region of rifd phages and a homologous region of Escherichia coli chromosome. The integration of transdusing phage DNA into the chromosome of recA host is significantly more sensitive to the inhibitory effect of coumermycin A1 than the two other processes (by one -- three orders or magnitude). Another inhibitor of DNA gyrase, nalidixic acid, does not depress the integration of both lambda and rifd phages DNA. The differential effect of coumermycin A1 on the integration of lambda and rifd phages correlates with our concept of the int-idependent mechanism of rifd phage DNA integration in recA bacteria. According to preliminary results, coumermycin A1, in contrast to its action on integration, does not inhibit the exclusion of lambda prophage from E. coli chromosome, while nalidixic acid produces a slight inhibitory effect.