Characterisation of transfer-deficient mutants of the R100-1TcS plasmid pDU202, caused by insertion of Tn10

Molecular Genetics and Genomics - The transposition of Tn10 from the E. coli chromosome to pDU202 (a TcS deletion mutant of R100-1) was selected by a mating technique: it took place at a frequency...     

Characterisation of transfer-deficient mutants of the R100-1TcS plasmid pDU202, caused by insertion of Tn10

Summary The transposition of Tn10 from the E. coli chromosome to pDU202 (a Tc^S deletion mutant of R100-1) was selected by a mating technique: it took place at a frequency of 10^-7 in both rec ⁺ and recA hosts. Seventeen transfer deficient mutants of pDU202 caused by the insertion of Tn10 into the R factor's transfer genes were analysed. Insertion of Tn10 occurred at at least eight sites, with some apparent clustering in the second part of the transfer operon, and with equal numbers in each orientation. Both strongly and weakly polar insertions were observed, and the strength of the polarity was correlated with the Tn10 orientation. The map order of the second part of the transfer operon was confirmed as traC traF traH traG traS traD, analogous to that of F. The strongly polar insertion mutants still expressed traI at wild-type levels, providing further evidence that this gene does not belong to the transfer operon of R100.     

Pili determined by the tra genes of F-like plasmids

The study of structural and functional features of plasmid-specific pili synthetized by E. coli cells under control of 27 F-like plasmids was performed. All the plasmids determined the pili of "flexible" type which were classified into 3 groups on the basis of difference in cell sensitivity to pili-specific phages f1, f2, Q. The possible role of transposons in the variation of pili functional properties was supposed.     

Genetic syndromes caused by mutations in epigenetic genes

The orchestrated organization of epigenetic factors that control chromatin dynamism, including DNA methylation, histone marks, non-coding RNAs (ncRNAs) and chromatin-remodeling proteins, is essential for the proper function of tissue homeostasis, cell identity and development. Indeed, deregulation of epigenetic profiles has been described in several human pathologies, including complex diseases (such as cancer, cardiovascular and neurological diseases), metabolic pathologies (type 2 diabetes and obesity) and imprinting disorders. Over the last decade it has become increasingly clear that mutations of genes involved in epigenetic mechanism, such as DNA methyltransferases, methyl-binding domain proteins, histone deacetylases, histone methylases and members of the SWI/SNF family of chromatin remodelers are linked to human disorders, including Immunodeficiency Centromeric instability Facial syndrome 1, Rett syndrome, Rubinstein–Taybi syndrome, Sotos syndrome or alpha-thalassemia/mental retardation X-linked syndrome, among others. As new members of the epigenetic machinery are described, the number of human syndromes associated with epigenetic alterations increases. As recent examples, mutations of histone demethylases and members of the non-coding RNA machinery have recently been associated with Kabuki syndrome, Claes-Jensen X-linked mental retardation syndrome and Goiter syndrome. In this review, we describe the variety of germline mutations of epigenetic modifiers that are known to be associated with human disorders, and discuss the therapeutic potential of epigenetic drugs as palliative care strategies in the treatment of such disorders.     

Precise excision of Tn10 in Salmonella typhimurium: effects of mutations in the polA, dam, mutH and mutB genes and of methionine or ethionine in the plating medium.

Precise excision of Tn10 occurs at significantly elevated frequencies in cultures of the polA7 mutant strain of Salmonella typhimurium, is further increased in polA7 dam-1 and polA7 mutB strains and decreased in a polA7 mutH background. The numbers of precise excision events occurring in polA7 strains are also significantly increased when methionine (20 micrograms/ml or less) is present in the medium but decreased when ethionine (again, 20 micrograms/ml or less) is present. When both amino present, the outcome is about a 2-fold increase in precise excision events. The involvement of mismatch repair and methylation patterns in precise excision events is discussed.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

RecA-dependent increased precise excision of Tn10 in Salmonella typhimurium.

UV irradiation induced the precise excision of Tn10 inserted in met, trp or srl in a Salmonella typhimurium strain; mitomycin C was also found to induce the frequency of precise excision of Tn10 from srl or met. Precise excision of Tn10 was not increased by either UV or mitomycin C in a recA mutant. Similarly, a recA mutant derived from a uvrD strain showed a drastic reduction in the high spontaneous levels of precise excision of Tn10 of this strain. These results indicate that recA is involved in the increased precise excision of Tn10. In contrast to point mutations excision of Tn10 was found to be UV inducible in a top mutant.     

Specificity in the formation of delta tra F-prime plasmids.

Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E. coli chromosome. Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci. Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E. coli chromosomal map. Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.     

Heteroduplex analysis of tra delta f'' plasmids and the mechanism of their formation.

Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.     

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.     

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.     

Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12.

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.     

The effect of mutations in recB and recC genes on the precise excision of Tn5 from pNM1 plasmid genome

The affect of mutations in chromosomal genes determining the realization of RecBC and RecF pathways of recombination in E. coli K12 on the frequency of transposon Tn5 precise excision from the genome of the conjugative plasmid pNM1 has been demonstrated. The pNM1 plasmid is a derivative of R100.1 and differs from the latter in the presence of Tn5 inactivating the tet gene of transposon Tn10.     

Identification of additional genes on transposon Tn10: tetC and tetD.

Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R. Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively. The TetD protein was found to be membrane associated. Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested. The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K. Schollmeier and W. Hillen, J. Bacteriol. 160:499-503, 1984).     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.