RESUMO
Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Though most transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using single-stranded DNA, always inserts just 3' of a TTAC tetranucleotide. Our results from studies on the IS608 transposition mechanism demonstrated that the transposase recognizes its target site by co-opting an internal segment of transposon DNA and utilizes it for specific recognition of the target sites through base-pairing. This suggested a way to redirect IS608 transposition to novel target sites. As we demonstrate here, we can now direct insertions in a predictable way into a variety of different chosen target sequences, both in vitro and in vivo.
Assuntos
Proteínas de Bactérias/fisiologia , Elementos de DNA Transponíveis/fisiologia , DNA de Cadeia Simples/química , Helicobacter pylori/genética , Modelos Genéticos , Transposases/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Pareamento de Bases , Sequência de Bases , Mutação Puntual , Transposases/química , Transposases/genéticaRESUMO
Streptococcus uberis UCN60 was resistant to spiramycin (MIC = 8 microg/ml) but susceptible to erythromycin (MIC = 0.06 microg/ml), azithromycin (MIC = 0.12 microg/ml), josamycin (MIC = 0.25 microg/ml), and tylosin (MIC = 0.5 microg/ml). A 2.5-kb HindIII fragment was cloned from S. uberis UCN60 DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to spiramycin by inactivation. The sequence analysis of the fragment showed the presence of an rdmC-like gene that putatively encoded a protein belonging to the alpha/beta hydrolase family and of the first 196 nucleotides of the mph(B) gene putatively encoding a phosphotransferase known to inactivate 14-, 15-, and 16-membered macrolides in E. coli. The entire mph(B) gene was then identified in S. uberis UCN60. The two genes were expressed alone or in combination in E. coli, Staphylococcus aureus, and Enterococcus faecalis. Analysis of MICs revealed that rdmC-like alone did not confer resistance to erythromycin, tylosin, and josamycin in those three hosts. It conferred resistance to spiramycin in E. coli and E. faecalis but not in S. aureus. mph(B) conferred resistance in E. coli to erythromycin, tylosin, josamycin, and spiramycin but only low levels of resistance in E. faecalis and S. aureus to spiramycin (MIC = 8 microg/ml). The combination of mph(B) and rdmC-like genes resulted in a resistance to spiramycin and tylosin in the three hosts that significantly exceeded the mere addition of the resistance levels conferred by each resistance mechanism alone.
Assuntos
Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Macrolídeos/farmacologia , Streptococcus/efeitos dos fármacos , Streptococcus/genética , Azitromicina/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Josamicina/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Tilosina/farmacologiaRESUMO
Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 microg/ml) while remaining susceptible to clindamycin (MIC = 0.06 microg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinA(N2), and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2'') was identified.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Lincomicina/farmacologia , Mastite Bovina/microbiologia , Nucleotidiltransferases/genética , Streptococcus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bovinos , Feminino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Análise de Sequência de DNA , Streptococcus/enzimologia , Streptococcus/genética , Streptococcus/isolamento & purificaçãoRESUMO
The transposon MTnSag1 from Streptococcus agalactiae carried an IS1-like transposase gene and the lnu(C) gene, which encoded a lincosamide nucleotidyltransferase. MTnSag1 could be mobilized by the conjugative transposon Tn916. An intermediate circular form of MTnSag1 and a putative origin of transfer at the 3' end of the lnu(C) gene were characterized.
Assuntos
Elementos de DNA Transponíveis/genética , Streptococcus agalactiae/genética , Sequência de Aminoácidos , Sequência de Bases , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Insercional , Alinhamento de Sequência , Análise de Sequência de DNA , Transposases/genéticaRESUMO
Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 microg/ml) but susceptible to clindamycin (MIC = 0.12 microg/ml) and erythromycin (MIC = 0.06 microg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA(N2), LnuA, and LnuA' lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.
