RESUMO
pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer. A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer. Initiation (i.e. excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain. pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome. Transfer generally involves single-stranded DNA. In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer. Using the differential sensitivity to the SalI restriction-modification system of transfers involving single- and double-stranded DNA, we found that pSAM2 was probably transferred to the recipient as double-stranded DNA. This provides the first experimental evidence for the transfer of double-stranded DNA during bacterial conjugation. Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double-stranded DNA actively, either from donor to recipient or from mother cell to prespore.
Assuntos
Conjugação Genética , Elementos de DNA Transponíveis/genética , Fator sigma , Streptomyces/genética , Fatores de Transcrição , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Dados de Sequência Molecular , Streptomyces/fisiologia , Transformação BacterianaRESUMO
Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.
Assuntos
Genes Bacterianos/genética , Streptomyces/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Clonagem Molecular , Códon/genética , DNA Bacteriano/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Regiões Promotoras Genéticas/genética , RNA Bacteriano/análise , RNA Mensageiro/análise , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia , Transcrição Gênica/genética , alfa-Amilases/metabolismoRESUMO
In glycerol-grown, but not in glucose-grown cultures, expression in Streptomyces lividans TK24 of the cloned alpha-amylase gene (aml) of Streptomyces limosus is switched on toward the end of exponential growth. During this period, aml expression is further inducible by maltotriose. We showed that a 378 bp fragment, extending from position -204 through to +174 (relative to the transcriptional start site), included cis-acting sequences involved in aml regulation. When this fragment was present on a multicopy plasmid, the growth-phase-dependent aml expression conferred by a DNA fragment cloned on a compatible low-copy-number plasmid was greatly enhanced, as if negative regulators were being titrated. A study of the regulation of aml expression in variants with deletions in the aml promoter region indicated that a direct repeat (DR) between positions -124 and -106 (relative to the transcriptional start site) and an inverted repeat (IR) between positions +9 and +24 were good candidates for secondary and primary operator sites, respectively. Deletion of a 29 bp fragment containing the IR rendered aml expression partly growth-phase-independent, resistant to glucose repression, and insensitive to maltotriose induction.
Assuntos
Repressão Enzimática/genética , Regulação Bacteriana da Expressão Gênica , Streptomyces/genética , alfa-Amilases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Glucose/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Deleção de Sequência , Streptomyces/crescimento & desenvolvimento , Transcrição Gênica , Trissacarídeos/farmacologia , alfa-Amilases/genéticaRESUMO
The Streptomyces ambofaciens genome contains four rRNA gene clusters. These copies are called rrnA, B, C and D. The complete nucleotide (nt) sequence of rrnD has been determined. These genes possess striking similarity with other eubacterial rRNA genes. Comparison with other rRNA sequences allowed the putative localization of the sequences encoding mature rRNAs. The structural genes are arranged in the order 16S-23S-5S and are tightly linked. The mature rRNAs are predicted to contain 1528, 3120 and 120 nt, for the 16S, 23S and 5S rRNAs, respectively. The 23S rRNA is, to our knowledge, the longest of all sequenced prokaryotic 23S rRNAs. When compared to other large rRNAs it shows insertions at positions where they are also present in archaebacterial and in eukaryotic large rRNAs. Secondary structure models of S. ambofaciens rRNAs are proposed, based upon those existing for other bacterial rRNAs. Positions of putative transcription start points and of a termination signal are suggested. The corresponding putative primary transcript, containing the 16S, 23S and 5S rRNAs plus flanking regions, was folded into a secondary structure, and sequences possibly involved in rRNA maturation are described. The G + C content of the rRNA gene cluster is low (57%) compared with the overall G + C content of Streptomyces DNA (73%).
Assuntos
Genes Bacterianos , Família Multigênica , RNA Ribossômico/genética , Streptomyces/genética , Composição de Bases , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Mapeamento por Restrição , Transcrição GênicaRESUMO
When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.
