RESUMO
Group IIC introns insert next to the stem-loop structure of rho-independent transcription terminators, thus avoiding intact genes. The insertion sites of 17 copies of the G.st.I1 intron from Geobacillus stearothermophilus were compared. One copy of the intron was found to interrupt an open reading frame (ORF) encoding an rRNA methylase.
Assuntos
Proteínas de Bactérias/genética , Geobacillus stearothermophilus/genética , Íntrons/genética , Metiltransferases/genética , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Complete genome sequencing of dozens of strains of the soil bacterium Rhodococcus has revealed the presence of many cryptic biosynthetic gene clusters, presumably dedicated to the production of small molecules. This has sparked a renewed interest in this underexplored member of the Actinobacteria as a potential source of new bioactive compounds. Reported here is the discovery of a potent inhibitory molecule produced by a newly isolated strain of Rhodococcus, strain MTM3W5.2. This small inhibitory molecule shows strong activity against all Rhodococcus species tested, including the veterinary pathogen R. equi, and some closely related genera. It is not active against other Gram positive or Gram negative bacteria. A screen of random transposon mutants identified a gene required to produce this inhibitory compound. This gene is a large multi-domain, type I polyketide synthase that is part of a very large multi-gene biosynthetic gene cluster in the chromosome of strain MTM3W5.2. The high resolution mass spectrum of a major chromatogram peak from a broth culture extract of MTM3W5.2 shows the presence of a compound at m/z 911.5490 atomic mass units. This compound is not detected in the culture extracts from a non-producing mutant strain of MTM3W5.2. A large gene cluster containing at least 14 different type I polyketide synthase genes is proposed to be required to synthesize this antibiotic-like compound.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Rhodococcus/efeitos dos fármacos , Rhodococcus/metabolismo , Infecções por Actinomycetales/tratamento farmacológico , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Elementos de DNA Transponíveis , Genoma Bacteriano , Mutação , Rhodococcus/genética , Rhodococcus/isolamento & purificação , SoloRESUMO
Retrons are reverse transcriptase (RT) encoding genetic elements usually located on the chromosome of a wide variety of mostly Gram-negative bacteria. Here we describe a new retron, designated Ne144, found in the chromosome of the myxobacterium Nannocystis exedens. This element codes for a 515-amino-acid RT that is most closely related to those found in other myxobacterial retrons. The RT is responsible for the production of a small satellite DNA called msDNA. This msDNA is composed of a 144 base, single-stranded DNA that is linked to a 72 base single-stranded RNA. The RNA strand is joined to the 5' end of the DNA chain via a 2'-5' linkage that occurs from the 2' position of an internal guanosine residue in the RNA. In addition to the retron element, the chromosome of N. exedens also contains several partial copies of the msDNA sequence as revealed by DNA hybridization experiments using msDNA as a probe. One of these partial copies was characterized from a chromosome restriction fragment and found to contain a sequence that matches the last 82 bases of the DNA strand and five bases of the RNA strand in msDNA-Ne144. This partial copy of msDNA is very likely a retrotransposed sequence that was generated by reverse transcription using an RNA (the primer-template RNA for msDNA) as a template and the 3' end of a nick in the chromosome as a primer, followed by incorporation into an open reading frame. The presence of this truncated copy of msDNA is strong evidence of retrotransposition in N. exedens causing an alteration in the bacterial genome.
Assuntos
DNA Bacteriano/genética , Myxococcales/genética , Retroelementos/genética , Sequência de Aminoácidos , Sequência de Bases , Sondas de DNA/genética , DNA Bacteriano/química , Duplicação Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Filogenia , DNA Polimerase Dirigida por RNA/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75 degrees C.