RESUMO
Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.
Assuntos
Lacticaseibacillus casei/genética , Lacticaseibacillus casei/virologia , Prófagos/genética , Prófagos/fisiologia , Ativação Viral , Microbiologia de Alimentos , Mitomicina/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , Proteínas da Cauda Viral/genéticaRESUMO
Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation.
Assuntos
Acidithiobacillus/genética , Transferência Genética Horizontal , Sequências Repetitivas Dispersas , Acidithiobacillus/efeitos dos fármacos , Biologia Computacional , Expressão Gênica , Mitomicina/metabolismo , Recombinação Genética , Ativação Transcricional/efeitos dos fármacosRESUMO
In this work the involvement of polymerase II (Pol II) in the precise excision of Tn10 stimulated by a dnaB252 thermosensitive (Ts) mutant at the permissive temperature, by a uvrD mutant, or by mitomycin C (MMC) or ultraviolet (UV) light treatment, was investigated. A deltapolB::kan mutant showed a significant decrease in the excision of Tn10 induced by the dnaB mutation, or by MMC or UV treatment, indicating the participation of Pol II in this type of deletion process. However, no effect of Pol II was evidenced in the excision of Tn10 stimulated by the uvrD mutation. The effect of the polB mutation on Tn10 precise excision induced by all these treatments was compared to that of mutations in repair-recombination genes recF and recA. The results reveal that the degree of participation of these genes varies depending on the agent that stimulates the deletion event.