RESUMEN
It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC50) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease.
Asunto(s)
Actinomycetales/efectos de los fármacos , Actinomycetales/metabolismo , Antiinfecciosos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Actinomycetales/genética , Antiinfecciosos/toxicidad , Proteínas Bacterianas/toxicidad , Concentración 50 Inhibidora , Solanum lycopersicum , Pruebas de Sensibilidad Microbiana , Hojas de la Planta/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Solanum tuberosum/efectos de los fármacosRESUMEN
BACKGROUND: Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae. RESULTS: Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels. CONCLUSION: Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens.