RESUMEN
Bacterial two-component regulatory systems (TCS) are common components of complex regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI controls exopolysaccharide succinoglycan production and flagellum biosynthesis. Although this system plays a crucial role in establishing the symbiosis between S. meliloti and its host plant, it is not well characterized. Attempts to generate complete loss-of-function mutations in either exoS or chvI in S. meliloti have been unsuccessful; thus, it was previously suggested that exoS or chvI are essential genes for bacterial cell growth. We constructed a chvI mutant by completely deleting the open reading frame encoding this gene. The mutant strain failed to grow on complex medium, exhibited lower tolerance to acidic condition, produced significantly less poly-3-hydroxybutyrate than the wild type, was hypermotile, and exhibited an altered lipopolysaccharide profile. In addition, this mutant was defective in symbiosis with Medicago truncatula and M. sativa (alfalfa), although it induced root hair deformation as efficiently as the wild type. Together, our results demonstrate that ChvI is intimately involved in regulatory networks involving the cell envelope and metabolism; however, its precise role within the regulatory network remains to be determined.
Asunto(s)
Medicago sativa/microbiología , Medicago truncatula/microbiología , Polisacáridos Bacterianos/biosíntesis , Rizoma/microbiología , Eliminación de Secuencia , Sinorhizobium meliloti/metabolismo , Simbiosis/fisiología , Flagelos/genética , Flagelos/metabolismo , Fenotipo , Polisacáridos Bacterianos/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crecimiento & desarrolloRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now "super strains," resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47U mg(-1) under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Mycobacterium tuberculosis/enzimología , 3-Desoxi-7-Fosfoheptulonato Sintasa/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Tuberculosis (TB) still remains a worldwide health concern. Efforts to understand the complex biology of Mycobacterium tuberculosis, the causative agent of TB, are important for new antitubercular drug development. Despite the completion of the genome sequence and the development of new genetic tools to manipulate this organism, the availability of sufficient amounts of mycobacterial proteins still remains an essential and laborious step to study the biochemical features of this pathogen. The T7-RNA polymerase-based pET system has been largely employed to express mycobacterial proteins in Escherichia coli, but it presents some limitations. To overcome problems with unstable expression of an M. tuberculosis inhA-encoded enoyl reductase mutant protein and lack of expression of two mabA-encoded ketoacyl reductase mutants, a sub-population of E. coli BL21(DE3) host cells was selected from a small-opaque colony. This empirically selected host, named BL21(DE3)NH, allowed stable expression of these mutant proteins. Although the mechanism that led the BL21(DE3)NH host to express the recombinant mutant proteins remains unknown, the persistent phenotype points to a stable genetic switch. This genetic alteration resulted in a tight control of the highly processive T7 RNA polymerase. Moreover, the absolute requirement for IPTG to obtain protein expression in the BL21(DE3)NH host cells suggests that no inherent defect in the transcriptional activity of the T7 promoter is present. Empirical host selection requires no further genetic manipulation of recombinant plasmids and may represent a means of obtaining tailor-made E. coli strains that overcome toxic effects associated with heterologous protein expression.