RESUMEN
Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.
Asunto(s)
Agrobacterium tumefaciens/patogenicidad , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Homeostasis , Lisina/metabolismo , Fosfatidilgliceroles/metabolismo , Factores de Virulencia/metabolismo , Agrobacterium tumefaciens/crecimiento & desarrollo , Proteínas Bacterianas/genética , Dominio Catalítico , Análisis Mutacional de ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Eliminación de Gen , Prueba de Complementación Genética , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Enfermedades de las Plantas/microbiología , Solanum tuberosum/microbiología , Transformación Genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.
Asunto(s)
Aminoaciltransferasas/química , Bacillus/enzimología , Proteínas Bacterianas/química , Fosfatidilgliceroles/metabolismo , Pseudomonas aeruginosa/enzimología , Factores R , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Lisina/metabolismo , Aminoacilación , Aminoaciltransferasas/metabolismo , Bacillus/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Lisina/biosíntesis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Fosfatidilgliceroles/biosíntesis , Conformación Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusión/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.