RESUMEN
BACKGROUND: Lactococcus lactis strain Plasma (LC-Plasma) possesses strong stimulatory activity for plasmacytoid dendritic cells (pDCs) via the TLR9-Myd88 pathway. To reveal the effective lactic acid bacteria (LAB) genome structure for pDCs stimulatory activity, we performed in vitro screening, using randomly selected 200 bp DNA fragments from the LC-Plasma genome. RESULTS: We found that the CpG motif copy number in the fragments was positively and significantly correlated with pDCs stimulatory activity (R = 0.491, p < 0.01). However, the determination coefficient (R2) was 0.24, which means other factors affecte activity. We found that the G + C contents of the fragment showed a significant negative correlation with activity (R = - 0.474, p < 0.01). The correlation between pDCs stimulatory activity and the copy number of CpG motifs was greatly increased when DNA fragments were stratified by G + C contents. We also performed bioinformatics analysis and a screening of LAB strains with high pDCs stimulatory activity. Species with a high copy number of CpG motifs in the low-G + C region of their genomes had higher probability of inducing high-pDCs stimulatory activity. L. lactis subsp. lactis, Leuconostoc mesenteroides, and Pediococcus pentosaceus were three typical examples of LAB that had high pDCs stimulatory activity. CONCLUSIONS: Our data suggested that the G + C content of DNA is one of the critical factors for pDCs stimulatory activity by DNA fragments. Furthermore, we found that the copy number in the low-G + C regions strongly affected the pDCs stimulatory activity of whole cells of LAB strains. These results should be useful for the design of new DNA fragments containing CpG motifs. This study also demonstrated an in silico screening method for identifying bacterial species that are able to activate pDCs.
Asunto(s)
ADN Bacteriano/inmunología , Células Dendríticas/inmunología , Genoma Bacteriano , Lactobacillales/genética , Animales , Composición de Base , Células Cultivadas , Islas de CpG , Femenino , Genómica , Ratones , Ratones de la Cepa 129 , OligodesoxirribonucleótidosRESUMEN
LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.
Asunto(s)
Proteínas Arqueales/metabolismo , Arginina/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Lisina/biosíntesis , Sulfolobus acidocaldarius/metabolismo , Ácido 2-Aminoadípico/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Evolución Molecular , Duplicación de Gen , Ácido Glutámico/metabolismo , Modelos Moleculares , Ornitina/metabolismo , Filogenia , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Sulfolobus acidocaldarius/genética , Thermus/genética , Thermus/metabolismoRESUMEN
Although the latter portion of lysine biosynthesis, the conversion of alpha-aminoadipate (AAA) to lysine, in Thermus thermophilus is similar to the latter portion of arginine biosynthesis, enzymes homologous to ArgA and ArgJ are absent from the lysine pathway. Because ArgA and ArgJ are known to modify the amino group of glutamate to avoid intramolecular cyclization of intermediates, their absence suggests that the pathway includes an alternative N-modification system. We reconstituted the conversion of AAA to lysine and found that the amino group of AAA is modified by attachment to the gamma-carboxyl group of the C-terminal Glu54 of a small protein, LysW; that the side chain of AAA is converted to the lysyl side chain while still attached to LysW; and that lysine is subsequently liberated from the LysW-lysine fusion. The fact that biosynthetic enzymes recognize the acidic globular domain of LysW indicates that LysW acts as a carrier protein or protein scaffold for the biosynthetic enzymes. This study thus reveals the previously unknown function of a small protein in primary metabolism.
Asunto(s)
Ácido 2-Aminoadípico/metabolismo , Proteínas Bacterianas/biosíntesis , Lisina/biosíntesis , Thermus thermophilus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Lisina/química , Lisina/genética , Datos de Secuencia Molecular , Familia de Multigenes , Plásmidos , Conformación Proteica , Alineación de Secuencia , Thermus thermophilus/enzimologíaRESUMEN
Lactococcus lactis subsp. lactis JCM 5805(T) is a dairy lactic acid bacterium that induces plasmacytoid dendritic cell (pDC) activation. Here, we report the 2.55-Mb draft genome and annotation of Lactococcus lactis JCM 5805(T). This genome information will provide further insights into the mechanisms underlying the immunomodulatory function of this strain.