Osmotically induced synthesis of the dipeptide N-acetylglutaminylglutamine amide is mediated by a new pathway conserved among bacteria

Abstract

The dipeptide N-acetylglutaminylglutamine amide (NAGGN) was discovered in the bacterium Sinorhizobium meliloti grown at high osmolarity, and subsequently shown to be synthesized and accumulated by a few osmotically challenged bacteria. However, its biosynthetic pathway remained unknown. Recently, two genes, which putatively encode a glutamine amidotransferase and an acetyltransferase and are up-regulated by osmotic stress, were identified in Pseudomonas aeruginosa. In this work, a locus carrying the orthologous genes in S. meliloti, asnO and ngg, was identified, and the genetic and molecular characterization of the NAGGN biosynthetic pathway is reported. By using NMR experiments, it was found that strains inactivated in asnO and ngg were unable to produce the dipeptide. Such inability has a deleterious effect on S. meliloti growth at high osmolarity, demonstrating the key role of NAGGN biosynthesis in cell osmoprotection. β-Glucuronidase activity from transcriptional fusion revealed strong induction of asnO expression in cells grown in increased NaCl concentration, in good agreement with the NAGGN accumulation. The asnO-ngg cluster encodes a unique enzymatic machinery mediating nonribosomal peptide synthesis. This pathway first involves Ngg, a bifunctional enzyme that catalyzes the formation of the intermediate Nacetylglutaminylglutamine, and second AsnO, required for subsequent addition of an amide group and the conversion of Nacetylglutaminylglutamine into NAGGN. Interestingly, a strong conservation of the asnO-ngg cluster is observed in a large number of bacteria with different lifestyles, such as marine, symbiotic, and pathogenic bacteria, highlighting the ecological importance of NAGGN synthesis capability in osmoprotection and also potentially in bacteria host-cell interactions.osmotic stress | rhizobium | peptide synthesis | GCN5-related N-acetyltransferase | glutamine amidotransferase

Physiological and Transcriptional Responses to Osmotic Stress of Two Pseudomonas syringae Strains That Differ in Epiphytic Fitness and Osmotolerance

“…S3 in the supplemental material) (7). A B728a mutant that lacked the ggnAB genes required for NAGGN synthesis (27,34), designated B7⌬ggn, exhibited impaired osmotolerance but only at high NaCl concentrations (Ͼ0.6 M) ( Fig. 4A and B).…”

“…4C; see Fig. S4A); the operon contained two putative genes downstream of ggnAB that are not required for NAGGN synthesis (34). A trehalose-deficient DC3000 mutant, designated DC⌬tre (Table 1), showed a much greater growth delay than DC⌬ggn in MinAS medium, with a mutant that lacked the loci for both NAGGN and trehalose production showing the most severe reduction in osmotolerance ( Fig.…”

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

“…We assayed the trehalose content of P. stutzeri A1501 cells grown in MM63 containing either 50 mM (low osmolarity) or 700 mM (high osmolarity) NaCl and found increased amounts of trehalose in the high-osmolaritystressed cells (data not shown), in accord with the data reported by Seip et al (58) for the P. stutzeri DSM5190 type strain. Genes (asnO and ngg) encoding a glutamine amidotransferase (AsnO) and N-␥-acetylglutaminyl glutamine acetyltransferase (Ngg) for the synthesis of NAGGN (52) are also present in the genome sequence of P. stutzeri A1501, but we have not assessed the formation of this peptide in osmotically stressed cells.…”

Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

“…The uptake of compatible solutes by microorganisms is typically preferred, possibly for energetic reasons (48), over their de novo synthesis. It is thus often observed that the uptake of compatible solutes suppresses the expression of biosynthetic genes for these compounds (53). To test whether this was also the case for the proHJ genes, we grew the ⌽(proHtreA)1 reporter gene fusion strain JSB7 in SMM with increasing concentrations of NaCl in either the absence or presence of glycine betaine (1 mM).…”