The Physiological Metabolism Of The Curdlan-producing Agrobacterium SP. And Its NtrC Mutant

Curdlan is a kind of microbial exopolysaccharide synthesized by Agrobacterium sp. after nitrogen exhaustion. Nitrogen-limited condition during curdlan synthesis leads to the decreasing productivity of curdlan and is also detrimental to curdlan synthesis. It is necessary to investigate the relationship between nitrogen regulatory and curdlan production and to relieve nitrogen limitation has important significance for incresing curdlan yield and simplifying fermentation process.In this study, we first investigated the influence of signal moleculeα-ketoglutaric acid and different nitrogen sources on curdlan production of Agrobacterium sp. and its ntrC mutant. Then different expression level of nitrogen and carbon metabolism-associated genes between Agrobacterium sp. and its ntrC mutant under nitrogen sufficient and nitrogen limitation were determined by real-time PCR. Finally, structure of the unknown compound from ntrC mutant was preliminary studied.Signal moleculeα-ketoglutaric acid was added at Agrobacterium sp. and its ntrC mutant fermentation process. The results indicated thatα-ketoglutaric acid increased curdlan production by Agrobacterium sp. Curdlan production of ntrC mutant was still low whenα-ketoglutaric acid was added. The results showed that nitrogen regulation protein NtrC was important for curdlan production. The ntrC mutant produced an unknown compound different from curdlan as energy storage compound.Influence of different nitrogen souces on cell growth and fermentation of Agrobacterium sp. and its ntrC mutant was investigated. The ntrC mutant grew slower with NH4Cl, KNO3, glycine, alanine, methionine, lysine, asparagic acid and urea than Agrobacterium sp. and grew faster in cysteine than Agrobacterium sp.. The results showed that the regulation of NtrC on different nitrogen sources absorption and utilization were not the same. For Agrobacterium sp., curdlan production with glycine, asparagic acid and urea as nitrogen sources were higher than that with NH4Cl as nitrogen source while curdlan yield was extremely low in medium with KNO3, cysteine, alanine and methionine as nitrogen sources. The ntrC mutant with all those nitrogen sources hardly produced curdlan.In addition, different expression level between Agrobacterium sp. and the ntrC mutant under nitrogen sufficient and nitrogen limitation were determined by real-time PCR. The results indicated that Agrobacterium sp. not only regulated nitrogen assimilation, but also channeled the carbon flux into curdlan synthesis for energy storage under adverse condition. NtrC protein was important for nitrogen assimilation, but it is not the only regulation protein, the other ntrBC-like regulation protein such as ntrYX may exist. Curdlan production capacity of the ntrC mutant decreased severely although relative expression level of key enzymes coden genes in the curdlan synthesis process increased, the experiment showed that NtrC protein was involed in regulating curdlan synthesis, and key enzymes in the curdlan synthesis were regulated not only at transcriptional level but also at translational level.Finally, the structure of the unknown compound from the ntrC mutant was preliminary studied. The results revealed that the unknown compound A3 may be a glycoprotein. The β-elimination reaction showed that it contained O-cardohydrata-peptide linkage. The polysaccharide part comprised mannose, glucose, galactose and an unknown monosaccharide. Amino acids analysis showed glutamic acid content was the highest, while it contained a certain amount of serine and threonine. Infrared spectroscopic analysis showed that it consisted ofβglycosidic bonds and sulfate may be present in this matter.