Enzymatic Function And Biological Roles Of WSPA Protein In Nostoc Flagelliforme

Nostoc flagelliforme is a terrestrial filamentous nitrogen-fixing cyanobacterium,usually called 'Facai' in Chinese.It is distributed in arid or semiarid steppes of the west and west-northern parts of China.The native habitats are characterized by high evaporation rates,substantial temperature differences,intense solar radiation and poor nutrient availability.The individual filament(thallus)consists of numerous parallel arranged trichomes surrounded by a common exopolysaccharide(EPS)matrix.The filaments undergo frequent expansion and shrinkage in native environments,accompanying the repeated desiccation and rehydration processes.Water stress protein(WSPA)is an acidic protein,found in N.flagelliforme,Nostoc commune and Nostoc sphaeroides.This protein can be induced by desiccation and UV irradiation,and secretes into the EPS matrix upon rehydration.However,the biochemical identity and biological roles of WSPA protein remains still unclear.In this study,the wspa gene was divided into three segments,with the N-terminus,the middle segment and C-terminus being 35,92 and 234 amino acids,respectively.The C-terminus was referred as WSP-A,while the truncated WSPA lacking of the 35-aa N-terminus was referred as WSP-B.Wsp-a and wsp-b sequences were respectively ligated into pET28a vector and transformed into Escherichia coli BL21(DE3)for in vitro protein expression,followed by purification and enzymatic analysis.The results showed that WSP-A possesed significant ?-galactosidase activity and this activity was nearly 10 times higher than that of WSP-B.Thus,WSP-A represented the central activity region and the middle segment(90 aa)possibly played an inhibitory or regulative role in the enzymatic activity.In addition,the ?-galactosidase activity of WSP-A could be inhibited by Ca2+and Zn2+,and promoted by Mg2+and K+.The liquid-cultured N.flagelliforme samples were placed in perforated petri plates for 3-d drying,and then were subjected to various treatments,including by WSP-A,boiled WSP-A,bovine serum albumin and Ca2+.The ratio of EPS to chlorophyll a(indicating the synthesis level of EPS)was determined at 5th and 10th d.It was found that WSP-A could result in a relatively higher increase of EPS/chlorophyll a ratio than other controls,which suggested that WSPA contributed to the thickening of the EPS matrix via transgalactosylation activity to combate the desiccation resistance of N.flagelliforme.In addition,natural N.flagelliforme samples were also treated by WSP-A.We found that it could hydrolyze the EPS matrix,leading to its softening,which is beneficial for the cell growth and trichome elongation.Therefore,along with the moisture rhythm,WSPA plays biological roles in the dynamic regulation of the elasticity and rigidity of the EPS matrix,thus maintaining a favourable balance of cell growth and stress resistance.Finally,we also performed proteomic analysis of liquid-cultured N.flagelliforme upon dying induction.The up-regulated proteins were subjected to the clustering,including secretory or regulatory proteins,nitrogen metabolism proteins,carbon or energy metabolism proteins,antioxidant proteins,and chaperone proteins,etc.The preliminary proteomic analysis will also to a certain extent contribute to the understanding of desiccation resistance mechanism in N.flagelliforme.