| Because of its unique edible value,medicinal value and ecological value,Nostoc flagelliforme has attracted more and more attention.During the long-term cultivation of Nostoc flagelliforme,it was found that other microorganisms were accompanied around the cells.It is helpful to further understand the growth system of Nostoc flagelliforme by exploring the effect of associated bacteria on growth and metabolism of Nostoc flagelliforme.and it is of guiding significance to investigate the interaction between Nostoc flagelliformee and associated bacteria.In this paper,the high-throughput sequencing method was used to determine the community structure of the associated bacteria in Nostoc flagelliforme.Eleven associated bacteria were isolated by the traditional method of flat streaking.The effect of exogenous addition of N-acyl-homoserine lactones(AHLs)on the growth of Nostoc flagelliforme cells was detected.The types of AHLs produced by associated bacteria were identified by LC-qToF-MS,and the metabolite changes during the co-cultivation of Nostoc flagelliforme and AHLs-producing bacteria were analyzed using metabolomics.The results are as follows:(1)The microbial community structure of the culture of Nostoc flagelliforme was analyzed by high-throughput sequencing.Under the four liquid culture conditions,the nitrogen-containing medium(NC),nitrogen-free medium(NN),glucose-added medium(AG),antibiotic-added medium(AA),all contained a certain high abundance of Gemmata,Nostoc,Aquimonas,Pseudomonas,and changes in culture conditions would make the flora abundance a difference.Compared with the other groups,the relative abundance of Aquimonas in the NN group was significantly reduced,indicating that the nitrogen source of the culture medium affected the amount of Aquimonas in Nostoc flagelliforme culture.The relative abundance of Pseudomonas in the AG group of exogenously added glucose was significantly increased compared with other experimental groups.It was speculated that the direct carbon source glucose had a significant effect on Pseudomonas in the associated bacteria of Nostoc flagelliforme.The main microbial communities of NN and AA group are similar,containing higher abundance of Gemmata and Nostoc.(2)The associated bacteria were isolated and cultured on the solid medium of Nostoc flagelliform by the flat-line method.After 16S rDNA identification,11 strains were isolated,including ABNF-1 Microbacterium,ABNF-2 Gordonia terrae,ABNF-3 Ochrobactrum,ABNF-4 Rhizobium,ABNF-5 Sphingopyxi,ABNF-6 Variovorax,ABNF-7 Blastomonas natatoria,ABNF-8 Agrobacterium,ABNF-9 Gordoniau,ABNF-10 Pseudomonas,ABNF-11 Ochrobactrum.(3)Bacteria can complete cell-to-cell communication by secreting quorum-sensing signal molecules AHLs to regulate the group behavior of microorganisms.By adding different concentrations of N-acyl homoserine lactone molecules,C6-HSL,C7-HSL,C8-HSL,C10-HSL and C12-HSL to the medium of Nostoc flagelliforme,the effect of them on the growth of Nostoc flagelliforme was determined.The experimental results showed that AHLs had little effect on biomass and pigment content,but when the concentration of AHLs reached 5 μM or more,it had a significant effect on the yield of polysaccharides,promoted the accumulation of capsular polysaccharides,and increased the total sugar content of cells.(4)Based on the characteristics that Agrobacterium tumefaciens A136 and AHLs producing bacteria can make the medium react in blue,we preliminarily identified the following 11 associated bacteria,the results show that,the ABNF-1 Microbacterium,ABNF-4 Rhizobium,ABNF-9 Gordoniau,ABNF-10 Pseudomonas balearica can produce acyl-homoserine lactone molecules.Using LC-qToF-MS to identify the type of quorum-sensing signal molecules secreted by AHLs-producing bacteria.From the mass spectrum,we can see that ABNF-1 has C12-HSL;ABNF-4 produces C6-HSL,C10-HSL,C12-HSL;ABNF-9 produces C6-HSL,C10-HSL;ABNF-10 produces C10-HSL,C12-HSL.(5)GC-MS and related software were used to detect,qualitatively and quantitatively analyze the metabolites of Nostoc flagelliforme co-cultured with AHLs producing bacteria.There are 16 kinds of sugars,15 kinds of alcohols,16 kinds of organic acids,7 kinds of amino acids,13 kinds of alkanes,33 kinds of lipids,6 kinds of amines,7 kinds of other kinds in Nostoc flagelliforme metabolites.By analyzing the metabolites of samples,PCA-X and PLS-DA score maps showed obvious discrete phenomenon,indicating that the intracellular metabolites changed,and the basic metabolism fatty acid metabolism and carbon metabolism of Nostoc flagelliforme in co-cultureed group changed significantly. |
