Functional Analysis Of The Fengycin Biosynthesis And Biofilm Formation Related Genes In Bacillus Subtilis Strain NCD-2

Bacillus subtilis NCD-2 is the key strain of the microbial source fungicide--the 109/g B. subtilis wettable powder, and had an excellent biocontrol effect on diseases caused by Botrytis cinerea, Rhizoctonia solani, Verticillium dahlia and Fusarium oxysporum. A privious study revealed that both the production of fengycin lipopeptides and effective colonization played important roles in the strain NCD-2 control of cotton seedling damping-off. The root colonization ability of B. subtilis is associated with its ability to form biofilm. To understand the mechanism of the biological control of soil-born disease, we used the methods of molecular genetics technology, DNA Microarray and phenotype microarrays to study the genes of fengycin biosynthesis and biofilm formation related in B. subtilis NCD-2. Main results in this paper were as follows: 1. Com A is an important regulator on the fengycin biosynthesis and biofilm formationTo understand the effect of the quorum sensing on antifungal activity and biofilm formation in B. subtilis NCD-2, we coloned the genes of com Q,com X,com P,com A(Gene Bank number are GQ924946, GQ924947, GQ924948, GQ924945). We constructed a com A in-frame deletion mutant strain, and evaluated its activities of extracellular enzymes, antifungal activity, fengycin production and biofilm formation comparing with the wild-type strain NCD-2. Expressions of biofilm-associated genes eps A and tas A were analyzed by using reverse transcription quantitative real-time polymerase chain reaction(RT-q PCR). Results showed that the com A deletion led to a less production of protease, cellulose and fengycin, reduced the biofilm formation and the inhibition of mycelial growth of Botrytis cinerea. RT-q PCR analysis revealed that a 70% decrease of the expression of the tas A gene in the com A null-mutant, but the eps A gene was not affected by the deletion of com A. Our results suggested that Com A is an important regulatory factor on fengycin biosynthesis and biofilm formation in B. subtilis strain NCD-2.2. Deg Q is an important regulator on the fengycin biosynthesis and biofilm formationTo understand the effect of the Deg Q on antifungal activity and biofilm formation in B. subtilis NCD-2, the deg Q genes(141 bp, Gene Bank number is GQ924949) was coloned.We constructed a deg Q in-frame deletion mutant strain and complementary strain by thehomelogens DNA technology and complementary technology, and evaluated theiractivities of extracellular enzymes, antifungal activity, fengycin production and biofilmformation comparing with the wild-type strain NCD-2. Expression of fengycin synthetasegene fen A was analyzed by using RT-q PCR. Results showed that the deg Q deletion led to aless production of protease, cellulose and fengycin, reduced the biofilm formation and theinhibition of mycelial growth of B. cinerea. RT-q PCR analysis revealed that a 50%decrease of the expression of the fen A gene in the deg Q null-mutant. Complementation ofthe intact deg Q gene in the deg Q-null mutant restored the wild-type levels. Our resultssuggested that Deg Q is an important regulatory factor on fengycin biosynthesis and biofilmformation in B. subtilis strain NCD-2.3. Deg Q influences osmolytes resistance and decarboxylase production in B. subtilisNCD-2Using the Phenotype Microarray(PM) system, we compared the metaboliccapabilities of the wild type strain NCD-2 and the deg Q-null mutant. The results of PManalysis showed that fewer differences involved carbon, nitrogen, phosphorus, and sulfursource utilization and biosynthetic pathways while phenotypic differences in tolerance toosmolytes and p H between the wild type strain NCD-2 and the deg Q-null mutant. Whenthe substrate is 100 m M sodium benzoate(p H 5.2), the deg Q-null mutant showed strongertolerance to osmolytes than the wild type strain NCD-2. The deg Q-null mutant lost theactivity of decarboxylase when the substrate is L-Leucine(p H 4.5), but increased theactivities of L-Ileucine, L-Tryptophan, L-Norleucine decarboxylase compared to the wildtype strain NCD-2. The results indicated that Deg Q maybe relate to the resistance ofosmolysts and maybe positive regulate the activity of L-Leucine decarboxylase andnegative regulate the activities of L-Ileucine, L-tryptophan, and L-Norleucinedecarboxylase.4. ywb B positively regulate the biofilm formation and extracellular matrix genesexpressionPrevious study indicated that the ywb B-null mutant lost the ability of biofilmformation. Expressions of extracellular matrix genes tas A and eps A were analyzed by usingRT-q PCR. Results showed that 64% and 85% decrease of the expression of the tas A andeps A gene in the ywb B null-mutant. Results suggested that ywb B positively regulate the tas A and eps A expression in B. subtilis strain NCD-2. 2. Deg Q is an important regulator on the fengycin biosynthesis and biofilm formation 5. DNA microarray analysis and obtained the ywb B regulated and biofilm formation related genesAnalysing the 4206 genes of the wild type strain NCD-2 and the ywb B-null mutant at biofilm state by DNA microarray, we obtained the genes which were biofilm formation related and ywb B regulated. Results showed that 37 genes were up-regulated and 42 genes were down-regulated. Expressions of genes tas A, ssp B, sip W, spo IIAA and cot JC were analyzed by using RT-q PCR. Results showed that 64%, 99%, 77%, 79% and 97% decrease of the expression of the tas A, ssp B, sip W, spo IIAA and cot JC genes in the ywb B null-mutant, and this is consist with the DNA microarray results. 6. Identified the function of sip W, spo IIAA and cot JC regulated by ywb B and biofilm formation related genes in Bacillus subtilis NCD-2Through analysising the whole genome of B. subtilis NCD-2, we cloned the sip W(573 bp), spo IIAA(354 bp) and cot JC(570 bp) genes which regulated by ywb B and related to biofilm formation. We constructed spo IIAA,sip W and cot JC deletion mutants by the homelogens DNA technology, and evaluated their biofilm formation, antifungal activity, and colonization comparing with the wild-type strain NCD-2. Results showed that the deletion led to a reduced biofilm formation, the inhibition of mycelial growth of B. cinerea, colonization and biocontrol efficiency, but the cot JC mutant strain had a less difference compared to strain NCD-2.The above results can summarize the function of these gene:(1) com A and deg Q were involved in extracellulase production, fengycin biosynthesis, biofilm formation in B. subtilis strain NCD-2.(2) deg Q gene was involved in the osmotic resistance and amino acid decarboxylase in B. subtilis strain NCD-2.(3) ywb B gene positively regulates the biofilm formation and tas A、eps A、ssp B、sip W、spo IIAA、cot JC genes expression.(4) sip W and spo IIAA genes were involved in the antifungal activity, biofilm formation, colonization in B. subtilis strain NCD-2.