| Mineral weathering is one of the most important geological processes on the earth's surface,which is the basis for the formation of various landforms and soil.Silicate weathering also plays important roles in terrestrial ecosystems.Until now,large numbers of silicate-weathering bacteria have been isolated;however,the mechanisms on silicate weathering have still not been understood clearly.In addition,there has not been reported on the bacterial quorum sensing(QS)regulation involved in silicate-weathering process.Therefore,in this study,several mineral-weathering bacteria were screened for their ownership of QS systems(based on the activity of acyl-homoserine lactones,AHLs)and high weathering mineral efficiency.Bacterial QS regulation on silicate weathering process will provide theoretical and experimental bases for the mechanisms involved in the interaction between silicate mineral and bacteria.One highly effective mineral-weathering bacterium Rhizobium pusense S41 was screened from 18 mineral-weathering bacteria preserved by our laboratory and a series of molecular biological techniques such as genome sequencing analysis,gene knockout and complementation and transcriptome sequencing analysis were used to study a set of QS system TraI-TraR in strain S41 and the system's effects on bacterial biotite-weathering effects and mechanisms.The key genes which regulated by the Tral-TraR system in strain S41 and had influence on biotite weathering efficiency were also identified.Eighteen strains were detected for their production of AHLs,mineral weathering effectiveness and antibiotics resistance.Among these strains,10 of them could produce AHLs,and four AHLs-producing strains had higher ability to weather biotite and potassium feldspar;furthermore,only strain S41 showed sensitivity to high concentrations of gentamicin and kanamycin and was used for the tested strain.Meanwhile,the effect of strain S41 on biotite weathering was evaluated in nutrient-poor medium(BHm).The release of Si,Al and Fe in the culture medium were found to increase significantly(ranging from 3.3-fold to 57-fold)in the presence of strain S41 compared to the controls.In addition,the expolysaccharides' concentration in the culture medium and the number of adsorbed cells on the biotite surfaces were 1.3 mg mL-1 and 108 cfu mL-1,respectively in the presence of strain S41.The complete genome sequencing analysis of strain S41 showed two independent chromosomes with one being circular of 2.95 Mb and one being linear of 2.57 Mb.Many identified genes(clusters)related to bacterial adsorption and expolysaccharide generation were identified.Two key genes in quorum sensing systems traI(the max-similarity to type strain was 73%)and traR(the max-similarity to type strain was 77%)were found in the linear chromosome.Mutants S41 ?tral and S41 ?traR were constructed via homologous recombination.The activities of AHLs produced by mutants S41 ?traI and S41 ?traR decreased significantly by 90-92%compared to the wild-type strain.Strain S41 could produce at least 6 different kinds of AHLs(C4-HSL,3-Oxo-C6-HSL,3-Oxo-C8-HSL,C8-HSL,3-Oxo-C10-HSL and 3-OH-C10-HSL),in which the major one was 3-Oxo-C8-HSL.However,strain S41?traI and S41 AtraR could only produce a little 3-Oxo-C8-HSL.The concentrations of mobilized Si,Al and Fe were significantly decreased by 12-68%during the biotite weathering process in the presence of the mutant strains compared to strain S41.Furthermore,the biofilm formation,expolysaccharides production and the number of adsorbed cells on the mineral surface of the mutant strains decreased significantly by 12-75%compared to strain S41.When genes tral and traR were complemented into the mutants,no significant differences in the AHLs synthesis and the contents of Si,A1 and Fe released from biotite were found.The relative expression of genes tral and traR in strain S41,S41-pBB2,S41?traIH and S41?traRH was analyzed by qRT-PCR,and the expression of genes tral and traR in the BHm with biotite was found to be up-regulated(1.7-4.9-fold).A double-genes mutant S41 ?traI?traR was constructed.The concentrations of mobilized Si,AL and Fe were significantly decreased by 26-70%during the biotite weathering process in the presence of mutant S41?traI?traR compared to strain S41.Transcriptome sequencing was used to compare the gene expression level of the wild-type strain with that of the mutant during their biotite weathering process.The results showed that 47 genes were up-regulated and 106 genes were down-regulated in the presence of mutant S41?traI?traR compared to the wild-type strain at 2 h of incubation.Only 35 genes were up-regulated and 62 genes down-regulated in the presence of mutant S41?traI?traR compared to the wild-type strain at 9 h of incubation.Based on genes annotation and KEGG pathway enrichment analysis,among these down-regulated genes,some of them were found to be related to iron transport,sugar transport,pilis biosynthesis and ATPase in strain S41.Moreover,the most down-regulating genes were found to belong to the type IV secretion system in strain S41,including genes virB4(down-regulated 6.2-fold),virBll(down-regulated 4.9-fold)and virD4(down-regulated 4.4-fold)both encoding ATP hydrolase,genes virB2(down-regulated 6.3-fold),virB3(down-regulated 15-fold)and virB5(down-regulated 5.5-fold)responsible for pili proteins,gene virB10(down-regulated 5.4-fold)for inner membrane protein and gene virB9(down-regulated 6.0-fold)for outer membrane protein.The genes virB2,virB3 and virB4 were knocked out at the same time by homologous recombination technology,and the mutant strain S41?virB234 was constructed.Compared with the wild-type strain,the mutant's ability of twitching motility decreased significantly and the total biofilm biomass significantly decreased by 71%.In addition,the concentrations of mobilized Si,Al,Fe and the number of adsorbed cells on the mineral surface were significantly decreased by 12-75%during the biotite weathering process in the presence of the mutant strain compared to strain S41.When genes virB2,virB3 and virB4 were complemented into mutant S41 ?virB234,there were no significant differences in the ability of biofilm formation and the contents of Si,A1 and Fe released from biotite.The relative expression of genes virB2,virB3 and virB4 in strain S41,S41-pBB2 and S41?virB234H was analyzed by qRT-PCR,and the expression of those genes in the BHm with biotite was found to be up-regulated(3.4-21-fold).During the screening process,one mineral-weathering bacterium Z29 was idenfied based on polyphacis taxonomical methods.Strain Z29 was mostly close to Chitinophaga polysaccharea MRP-15T,Chitinophaga arvensicola M64T and other three type strains belonging to the genus of Chitinophaga,and its homology with those type strains were ranged from 96-98.2%.The major fatty acids produced by strain Z29 were iso-C15:0,C16:?5c and iso-C17:0 3-OH;the mainly polar lipid was phosphatidylethanolamine(PE);the predominant quinone was MK-7 and the major polyamine was homospermidine.The DNA G+C mol%content of strain Z29 was 51.3%while the values of DNA-DNA hybridization were ranged from 14.6-29.8%.These results indicated strain Z29T represents a novel species of the genus Chitinophaga,for which the name Chitinophaga longshanensis sp.nov.was proposed. |
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