The Study On Mechanisms Of Pellicle Formation By Shewanella Oneidensis

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities.In recent years,biofilm development of S.oneidensis has been extensively studied because it plays an important role in solid metals reduction and microbial fuel cell.As a special form of biofilm,however,pellicles are largely overlooked.The goal of this work was to understand requirements of S. oneidensis MR-1 pellicle formation and the molecular basis of pellicle formation with the method combined with microbiological physiology and molecular biology.The main conclusions in this thesis were summarized as follows:(1) Growth kinetic studies of pellicle formation by S.oneidensis MR-1Pellicle formation of S.oneidensis MR-1 was determined to occur when oxygen and the cell density of cultures reached the threshold level. S.oneidensis MR-1 was capable of forming pellicle structure at the liquid-air interface of a standing culture while formed solid surface associated biofilm at the solid-liquid-air interface when aeration of the media was provided by vigorously shaking.Cells lived in the planktonic form only when the cell density was below -0.23 of OD₆₀₀(pellicle formation threshold,PFT).Once the OD₆₀₀ readings of the cultures passed over PFT,cells started to attach to the wall and then quickly spread across the surface to form a layer of pellicleat the liquid-air interface.This demonstrated that successful pellicle formation and survival was likely to require the threshold level of cell density.The pellicles were not found under anaerobic conditions with various electron acceptors,even when the cell densities significantly exceeded PFT established under aerobic conditions indicating that oxygen is the key agent in pellicle formation. (2) Characterization of physicochemical properties of S.oneidensis MR-1 pellicleExtracellular polyer substances,including protein,polysaccharide, extracellular DNA,are important in S.oneidensis MR-1 biofilm formation.The pellicles were prevented from formation in the presence of 100μg/mL proteinase K.Consistently,100μg/mL of the proteinase K was able to degrade the developed pellicles in 24 h.On the contrary, DNaseâ… (up to 1000 U/mL) and cellulase(up to 100 U/mL) had little effect on pellicle formation or developed pellicles degradation.These data suggested that proteins but not DNA or cellulose play an essential role in both pellicle formation and developed pellicles.Scanning electron microscopy showed pellicles consisted of a dense layer of MR-1 cells, which generated tens of nanometer long nanowire.(3) cDNA microarray analysis of pellicle cells relative to planktonic cells of S.oneidensis MR-1DNA microarray experiments were used to study the gene expression profile of young air-liquid interface pellicle relative to planktonic cells of S.oneidensis MR-1,which indicated that approximately 19.0%of the 4,648 ORFs spotted on the array was transcription-altered.All of genes encoding TCA enzymes were up-transcribed in pellicle cells.All of 9 atpA-I genes encoding subunits of ATP synthase and 12 out of 13 nuoA-N genes encoding subunits of NADH dehydrogenase were consistently up-transcribed.The result indicated the air-liquid interface pellicle was more metabolically active than the planktonic cells.Consistently up-regulation of iron or heme uptake and transportation proteins was observed in the S.oneidensis MR-1 pellicle,which indicated iron may play a role in pellicle formation. DNA microarray results were verified by q-RT-PCR.(4) The effect of various metals on pellicle formation by S. oneidensis MR-1 Neither the hmuT nor hugA heme transport mutant was defective in pellicle formation indicating that they are not closely related to the pellicle development of S.oneidensis.Up-regulation of iron or heme uptake and transportation proteins in pellicle cells was in response to the changing environment cells experience within a pellicle,and that few of these are involved in the pellicle-development pathway directly.0.3 mM EDTA completely blocked pellicle formation in S. oneidensis MR-1 even when the cell density was over pellicle formation threshold.An examination of the influence of several metal cations on the anti-pellicle activity of EDTA showed that Ca(â…¡),Mn(â…¡),Cu(â…¡) and Zn(â…¡) but not Mg(â…¡) fully protected S.oneidensis MR-1 pellicle against EDTA treatment.Additional of iron enabled the initiation of pellicle formation but maturation was significantly impaired.Collectively, iron was less important than Ca(â…¡),Mn(â…¡),Cu(â…¡) and Zn(â…¡) with respect to pellicle formation in S.oneidensis.(5) The influence of typeâ… protein secretion system of S.oneidensis MR-1 on pellicle formationIn order to investigate pellicle formation mechanism,we continued our efforts to elucidate the functions of typeâ… secretion gene rtxB,emrA and aggA,its upstream gene rtx and downstream gene ompA by generating inframe deletion mutants of these genes.Reverse transcription-PCR(RT-PCR) validated that rtxB,emrA,aggA and ompA were located in rtxB operon in MR-1.The possible promoter sequence of rtxB operon was finally predicted based on bioinformatic analyses.We consistently observed that the pellicle was severly deficient in Artx,ArtxB,AemrA and AaggA which indicated that Rtx protein and typeâ… secretion system are essential for pellicle formation.The motility abilities and surface properties ofâ–³rtx,â–³rtxB,â–³emrA andâ–³aggA were similar to the wild type.Efficient rescue ofâ–³rtx,â–³rtxB,â–³emrA andâ–³aggA phenotype can not be achieved by extracellular complementation from proteins secreted by the wild type,Under all conditions,no phenotypic difference was observed between Artx and typeâ… protein secretion system mutants,suggesting that the typeâ… protein secretion system of MR-1 is responsible for secretion of the RTX protein.We propose a model for MR-1 in which secretion of the RTX protein leads the formation of a biofilm in a calcium-dependent manner.(6) The role of S.oneidensis MR-1 C-type cytochromes in pellicle formationC-type cytochromes are essential for energy metabolism,however, the role of most cytochromes in pellicle formation remains unknown although Approximately 42 cytochrome c genes in S.oneidensis MR-1 were annotated based on sequence analysis.12 c-type cytochromes deletion mutants of S.oneidensis MR-1 were generated and tested if they were involved in pellicle formation.The results showed thatâ–³SO4666,â–³SO1777,â–³SO1782,â–³SO2361 andâ–³SO2363 had a range of deficiency in pellicle formation.A growth competition in another set of experiment revealed slower-growing strains in pellicle such asâ–³SO4666,â–³SO1777,â–³SO1782,â–³SO2361 andâ–³SO 2363 also grew slowly under planktonic growth condition.It is possible thatâ–³SO2361,â–³SO2363,â–³SO1777,â–³SO1782 reduced pellicle formation by affecting growth rates under static growth conditions.The deletion mutantâ–³SO4666 could not form a pellicle under non-shake conditions, suggesting that it may play an important role in pellicle formation in S. oneidensis MR-1.â–³SO4666 not only affect planktonic growth but also blocked the pellicle matrix formation at the air-liquid interface.Scanning electron microscopy showedâ–³SO4666 defects in nanowire formation.â–³SO4666 showed reduced swarming motility abilities compared to the wild type.The results indicated that nanowire was essential for pellicle formation.