Study Of ECF ? Factors And Their Mechanisms In Controlling Cytophaga Hutcbinsonii Cellulose Assimilation

Lignocellulose,derived from plant cells,is most likely to become one of the major new energy in the 21st century since its enormous production and extensive distribution.But the low efficiency of cellulose degradation hindered the large-scale industrialization application.Following the research progresses,they found a great many of cellulolytic microbe and hydrolytic enzymes,which made our preferably understanding for cellulose biodegradation mechanism.Thus it also layed the theoretical basis foundation for exploiting the renewable energy which utilized lignocellulose as raw materials.Cytophaga hutchinsonii,a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes,degrades cellulose in a substrate contact-dependent manner.But its cellulose degradation mechanism is different from the known free cellulase system model and the composite cellulosome model,which makes C.hutchinsonii a excellent material for exploring the theory of cellulose degradation and application.It is postulated to adopt a novel strategy to digest cellulose,which is still a mystery so far.At present,the study of the mechanism for cellulose degradation mainly concentrated on the cellulase component,important outer membrane proteins and genes related with slide and movement.The research associated with the regulatory mechanism is relatively scarce.Extracytoplasmic function?ECF?sigma???factor represents one of the simplest sigma factor and varies numerously.Previous studies found that the ECF ? factor was participated in the regulation of perception and response for external stress from the environment in prokaryotes.In C.hutchinsonii,given its high efficiency for cellulose degradation and the degradation process is absolutely depend on the contact of substrate,we speculated that the response for cellulose was related to a series of regulatory factors,which achieved the rapid conversion of carbon source utilization by changing the global transcription pattern.Currently,ECF ? factors participated in the utilization of cellulose has not been identified in C.hutchinsonii,which restrained our exploration for the mechanism for cellulose degradation.Therefore,the study focused on the identification and functional analysis of ECF ? factors which associated with cellulose degradation is a significant step to comprehensively clarify the mechanism for cellulose degradation.In our current study,we used C.hutchinsonii as the research object and obtained a mutant with defects in cellulose utilization through the transposon mutagenesis technique.According to the bioinformatic analysis,we found the transposon insertion site was an ECF a factor encoding gene.On one hand,the interaction model between ECF ? factor and the anti-? factor was analyzed through heterologous expression in vitro and the factors which affected this model was verified.Meanwhile,the influence derived from changing this model to cellulose degradation was explored.On the other hand,we evaluated some downstream target genes regulated by the ECF ? factor based on the transcriptome analysis.In addition,we found another ECF ?/anti-? pair participated in cellulose degradation process and identified a putative type ? secretion system which was important for cellulose degradation in C.hutchinsonii.The major contents and results of this study are as follows:1.SigE was identified as a ECF ? transcriptional factor which associated with cellulose degradation by screening and identification.The SigE and the potential HtrA family homologous protein HtrACh was essential to cellulose degradation by phenotypic analysis.A mutant MT2405?M3097?,whose absence severely compromised cellulose assimilation,was obtained by transposon mutagenesis screening.The transposon insertion site was determined to be within the gene locus CHU₃097 by plasmid rescue and DNA sequencing.The gene was annotated as an ECF sigma factor SigE.Complementation of M3097 restored the ability to digest cellulose,which verified that CHU₃097 plays an important role in cellulose assimilation.According to the bioinformatic analysis,we found the downstream gene CHU₃096 encoded a potential anti-? factor anti-SigE.However,targeted deletion of the full-length anti-SigE did not affect cellulose utilization.In addition,we found HtrACh,which belonged to the HtrA family,may related to the function of SigE.The phenotypic analysis of the inactivated HtrACh mutant and the complemented strain demonstrated that the HtrACh was also essential to cellulose degradation.We found that constitutively expression of SigE was capable of recovering the utilization defect caused by the absence of HtrACh.However,the double deletion mutant cells carrying plasmid expressing SigE failed to utilize cellulose.Taken all the genetic analysis above,we speculated SigE represents a critical downstream regulatory target of HtrACh.The double deletion mutant which derived though knocking out of anti-SigE and inactivation of HtrACh possibly changed the state of SigE,leading to the failure of recovering the double deletion mutant cells by the expression of SigE.2.The interaction between anti-SigE factor and SigE was identified by co-purified two proteins in vitro.Both of them located on the cell membrane in different status.Anti-SigE anchored tightly on membranes in polymers and SigE was detected in cytoplasm fractions when cells were induced by Avicel.The state of SigE was influenced by anti-SigE and HtrACh.To test whether the putative anti-SigE factor could bind to SigE,these two proteins were co-expressed in E.coli with N-terminal His-tag and C-terminal S-tag,respectively.These two proteins were always shown to co-purified using affinity chromatography against either His-tag or S-tag,suggesting that anti-SigE is a putative anti-? factor against SigE.To study the acting mechanism of SigE,we examined its cellular localization by cell-fractionation and Western blot using c-Myc antibody.Anti-SigE and SigE were detected in membrane fractions,but anti-SigE was bind on membranes in polymers tightly.SigE was released into cytoplasm fractions when cells were induced by Avicel.Partial release of membrane-localized SigE into the cytoplasm in anti-SigE absence mutant was not only observed under cellulose condition,but also occurred during cultivation on glucose.These results suggested that anti-SigE was involved in regulating the partial release of SigE from the cytoplasmic membrane in response to cellulose.Further analysis showed that in HtrACh inactivated mutant cells,SigE was only detected in total membranes regardless whether glucose or cellulose was provided as the sole carbon source.Howere,different signals could be detected in anti-SigE and HtrACh inactivated double mutant.To sum up the genetic phenotype results,we speculate that,although release of SigE into the cytoplasm is not a prerequisite for its function,HtrACh may not regulate the activity of SigE by eliminating anti-SigE.It is more likely to regulate the response for cellulose through changing the interaction state of anti-SigE and SigE.The simultaneous absence of HtrACh and anti-SigE resulted in the change of the stability and active state of SigE,thus affect the response for cellulose.3.SigE regulons were analyzed by transcriptome sequencing and CHU₁276 was found as a critical downstream target gene regulated by SigE during cellulose utilization.We performed RNA sequencing?RNA-seq?to profile genome-wide mRNA abundance in the WT and M3097 strains when exposed to different culture conditions including glucose,no-carbon and Avicel.Firstly,the numbers of genes are indicated and subdivided by genes repressed and genes activated due to the absence of SigE compared to wild-type under three different culture conditions.We found that the up-regulated and down-regulated gene number was more on Avicel condition than on glucose and no carbon conditions.The results indicated that SigE was responsed to cellulose carbon source.To identify genes regulated by SigE and better understand the mechanism by which SigE regulates the cellulolytic process in C.hutchinsonii,we screened the differentially expressed genes between WT and the mutant strain that were specifically responsive to Avicel.Some SigE regulons were identified from the specifically down-regulated genes that response to Avicel.Some SigE regulons of known function and hypothetical protein were inserted inactivated or knocked out,and the growth phenotype were analyzed.CHU 1276 was found as a critical downstream target regulated by SigE during cellulose utilization based on recovering of the cellulose utilization defect caused by the absence of SigE by expressing CHU₁276.4.The transcriptome data showed that SigF,another gene encoding an ECF ?factor,was specifically down-regulated on Avicel.Its downstream gene encoded a possible anti-? factor anti-SigF,which was interacted with SigF according to the bioinformatic analysis.SigF and anti-SigF were identified to play a significant role in cellulose utilization in C.hutchinsonii.SigF,which encodes an ECF ? factor,was potentially regulated by SigE and was found specifically down-regulated on Avicel by analyzing the transcriptome databetween the mutant and wild-type strains.According to the bioinformatic analysis,its downstream gene encoded a predicted anti-? factor related to SigF,named anti-SigF.SigF and anti-SigF were identified to play a significant role in cellulose utilization by gene knocked out and inserted inactivation.In vitro,we proved that anti-SigF was interacted with SigF by bacterial two-hybrid system.In order to identify the key site of SigF which was against with anti-SigF,several sites were mutated including T4A,L9P,R15G,A23G,W35G,W41V and S59G.However,we could not find the key site.The specific interaction model and its function in the signal transduction process need to explore further.5.A putative type ? secretion system which was important for cellulose utilization was identified in C.hutchinsonii.Previous study revealed that some specific proteins are speculated to participate in adhesion to cellulose and further digestion by altering its extracellular milieu or outer membrane surface.We found a putative type ? secretion system component T2S-F,which was specifically down-regulated on Avicel according to the transcriptome data between the mutant and wild-type strains.In this study,we found three orthologous genes encoding the major components?T2S-D,-F,and-G?of type? secretion system?T2SS?by bioinformatics analysis.Individual disruption of these three t2s genes resulted in a significantly retarded growth on cellobiose,regenerated amorphous cellulose and Avicel.Enzymatic analyses demonstrated that,whereas the endoglucanase activity of the T2S mutant cells was increased,the ?-glucosidase activity was remarkably reduced compared to that of WT cells.Further analysis revealed that the T2S mutant cells are involved in cellulose degradation but not in cell motility.However,the T2S mutant cells exhibited a different profile of cellulose-bound outer membrane proteins from that of wild-type cells and displayed a significant decrease in their capability to adhere cellulose.These results indicated that a functional link exits between the putative T2SS and cellulose utilization in C.hutchinsonii,thus provided a conceptual framework to understand the unique strategy to assimilate cellulose deployed by C.hutchinsonii.