Mechanisms Involved In The Coordination Of Function And Transcription Of Duplicate GroELs In Myxococcus Xanthus

Gene duplication is the main mechanism for the production of new genetic material in molecular evolution.As an important evolutionary model,it can provide the raw material basis for new functional genes.In prokaryotes,gene duplication can increase copy number of genes,that results in novel function and adaptation to diverse ecological changes.GroEL proteins belong to the chaperonin of molecular chaperone that assist in protein folding,assembly,transport and degradation,and are essential for many physiological processes.GroEL depends on its co-chaperone?co-factor?GroES for proper function.Both GroEL and GroES belong to major heat shock proteins that can be over-expressed under non-permissive temperatures to help misfolded proteins renatured.The duplicate groEL genes are present widely among prokaryotes.Currently,study related to the structure and function of single groEL is well documented,however,there is the scarcity of knowledge regarding the function,molecular mechanism and transcriptional regulation of duplicate groELs.Myxobacteria are gram-negative bacteria that belong to ?-Proteobacteria.They are unique among bacteria due to the complex multicellular behavior,ability to produce novel secondary metabolites and wide environmental adaptability.Therefore,myxobacterium is an important model for the study of bacterial cell recognition and interaction,cell communication,multicellular morphogenesis and life evolution.Due to the complex lifestyle,myxobacteria have large genomes and a great number of duplicate genes.Study of these duplicate genes in myxobacteria will help in understanding the evolutionary significance of functional differentiation,relationship between the function and expression of duplicate genes,and will elucidate the molecular mechanism of transcriptional regulation.It will further provide a theoretical basis for transformation,understanding the behavior of the multicellular population and artificial reduction of the genome to decrease the burden of the host.Our previous studies showed the physiological function,functional differentiation and molecular mechanism of duplicate groELs in Myxococcus xanthus DK1622,the model strain of myxobacteria.In terms of nucleotide and amino acid sequence composition,the two groELs share 79%and 83%homology respectively,but there are obvious functional differences in cell physiology.groELl plays an essential role in development and sporulation ability,and that groEL2 is required for cell predation and the biosynthesis of the secondary metabolite myxovirescin?TA?.The domain replacement for GroEL1 and GroEL2 showed that the differences between terminal domain and C-terminal equatorial domain of GroEL lead to the differences of substrate selectivity between GroEL1 and GroEL2.Additionally,site-directed mutagenesis disclosed that six GGM repeats at the C-terminal of GroEL1 are significant for GroEL1 to accomplish its function.In this paper,the synergetic relationship between duplicate groEL and single groES genes in function and expression was further studied on the basis of the related former research using Mvxococcus xanthus DK1622 as the basic material.The underlying causes and molecular mechanisms of transcription differences of duplicate groELs were revealed and a novel regulatory factor related to groEL was discovered in order to explain its action mechanism and existence significance.In Myxococcus xanthus DK1622,groELl appears in a groESL operon,while groEL2 has no neighboring groES.In 4861 completely-sequenced prokaryotic genomes,duplication phenomenon exists in 19.5%of genomes.Among them,884 possess duplicate groEL genes and 770 possess groEL genes with no neighbouring groES.It is unclear whether stand-alone groEL requires groES in order to function and,if required,how duplicate groEL genes and unequal groES genes balance their expressions.In Myxococcus xanthus DK1622,we determined that,while duplicate groELs were alternatively deletable,the single groES that clusters with groELl was essential for cell survival.Either GroEL1 or GroEL2 required interactions with GroES for in vitro and in viivo functions.Deletion of groELl or groEL2 resulted in decreased expressions of both groEL and groES;and ectopic complementation of groEL recovered not only the groEL but also groES expressions.The addition of an extra groES gene upstream groEL2 to form a bicistronic operon had almost no influence on groES expression and the cell survival rate,whereas over-expression of groES using a self-replicating plasmid simultaneously increased the groEL expressions.The results indicated that M.xanthus DK1622 cells coordinate expressions of the duplicate groEL and single groES genes for synergistic functions of GroELs and GroES.We proposed a potential regulation mechanism for the expression coordination.Based on the synergetic function and expression of duplicate groELs and single groES,we further analyzed the regulation mechanism.The q-PCR analysis at different growth time points showed that there were significant differences in the transcription level of groELs in M.xanthus DK1622.In the composition analysis of promoters,we first reported that the controlling inverted repeat of chaperone expression?CIRCE?element?the HrcA-binding site located upstream of the promoter?evolved for the transcriptional regulation of duplicate groELs.CIRCE composition and locations were found to be phylogenetically conserved in bacterial taxa.DK1622 has two CIRCE elements?CIRCElg,-oESL1and CIRCE2groESL1?in the promoter region of groESLl and one CIRCE element?CIRCEgroEL2?before groEL2.We also found that negative HrcA and positive ?32 regulators coordinated the transcription of duplicate groELs,and that the double deletion in M.xanthus DK1622 eliminated transcriptional differences.In vitro binding assays showed that HrcA protein binding was biased toward CIRCE1g,-oESL1,followed by CIRCEgroEL2,but that HrcA proteins failed to bind with CIRCE2groESL1.Mutation experiments revealed that single-nucleotide mutations in the inverted repeat regions changed the HrcA-binding abilities of CIRCEs.We constructed an in vivo transcription-regulation system in Escherichia coli to pair each of the regulators with a groEL promoter.The results indicated that the transcriptional regulation performed by HrcA and ?32 was biased towards the groEL2 and groELl promoters,respectively.On the basis of promoter-sequence characteristics,we proposed a model of the coordinated regulation of the transcription of duplicate groELs in M.xanthus DK1622.The a32 protein combined with the core enzymes of RNA polymerase,leading it to bind to the-10 and-35 regions of the target gene,thus initiating transcription of the downstream gene,while HrcA bound CIRCE element to inhibit the transcription of the downstream gene.In M.xanthus DK1622,the binding ability of HrcA protein to CIRCE1groESL1 was stronger than that of CIRCEgroEL2,but not to CIRCE2groESL1.CIRCElgroESL1 was distributed between TSS and TLS of groESLl promoter,while CIRCEgroEL2 was located upstream of TSS of groEL2 and partially overlapped with-35 region.For the transcription of groESLl,HrcA and ?32 bound to the corresponding recognition sequence.The binding of HrcA and CIRCE lgroESL1 affected the binding of RNA polymerase to the promoter region and thus blocked transcription.The two regulatory did not interfere with each other.However,for the transcription of groEL2,HrcA and ?32 bound competitively in the promoter region,which may weaken the binding ability of ?32 to the promoter to a certain extent,and thus decreased the positive regulatory effect of ?32 on groEL2.The differences in the position of CIRCE element led to the regulation preference of HrcA and ?32 and resulted in the transcription differences of duplicate groELs.Under heat shock condition,although the transcrption of hrcA was up-regulated,the HrcA protein could not be completely renatured,which weakened the ability of HrcA to bind to the CIRCE element.The transcription of ?32 was also up-regulated under heat shock,which caused more RNA polymerase binding to the promoter region.The expression of two groELs was further up-regulated.That is,under heat shock condition,the transcription level of duplicate groELs was significantly higher than that of normal condition,but it also reached a dynamic equilibrium under the regulation of HrcA and ?32.In the process of studying the regulation mechanism of duplicate groELs,we also found that a CheY-homologous protein-coding gene?MXAN 4468?has an effect on the transcription of groES and groELs.The gene is located upstream of groEL2 and co-directional in the M.xanthus DK1622 genome.However,our results showed that the two genes are not co-transcribed.In addition,the genes encoding CheY homologue protein exist only in the upstream and downstream of stand-alone groEL2 in all sequenced myxobacteria,but not in the neighboring genes of groESL.In other locations of the M.xanthus DK1622 genome,we have also found multiple copies of CheY homologous protein-coding genes,suggesting that this class of genes may be important and diverse in function.Through the construction of a series of related mutants,we found that MXAN 4468 has a strong negative regulation on groES and groEL1/2,which may be achieved by HrcA protein.MXAN₄468 had a direct inhibitory effect on the transcription of groEL2.There was probably a negative regulatory sequence of groEL2 in the downstream region of MXAN₄468.Furthermore,in protein structure conservative analysis,we found that MXAN 4468 protein has a conserved phosphorylation site?61st,Asp?,which acts as a response regulator in two-component regulatory system.MXAN 4468 protein needs to be phosphorylated by histidine protein kinase before it can negatively regulate groES and groELl/2 or perform other functions.In conclusion,our study revealed the regulatory mechanism of duplicate chaperone groELs and the synergistic relationship with single co-chaperone groES in both function and expression.At the same time,a new regulatory factor was discovered and the preliminary exploration mechanisms were carried out.In addition,we speculated that the difference in the transcription level of groELs have occurred in the early stages of gene duplication,earlier than the occurrence of functional differentiation.The difference of transcriptional level may help duplicate groEL genes remain in the genome,which is the prerequisite for their functional differentiation.Therefore,we suggested that it is also important to focus on the non-coding region of duplicate genes as well as to pay attention to the gene sequence itself.The differences that occurred in the non-coding region between duplicate genes may provide important enlightenment to the differences of coding region in sequence and function.In myxobacteria with many duplicate genes,the transcription level of groELs was significantly higher than that of other duplicates.The functional differentiation,regulation mechanism and evolutionary significance of the two genes may provide a theoretical basis and guiding significance for us to study other duplicate genes.