Insights Into The Persistence Mechanisms Of An Endogenous Cryptic Plasmid In Its Native Myxobacterial Host

Bacterial plasmids, first reported by Joshua Lederberg （Lederberg,1952）, are a diverse group of self-replicating extra-chromosomal genetic elements.Basically, plasmid encompasses a series of essential genes,like independentreplication and segregationfor persistence in host, and often some accessory genes.Theyareof the vital contributors for bacteria evolution and adaptation. Generally, plasmidsfacilitate the gene communicationby transmittingbetween different bacterial species. Markedly, theyencodemany traits that may be beneficial for cellular survival in different stresses and niches.However, there are also many plasmids playing no noticeable physiologicalfunctionsto their hosts, thus termed cryptic plasmids, and, as detected by a large-scale survey on mobilization genetic modules of sequenced plasmids, about half are non-mobilizable plasmids.Carrying a plasmid is ametabolic burdento host, for example, requiring production of additional proteins for replication and partition of plasmid DNA,using up raw materials within the cell, occupying cellular machinery such as ribosomes and disrupting the cellular environment. So, the prerequisite for maintaining a plasmid in a bacterialpopulationis thus that the positive selection for plasmid-borne beneficial traits outweighs thecosts, but constant positive selections should make the beneficial genes integrate into host chromosomes.Nevertheless, plasmids are able to exist in their natural hostsrather stablyandwidely, even showing no significant benefits and in the absence of positive selection.Many hypotheses have been proposed for the retaining mechanisms of plasmids, such as site-specific resolution systems, active partitioning for low-copy-number plasmids, post-segregation killing systems,high mobility rate of conjugation plasmids, and positive selection and compensatory adaption of non-transmissible plasmids.However, the persistence mechanisms of cryptic plasmids in bacterial hosts,as well as their effects on host,are yet unclear.Myxobacteria are a group of gram-negative bacteria that possess complex multicellular social behavior and large genomes.Myxobacterial genomes are characterized byabundant duplications and horizontally transferred genes, for instance, approximately 40% genes of the 14.78-Mb genome inSorangium cellulosum So0157-2 may come from horizontal transfers, whichimplies that the myxobacterial genomes are rather subject to integration offoreign DNA sequences and self-reorganization.Integration requires some vehicles, such as plasmids, to importforeign DNA. However, extrachromosomal autonomously replicating genetic materials are normally absent in myxobacterial cells, and up to now, pMF1, which was discovered in Myxococcus fulvus 124B02, is still thesole endogenous plasmid that is able to replicate autonomously in myxobacterial cells.It is thus greatly interesting to investigate how the 124B02 strainis able to harbor the plasmid and what effectsthe plasmidimposes on host for its persistence.The thesis is focused on the persistence mechanism of myxobacterial endogenous plasmid pMFl in its native host M.fulvus 124B02, and the main research contents and results are as follows:1. The functional models analysis of genetic replication and segregation regions of pMF1Using PEG6000 precipitation, we extracted replication intermediates （RI） of M. xanthus DZl pZJY41 strain and proved that pMF1 replicates via theta mode, which is adopted by most Gram-negative bacteria. Different from the classic repABC plasmids, the replication and segregation functions of pMFl are realized by two independent operons（pMF1.13-pMFl.16, pMF1.21-pMF1.23）, and the genetic structure and regulation network are more complicated. Except for the genes encoding ATPase （pMF1.22, parA） and DNA-binding protein （pMF1.23, parB）, and parS sites, the par loci of pMF1 still has a new gene, named parC, which differs from other low copy number plasmids significantly, implying that the partition mode used by pMF1 is very novel. We discussed the function of ParC in the second chapter.parC locates between promoter andparA, and has a 4-bp overlap with parA, suggesting that maybe parC has some functions. The stability of parC-deletion recombinant plasmid decreased dramatically, to the level of pZJY41. The deletion of parC also affected the maximum growth quantity of M.xanthus DZ1 host. Above results shows that parC participatesinto the process of plasmid partition and host growth. Blastp found no homologous proteins with ParC in NCBI database, showing that ParC is a new protein. The secondary structure prediction indicates that there are large amounts of alpha helixes in ParC, account for 80% of total amino acids. The 3D structure by comparative protein modeling demonstrates that ParC forms a hairpin-like long helix, and rotates counterclockwise, just like the right-hand superhelix of DNA. The surface potential analysis shows that many amino acids with positive charge distributes widely on the N-terminal, and negative-charge amino acids on the C-terminal. Other results indicated ParC could form trimers. Accordingly, we can summarize that ParC could assemble into a rodlike structure with three helixes by disulfide bonds, and the N-terminal is rich in positive charges and C-terminal negative charges.EMSA experiments proved that ParC could enhance the binding affinity between ParB and ItA, but has no interaction with either ItA or total ori and par loci. In intro and in vivo experiments showed that ParC did not interact with ParB. During the segregation process of low copy number plasmids, the first step is the formation of partition complex by interaction of ParB with parS, and our results demonstrated that ParC doesn’t participate into the first step of pMFl segregation.We are still doing further research on the replication and segregation mechanisms. To a cryptic plasmid, only owning complete replication and segregation functions is not enough to ensure its stable persistence in host during the long evolution process. Next, we enlarge our vision to the whole plasmid and host to study the plasmid-host co-evolution in terms of genomics.2. The genomics analysis on the co-evolution between pMFl plasmid and M.fulvus 124B02 hostAccording to the predicted function and source, we classified the total 23 pMF1 genes into 4 categories. About 14 genes were derived from myxobacteria, including eight M.stipitatus DSM 14675-related 8 genes, one from Stigmatella aurantiaca, one from Anaeromyxobacter, one from Chondromyces crocatus or Sorangium cellulosum, and theother three from many species. In addition, nine genes, having no homologues in database, are specific to pMF1. Unfortunately, most genes have not been predicted clear functions. The transcriptome datum indicated that pMF1.17 and pMFl.18 genes have highest transcriptional levels, next is pMFl.12. Strand-specific transcriptomics and RT-PCR results showed that most pMFl genes organized into six operons, occupied 87% of total genes.The genome of M. fulvus 124B02 was sequenced with a combination of the Roche 454 GS FLX and Illumina GAII sequencing platforms. M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. The G+C contents of the chromosome and the plasmid are 69.96% and 68.7%, respectively. The phylogenomic analysis and genome synteny indicated that M.fulvus 124B02 is closer to M. stipitatus DSM 14675, whose genome size is more similar with M.fulvus 124B02, rather than M. xanthus DK1622or M. fulvus HW-l.In spite of the 1-2 Mb genomic expansion of M. fulvus 124B02, there is no obvious difference in the percentages of orthologous and paralogous genes among four myxococcus strains. Compared with other three myxococcus strains, the genomic defense systems of M.fulvus 124B02are much weaker, with less Cas proteins, spacers and methylation-restriction enzymes.Sequence alignments showed that some pMF1 genes were derived from other myxobacteria, suggesting that pMF1 had transferred horizontally between different myxobacteria. Another phenomenon is that pMF1 has most homologous genes with M. stipitatus DSM 14675. The myxococcus genus was generated before 47-51 million years, and M.fulvus 124B02and M.stipitatus DSM14675 were differentiated from their common ancestor about 41 million years ago.The weaker genomic defense system explained why pMFl was retained in M.fulvus 124B02 and evolved with its host.3. The presence of pMF1 contributes to the genome stability in hostIn order to get insight into the impacts of pMFl on its host, we cured the endogenous plasmid from the original M. fulvus 124B02/pMFl strain using pZJY4111, a shuttle plasmid constructed containing the ori and parloci regions of pMF1, based on the plasmid incompatibility principle. Curing pMF1 slightly affected cellular phenotype characteristics. Next, we conducted laboratory evolution experiments with M. fulvus 124B02 in three conditions:easily utilizednutrients （CYE medium）, living or autoclave-dead E. coli cells （L-cell and D-cell） as the sole nutrients. We checked routinelythe presence of pMF1 in the periodically preserved 124B02/pMF1 subculturesby PCR amplification of the pMF1 ori and par gene fragments. Interestingly, pMF1 was lost from the CYE and L-cell subcultures after eight and seven transfers respectively, whereas the plasmid was still available in the D-cell subcultures even after 40 transfers. We re-sequenced the strains sub-cultured under three conditions, deleted some potential genes, and tested the influence of these genes on pMF1 persistence using subculture again. We determined some genes-124B02₀438,124B02₃420,124B02₆993,124B02₇965 finally.To investigate whether the presence of pMFl has some long-term effects on its host, we performed laboratory evolution experiments with the124B02/pMFl and 124B02/free strains comparatively, under the same D-cell conditions as described above. Interestingly, compared with that of the 124B02/free subcultures, the 124B02/pMFl subcultures sustained rather normal development and sporulation abilities after similar transfer rounds. We re-sequenced the genomes of 124B02/pMFl and 124B02/free D-cell subcultures of differentrounds of transfer to investigate the underlyinggenome changes during the subcultivation processes. Interestingly, the SNP and Indel numbers in those plasmid-harboring subcultures were much lower than that in the plasmid-free subcultures. The increase of the genomic mutation numbers inthe 124B02/pMF1 and 124B02/free subcultures accumulated in an almost linear pattern with the transfer rounds. Because the pMF1 and pZJY4111 both had positive effects on the stabilization of genome by alleviating the spontaneous mutation in genome, we suggested that the shared par（pMF1.21-pMF1.23） regions might have the functions in keeping the genome stability of 124B02.pMF1 plasmid is of great importance for solving the genetic manipulation, and our understanding of myxobacteria evolution. Based on above results, we could speculate the mechanism model of how the cryptic plasmid pMF1 retained stably in its M.fulvus 124B02 host. In the long evolution process of myxobacteria, as a mobilizable vector for genes, pMFl transferred horizontally among different strains. Because of its simple structure and high copy numbers, plasmids are known as the rapid genetic evolution machine. The weaker genomic defense systems of M.fulvus 124B02 make it easier for pMF1 to access into cells and retained. On the other hand, pMF1 contributes to the genome stability in host to persist finally.