| This thesis includes two research projects. One part is on characterization of the partition system of Myxococcus plasmid pMF1. The other part is the the relation of ring formation of morphogenetic cytoskeleton proteins MreB, MreC, RodA, PBP2and RodZ to cell division protein FtsK.Myxobacteria are well known for their unique multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, autonomously replicating plasmids have not been reported until recently. In2008, we reported the discovery of the first and as yet only endogenous myxobacterial plasmid, pMF1, which was isolated from Myxococcusfulvus strain124B02. Except for a few ORFs that are highly homologous to those in myxobacterial genomes, most ORFs in the Myxococcus plasmid pMF1do not have homologs in the GenBank database, including the replication-associated sequence. We localized the replication region to the pMF1.14ORF using a vector that cannot replicate in M. xanthus, and shuttle vectors between Escherichia coli and M. xanthus were thus successfully constructed. Because they lack a stabilizing sequence, these low-copy-number shuttle vectors are not stably inherited in M. xanthus and are easily lost in the absence of selective antibiotics.Low-copy-number plasmids are dependent on plasmid-encoded partitioning (par) systems for stable segregation into daughter cells. The par loci of a plasmid typically consist of an operon containing an autogenously regulated gene pair, parA and parB, and one or more centromere-like iteron regions. ParA is an ATPase (other two classes of motor proteins are GTPases ParM and TubZ) involved in the segregation of plasmids or chromosomes while ParB is a DNA-binding protein that recognizes specific iteron sequences of the centromere-like sites that are normally located upstream or downstream in the par loci. In general, the two plasmid-encoded trans-acting partitioning proteins and the cis-acting centromeric site are all essential for stable segregation of plasmid. The plasmid partitioning systems have been identified into three types. The type Ⅱ par loci encode an ATPase containing Walker motifs, while types Ⅱ and Ⅲ encode actin-like and tubulin-like proteins, respectively. Type Ⅰ par system is further divided into two sub-groups, designated type la and type Ⅰb, based on the regulation manner of the operon, the properties of ParB, and the location of the cis-acting sites. In the type la par system, represented by P1and F plasmids, the sizes of ParA and ParB proteins are321-420aa and312-342aa, respectively. The la ParA contains an N-terminal DNA-binding domain, which plays an autoregulation role for the transcription of operon. In contrast to the type la par system, the type Ib, represented by pTAR, TP228, pB171and pSM19035, encodes a ParA protein of192-308aa, lacking the N-terminal DNA-binding domain. The ParB protein of this sub-group is46-131aa in size, and is able to bind to the par operon promoter and regulates the transcription of the operon.This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMFl.21-pMF1.23(parCAB).The pMF1.22ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that thepMF1.23(parB) product is a DNA-binding protein of iteron DNA sequences, while the product ofpMF1.21(parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ⅰb par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1parsystem has novel structural and functional characteristics.The MreB-associated helical cytoskeleton has been implicated in several cellular processed, including chromosome segregation, regulation of cell shape and determination of cell polarity. MreC, MreD, Pbp2and RodA which are also required for maintence of the cylindrical cell shape are also present as helical and ring structures. It has been reported that assembly of MreB, MreC, MreD, RodA and Pbp2into the cytoskeletal rings was dependent on the presence of FtsZ ring, which is an essential component of the cell division machinery. But the entry of these proteins except RodA to the cytoskeleton required no other known components of the division apparatus, while assembly of RodA into the cytoskeletal ring required one or more other additional components of the division machinery. The MreBCD and RodA proteins are able to entry of cytoskeletal rings and coiled structures independently of each other or Pbp2. However, the assembly of Pbp2rings and coiled elements requires the presence of MreC.The rodZ gene, which is another component of the cytoskeleton is required for determination of rod shape in Escherichia coli. Cells without rodZ gene became round or oval instead of rod shape. MreB determines the length of the short axis of the cell whereas Mbl determines the length of the long axis of the cell. RodZ mainly affects the length of the long axis of the cell other than that of the short axis of the cell. Over- production of RodZ resulted in an increase in the length of the long axis instead of the short axis. It might function in a different mode from both MreB and PBP2to maintain the rod shape of the E.coli. It was considered that MreB and PBP2regulate the length of the short axis of a cell while RodZ regulate the length of the long axis instead. The RodZ protein was also reported to form helical structure along the long axis of the cell. However, the dependence relation between the known cytoskeletal proteins and RodZ when they assembly to the coil and rings structure is unclear.Before cell division, MreB is incorporated into annular structures that located near midcell The MreB forms single rings around the cell cylinder. It forms doublets that flank the cytokinetic FtsZ ring. In the present study, we show that RodZ can also form coiled and rings structures in E.coli. It also shows poles localization in the cells. The RodZ ring formation requires the FtsZ protein which is similar to what MreB protein behaves. The ring structure formation of RodZ does not require any other cytoskeleton proteins. MreB protein is capable of assembling into cytoskeletal rings and coiled structures when rodZ gene is absent.RodA is a component of the prokaryotic cytoskeleton. RodA and the other components of the MreB-associated cytoskeleton proteins are organized into pole-to-pole helical structures in rod shaped cells. In pre-division cells the proteins also form circumferential rings located at the division site. It has been shown that assembly of MreB, MreC, MreD and Pbp2into the cytoskeletal rings requires the FtsZ ring but does not require other components of the cell division machinery. In contrast, RodA did not assemblu into the cytoskeletal rings in a temperature-sensitive ftsK mutant. In this study, we show that RodA protein does not assemble into rings when FtsK is absent in a ftsK deletion strain, Further more, PBP2, MreC and RodZ ring formation also require FtsK presence. However, MreB ring formation does not require FtsK. On the other hand, FtsK was not required for assembly of RodA, PBP2, MreC and RodZ into the cytoskeletal pole-to-pole helical elements. We further show that only the N-terminal domain of FtsK which is required for its cell division function, is required for RodA, PBP2and RodZ ring formation. These results suggest for the first time a link between the essential cell division protein FtsK and a component of the bacterial cytoskeleton. |
PDF Full Text Request