Research On Cytoskeleton-Related Genes From Spiroplasma Melliferum CH-1

Unlike other bacteria in Mollicutes, spiroplasma is a kind of wallless bacteria that can maintain their special spiral shape and motor function only with a unit membrane enveloping the cell and has become the model organism for researching motility. It is great significant to study their morphology and movement for knowing the basic properties of organisms. The cell’s morphology and movement is also related to cytoskeleton. The Fib protein encoded by fib gene is the principal component of cytoskeleton. And fib gene is a unique gene in spiroplasma. mreB gene is one of the relevant genes which encoding cytoskeletal proteins. MreB encoded by mreB gene can maintain the cell structure. So it is very important to study cytoskeleton-related genes mreB and fib of spiroplasma. The purpose of this study was analysing the cytoskeleton-related genes mreB and fib by applying bioinformatic methods based on the genome sequence of honeybee-associated bacteria Spiroplasma melliferum CH-1. We studied the prokaryotic expression of mreB gene and determined expression levels of these genes in spiral and non-spiral shape of spiroplasma. All these studys lay the foundation for future intensive research of the polymerization of MreB in vitro, and research of the aggregation characteristics in vivo and for the function analysis of mreB and fib gene. The main research results as follows:Firstly, S. melliferum CH-1 contains five mreB and one fib genes. We used bioinformatics methods to analysis the properties of proteins encoded by fib and mreBl-mreB5 genes. The results showed that:(a) The molecular weight of MreBl-MreB5 was 36、23、23、37、25 kD, respectively. Among them, MreBl protein was 36 kD and consistent with other bacterias (such as Escherichia coli). And the molecular weight of Fib was 58 kD which similar to some studies have reported; (b) MreB protein belongs to MreB/Mbl protein family, N-terminal residue of Fib (14-226 amino acid positions) was predicted as phosphorylase superfamily, the result was similar to the predictions by Cohen-Krausz etc; (c) We used SWISS-MODEL automated homology modeling tool to predict the three-dimensional structure of MreB and Fib proteins. For one hand, MreBl, MreB4 and MreB5 had all most the same structure, they just had subtle differences. For the other hand, the structure of MreB2 and MreB3 was similar, they had little differences. The three-dimensional structure of intact Fib protein can not be obtained by the method of homology modeling. Because Fib protein is the unique protein in spiroplasma; (d) The phylogenetic tree was constructed by Neighbor-Joining method based on the amino acid sequences of MreB from S. melliferum CH-1 and other bacterias. MreBl-MreB5 were closely related to corresponding MreB of other spiroplasma. respectively. But compared with spiroplasma. other wall-less bacteria such as Haloplasma contractile and Firmicutes (including of Bacillus subtilis and Clostridium botulinum) had more closer genetic relationship.Secondly, we designed primers based on the sequences of spiroplasma cytoskeleton-related gene mreB. And then we cloned mreB1-mreB5 genes and expressed the proteins by using E. coli expression system firstly. We purified MreB protein, and found MreB1-MreB5 proteins-present in the inclusion which can dissolve only in Binding Buffer containing 8 mol/L urea. They were hydrophobic proteins. Bioinformatics analysis suggested that all of the MreB proteins expressed from S. melliferum CH-1 may be hydrophobic proteins. This study lay the foundation for future intensive research of MreB polymerization in vitro, and further research of the aggregation characteristics in vivo.Thirdly, spiroplasma almost showed dot shape in the early lag phase and spiral shape in logarithmic phase, during culture process when observed by use of dark-field microscope. But when we observed them through electron microscope, we found the dot shape under dark-field microscope actually was nearly various irregular spherical or inflated terminal and slightly curved rod shape. Two-step real-time quantitative PCR method based on SYBR Green I dye was applied to compare the expression levels of mreB1-mreB5 and fib genes in spiral and non-spiral shape of S. melliferum CH-1. The expression levels of these genes in non-spiral shape relative to the spiral shape of spiroplasma showed significant difference. The results indicated that the expression levels of these genes in spiral shape of spiroplasma were relatively higher than that in non-spiral shape. These results showed that spiroplasma maintain the spiral shape may need mreB and fib genes. These cytoskeleton-related genes may be related to the morphology of spiroplasma. Spiroplasma cultured from non-spiral shape to spiral shape, the increase expression level of fib gene was greater than mreB gene. We speculated that fib gene play more important role than mreB gene. So the function of gene mreB and fib should be further verified.All results of this study play a role of revelation mreB and fib genes for shape and movement of spiroplasma, and lay the foundation for further studying the function of these genes.