Ultrastructure Of Cell Wall Peptidoglycans From Gram-positive Bacteria

Peptidoglycan is the unique fundamental structural constituent of the bacterial cell wall.It plays an important role in maintaining the specific shape of the bacterial cell,in cell growth and division and protecting the cell from rupture as a result of internal pressure.Research on peptidoglycan architecture has always been a fundamental topic in microbiology and is of great significance for understanding the growth and division mechanisms of cells,the anchoring forms of extracellular polymers on cell walls and the selection of antibiotic targets and so on.Peptidoglycans are supramolecular complex composed of glycan chains that are crosslinked by peptides.There is a high diversity in the composition and sequence of the peptides from different species.Despite many years of research,the architecture of peptidoglycan is still largely elusive.Up to now,the main topological models are:layered model,scaffolding model and coiled-coil model and so on.Atomic force microscopy(AFM)has been proven to be a powerful and effective method for studying the supramolecular structures of peptidoglycan.Various bacteria such as Escherichia coli,Staphylococcus aureus,and Bacillus subtilis have been studied using AFM.However,the structure of peptidoglycan is different between bacteria of different species,bacteria of the same strain and even the same strain of bacteria in different growth environments and growth periods.Thus,current data and models are still insufficient to decipher the complex cell wall architecture in bacteria.This project thesis focuses on the structure of peptidoglycans of cell walls and septa from two different shapes of typical gram-positive bacteria Bacillus subtilis and Staphylococcus warneri,by combining biochemistry with AFM and transmission electron microscope(TEM).We carried out systematic research and obtained the following research results:1.Ultrastructure of side wall and septa peptidoglycan from Bacillus subtilisWe investigated the high-resolution architecture of peptidoglycan from the typical gram-positive bacterium Bacillus subtilis using AFM and provided high-resolution evidence of peptidoglycan architecture remodeling at different growth stages at nanoscale.And found that the average cell length in stationary phase was 2.72±0.63?m(60 measurements from 3 replicates),while the measured average length between each adjacent septation sites was 3.78±1.28 ?m in mid exponential cells(70 measurements in 15 bacterial filamentous chains from 3 replicates),which was longer than the average cell length in stationary phase(p<0.05).Side wall peptidoglycan from strain B.subtilis AS1398 changed from an irregular architecture in exponential growth phase to an ordered cable-like architecture in stationary phase.Small cables entangled into larger ones could sometimes be noticed.The average width of these "cables" was 29.11±5.79 nm(n=32),which was much smaller as compared to that in another strain B.subtilis 168 HR in a previous report.Thickness of side wall peptidoglycan was found to be related with growth stages,with a slight increase after transition to stationary phase.The average thickness of single layered side wall peptidoglycan was 12.59 ±0.89 nm(n=59)in mid-exponential phase.Septal disks were synthesized progressively toward the center,while the surface features were less clear than those imaged with side walls.A thin interior leading edge could be noticed in incomplete septal disk.Except the interior leading edge,thicknesses at other parts of the septal disks were evenly distributed.A likely process was that a thin leading ring was formed at the interior edge and then thickened with the growth of the septa,until the septa were completely sealed.Compared with previous studies,our results revealed slight differences in architecture of peptidoglycan from different B.subtilis strains,expanding our knowledge about the architectural features of B.subtilis peptidoglycan.2.Investigation on formation of bacterial cell wall in poles from flat into hemispherical shape in Bacillus subtilisPeptidoglycan contributes to the maintenance of a defined cell shape and a rod bacterium is composed of a cylinder and two hemispherical poles.The molecular mechanism of morphogenesis of rod shaped bacteria has received extensive researches.However,most of the works focused on the cylinder part,but morphogenesis of the hemispherical poles is still not clear.To investigate the morphology and morphogenesis of Bacillus in poles,various technical means were used to explore the cell and isolated peptidoglycan.The transmission electron microscopy(TEM)images of ultrathin sections of B.subtilis cells showed hemispherical poles and it was confirmed by the observation of intact cells with scanning electron microscopy(SEM)and atomic force microscopy(AFM).A ring structure could be clearly seen in the pole area.But the curvatures of the poles were greatly reduced in isolated sacculi.These poles in isolated sacculi were relatively flat compared with the poles in intact bacterial cells.The results made people tend to speculate that the cell wall in poles is elastic and showing a hemispherical structure as a result of internal pressure.When internal pressure was reduced,cell walls in poles transformed into a relative flat form,internal pressure was responsible for the maintaining of the hemispherical structure in poles.Then we carried our works to check whether internal pressure was responsible for maintenance of the hemispherical shape in poles.B.subtilis cells were treated with high osmotic pressure with sucrose solution and it could be noticed that curvature of the bacterial poles was reduced when compared with control samples.When the cells with shrank poles were transferred back into low osmotic environment,the poles could change back into hemispherical shape again.In our study,we found that the poles both in the sacculi and cell walls of B.subtilis are relative flat.Thus,the formation and maintenance of hemispherical poles should most likely to be resulted from internal pressure.3.Supramolecular architecture of peptidoglycan from Staphylococcus warneriTo explore the ultrastructure of peptidoglycans in the spherical cell wall,the structure of a typical coccoidgram positive bacteria Staphylococcus warneri was investigated using AFM and other techniques.We found that the supramolecular architecture was similar to S.aureus.The rib-like ring belts separated the cell walls of different generation,the new cell walls(type ?)were composed of concentric ring structures,which were closely arranged.The thickness of type ? cell wall was about?10 nm(n=60).The old cell wall(type ?)sections showed loosely dotted structure,the thickness of which were about?20 nm(n=30).The intermediate state(type ?)of new cell wall to type ? old cell wall showed slightly scattered linear structure which were like ball-chains.And the thickness of type ? cell walls were from 15 nm to 20 nm(n=37),which had a large span.We then used pseudoalterin,an enzyme functioned as both an amidase and an endopeptidase which could degrade peptidoglycan with high efficiency to remove the peptide chains to digest the peptidoglycan of S.warneri.And found that glycan chains which were extremely long existed in S.warneri both under the observation of the AFM liquid phase and gas phase of the enzymatic samples.And the results were verified by HPLC chromatography.Regular linear glycan chain structures which may come from type ? cell wall and loosely ball-chains-like structure which were from type ? structure could be observed.The type III cell wall components may be dispersed in the enzymolysis process.It was the first time for long glycan chains to be observed in situ in the cell wall and there was a significant difference in structure of peptidoglycans in different species of Staphylococcus.4.Structure of septa from Staphylococcus warneriThe septa from Staphylococcus bacteria used to be considered as a monolayered cross wall.But the amazing work published on Sicence of Theriot et al.pointed out that bacteria from Staphylococcus divides in a quite fascinating way by fast mechanical cracking on septa,usually within milliseconds.Thus,the structure of septa was thought to be double layered,but there was still no clear experimental evidence reported.We isolated sacculi fragments including septa and cell wall components from S.warneri and carried out detailed works with AFM on the complete and incomplete septa,both intact and broken ones.The results provided clear evidence for the double-layered structure in the septa from S.warneri,which provided the structural basis for the mechanical cracking propagation mechanism in Staphylococcus.Although two septal layers were separable by mechanical forces,they were structurally connected at the interior edge in incomplete septa.The outer surface of septal peptidoglycan was relative featureless,while the peptidoglycan on inner surface of the double layers was organized into concentric rings.The spatial organization of the peptidoglycan on septa was retained when the septa were transformed into new cell walls after division.Further analysis revealed that the septum was not a fully complete structure,but a central depression was always present.The central depression had a severe negative impact on the formation of new septa in the next round of cell cycle.5.Capsular polysaccharides production from Zunongwangia profunda SM-A87 monitored at single cell level by atomic force microscopyWe investigated the capsular polysaccharide(CPS)production of a CPS producing bacterium Zunongwangia profunda SM-A87 isolated from deep-sea sediments with AFM.Z.profunda SM-A87 exhibited slow growth rates at 10?,which was lower than at optimum growth temperature(30?).But the CPS production at 10? was much higher than that at 30?.Single cell imaging with AFM revealed rod like cell morphology at both temperatures,but filamentous bacteria could sometimes be noticed at 10?.The CPSs surrounding bacterial cells were observed,and the fibrils of polysaccharides entangled into networks.The diameter of the capsules and the length of the polysaccharides fibrils increased as cultivation time increased.The average volume of the bacterial cells and capsular polysaccharides surrounding each cell at both temperatures were calculated.The average volume of a single bacterial cell was 0.2?0.3?m3,and it increased in exponential phase and slightly decreased in stationary phase,but the average volume of CPS produced by single cells increased during bacterial growth.CPS production increased to 0.097±0.051 ?m3 per cell at 10? in stationary phase,which is larger than that produced at 30?(0.055±0.013 ?m3 per cell).We report a novel method of estimating the average bacterial polysaccharide production with AFM.Since polysaccharides-producing bacteria are abundant in marine sediments,this method is potentially useful for studying the biological and ecological role of polysaccharides-producing bacteria from the deep sea in the future.