Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium

Abstract

] prompted this investigation. ]. sp. WH8113 provides new constraints on its motile mechanism. The spicules are well positioned to transduce energy at the cell membrane into mechanical work at the cell surface. One model is that an unidentified motor embedded in the cell membrane utilizes the spicules as oars to generate a traveling wave external to the surface layer in the manner of ciliated eukaryotes.

Background

] prompted this investigation.

Results

].

Conclusion

sp. WH8113 provides new constraints on its motile mechanism. The spicules are well positioned to transduce energy at the cell membrane into mechanical work at the cell surface. One model is that an unidentified motor embedded in the cell membrane utilizes the spicules as oars to generate a traveling wave external to the surface layer in the manner of ciliated eukaryotes.

Results and discussion

). Chemical fixation appears to cause detachment of the surface layer from the outer membrane whereas cryopreservation and freeze-substitution (data not shown) retain this layer.

(strain WH8113) Cross fracture revealing concentric layers of cell envelope. The inset corresponds to the outlined section of cell envelope comprising cell membrane (CM), peptidoglycan layer (P), outer membrane (OM), and surface layer (S). A thylakoid layer (T) is also indicated. Scale bar, 200 nm.

].

(strain WH8113) Complementary fracture plane showing the outer leaflet of the cell membrane (CMo) which has fewer intramembrane particles than the inner leaflet. The fracture then crosses to the outer leaflet of the outer membrane (OMo), and then turns to fracture across the surface layer (S). Scale bar, 100 nm.

).

(strain WH8113). (OMi) and a patch of the inner leaflet of cell membrane (CMi) where the outer membrane has been pulled away. Typical of such outer membrane fractures, a profusion of spicules lie about the perimeter or lie flat along the outer membrane surface. Consideration of the fracture process explains the disposition of these spicules. If the spicules are tightly rooted to their bases, and the bond energy of their composition exceeds that of the interaction between their surfaces and the ambient ice, then the spicules would be pulled out of the frozen material that is cleaved away by the knife fracture, and would then fall onto the newly exposed surface thereby demonstrating their original continuity with it. Differences in angles of cleavage may explain the relative sparseness of the spicules on the top of the exposed surface compared to the edges. Scale bar, 100 nm.

). These balls have the same size and spacing as those balls identified in cross-sections.

(strain WH8113) Patch of crystalline outer surface near fracture plane revealed by etching ambient ice. Since the bacterial surface is perpendicular to the line of sight, we used this image to measure the lattice arrangement: rhomboid with spacing ∼ 12 nm and obtuse angle ∼ 110°. Scale bar, 50 nm.

Spicules arise in profusion over the surface of the bacterium and extend up to 150 nm into the surrounding medium. The smallest observed separations between adjacent spicules are 12 nm and 24 nm, which corresponds to the spacing of the surface lattice. Spicules have uniform thickness of ∼ 5 nm along their length. Spicules are flexible; we have measured curvatures with radii as small as 30 nm.

). These facts taken together suggest that the spicules extend outward through channels in the surface layer and extend inward through underlying layers to contact the cell membrane.

].

]), these oscillations would be transduced into a rowing motion of the spicules. In this case, the crystalline surface layer could serve as oarlocks for the spicules, converting smaller motions at the bases of spicules into larger motions at the tips.

tend to aggregate.

does not.

, both motile and non-motile, that have been isolated. Comparative analysis of a selection of species could illuminate the motile mechanism of the cell envelope and the role of the structures described here.

]. This suggests that part, but not all, of the motility apparatus is disrupted in the absence of SwmA.

have been obtained for SwmA. However, fibers crossing from the cell membrane to higher layers that we interpret as the lower stems of spicules are evident for SwmA, and spicules are evident on some convex fractures of the outer membrane of SwmA (data not shown). It remains possible that the cell envelope of the SwmA mutant has subtle flaws consistent with a partial disturbance of its motility apparatus.

Conclusions

) provides new constraints on theoretical models for motility.

. Present observations taken together suggest that the spicules (SP) extend through the surface layer (S) and outer membrane (OM) to contact the cell membrane (CM) (as shown in the cutaway of the peptidoglycan layer (P)).

Bacterial strains, culture conditions

].

Sample preparation and viewing

]. In summary, a few microliters of cell suspension were spread on a layer of gelatin mounted on a freezing stage that was then slammed against a super-cooled copper block. For freeze-fracture/freeze-etch preparations, quick-frozen specimens were then knife fractured at nominally -108°C in a Balzer's 301 freeze-fracture apparatus equipped with a high-speed rotary stage. Fractures were within a few micrometers of the frozen surface. The fractured samples were lightly etched for 4 min at nominally -108°C. The high concentration of salt in the artificial sea water used as culture medium precluded deeper etching. The etched surface was rotary shadowed with platinum, and then rotary coated with a carbon backing. Replicas were cleaned in commercial bleach, rinsed in distilled water, and picked up on 400-mesh grids.

]. Additional staining was provided by immersion in 0.1% HfCl in acetone for 4 hr. Embedding was in araldite and sections were stained with uranyl acetate and lead citrate.

Samples were viewed in a JEOL 200CX electron microscope. Regions of the specimen with negligible ice crystal damage were chosen for analysis. Electron micrographs were scanned directly using an Agfa DuoScan scanner, and images were analyzed using stereo images generated in Adobe Photoshop.

Acknowledgements

We are indebted to J. Waterbury, F. Valois, and B. Brahamsha for kindly providing bacterial cultures and for helpful discussions. ADTS was supported by the Rowland Institute for Science and is an Amgen Fellow of the Life Sciences Research Foundation. ADTS dedicates his share of this work to his graduate advisor, Howard C. Berg.