Micron, 1980, Vol.: I 1, pp. 389-390 Pergamon Press Ltd. Printed in Great Britain

HIGH RESOLUTION STRUCTURE OF BACTERIAL REGULAR SURFACE LAYERS"

M. STEWART

CSIRO Division of Computing Research, P.O. Box 1800, Canberra Ciw, A.C.T. 2601, Australia

R. G. E. MURRAY

Department of Microbiology and Immunology, University of Western Ontario, London N6A 5C1, Canada

T. J. BEVERIDGE

Department of Microbiology, University of Guelph, Guelph, Canada

Many Gram positive and Gram negative bacteria have • regular array of protein subtmits (the RS layer) on their outermost surface. We have taken advantage of the high degree of resulerity shown by some of these layers to invesfijate their structure at high resolution using electron diffraction and electron microscopy in conjunction with computer irrmse prong.

Fralpmmts of the RS layer of the Gram positive S0orosarc/na urea¢ were prepm'ed and examined am deucribed in detail ebewhare'. Neptively stained preparations showed two dhginct I~tern~ which ¢orrespouded to the upper and lower surfaces o¢ the layer. Examples of each pattern were digitised and pngemed using the usual Fonrier-lmed methods o=! CDC Cyber 73 and 76 oomputers. Gley scale repreaentations of the filtered inuq~ were prod;_=~__ using an OpU'onics Colorwrite C-4300 system and FiS. 1 shows throe obtained from e,¢h type of p,ttern. In each data was collected to a resolution of 2.2 rim. Although the two filtered images appear at first Idght quite cl/ffenmt, dome inspe~ion reveals • common pattern of areas of high stain density (dark), which suumms that there are • number of holes or saps in the layer. Figure 2a shows the structure of the RS layer of the Gram negative AqumOirillum pu~.'_:_~onc~//um derived from couve~donal micrographs 2 by compmer Pro8 to 2 nm rmolmion. Again dark areas correspondinll to gap, or holes with • limiting diameter of about 2-3 nm are preaent. Larse f ~ t a of this layer can be obtained and this has allowed its structure to be solved to 1.4 nm resolution by combinim~ phase information derived from computer procesainll of low-dose micrographs with electron diffraction amplit~ ~,,~e,_ (Fig. 2b). This shows up the fine detail of the subunits and confirms the preuace of the A ~ u r n s~'~,nt strain MW5 has a more complex layer in which two hexasoual layers (and pow/bly aome additional material) overlap to produce a Moire pattern (Fig. 3). Althonsh fine detail is otmcured in the compmite pattern, the individual hexagonal layers clearly have gaps analogous to those found in S. urca¢ and A puo/d/¢onc~ylm~ Simil=r gaps can be seen in other layers studied to hilgh resolution.

One function of the RS layers appears to be to protect the underiyinS surface layers (cell wall, plasma membrane and, for Gram nqlatives, outer membtlme) of the bscterium, probably in a manner analoBom to a vinm cap~. But, unlike • virus, the bsctefinm has an active metabol/mn and so must obtain nutrients from and expel waste products to the cuviromnemt. We peopaee that here lies the. sijnhScance of the 8ape in the layers. They would enable the pamSe of *mall molecuks, such as nutrients, while mill scr,-m%--the ,_, ___,,,~4yi~ laym from Imljer ~__'_ _J~,_. meh as lysolg~dc enzymes, complememt, ~ and phqe. This implies that the ~ in the layec, may be as impecsant to the survival and evolutionary capm:ity of the orsanimm as the R8 Wc¢~-: The variety of ~ in different organisms would thus be of little cotmequence to function wovided the proc_~M and holm are suitable.

REFERENCES

I. T.J.BeverMle, Surface arrays on the wall of S~0omann~w ummt, ]. ~g~m'/o/. 139, 1039 ( 1 ~ ) .

2. T.J.kveridse & R.O.E.Murray, Superficial macromolecular arrays on the cell wall of Soirillum putnld/comob~n~/. Bec~mloL 119, 1019 (1974).

work wa, assisted by the Academic ~ t Fund of" the Universi/y d Westerns Omtario and the Medical Rmesrch Council of Canada.

389

390 M. Stewart, R G. E. Murray and T. J. Beveridge

O 0 013

a b

Fig. I. Fourier filtered patterns from S ureae (a) external side of the RS layer (b) inner (cytoplasmic) side. Note the correspondence between areas of high stain density (dark) which probably represent gaps in the layer. Bar is 10 nm

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tt tt

f

It

t~ It

. _ _ S t , l i t

a b

Fig. 2. Filtered inuqles from A. putTidiconchylium. (a) I~uted on conventional electron microllraphs to 2 nm resolution (b) baaed on low-dose micrographs and electron ditTraction to 1.4 nm resolution. Note the gaps between subunits in each case. Bar is 10 nm

m s

a~ It

a

Fig. 3. Filtered images from A. r~rOens MWS. (a) Moire pattern usually seen. (b) single hexagonal layer. The Moire ~ prodn__e~_ by the superpmition of two of these layers, pmsibly with some extra material. Again, there are gal~ in the layer. Bar is 10 am.