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Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis., Bacterial cell wall peptidoglycan is essential for viability and shape determination. Using high-resolution microscopy, Foster and colleagues elucidate the peptidoglycan architecture and insertion pattern in Escherichia coli and other Gram-negative bacteria, and propose a new model for cell wall elongation.

R.N. WATERHOUSE, E.J. ALLAN, F. AMIJEE, V.J. UNDRILL AND L.A. GLOVER. 1994. Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units (cfu)). The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers. Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts. In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance. The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.

Aloysius, S. K. D. & Paton, A.M. 1984. Artificially induced symbiotic associations of L-form bacteria and plants. Journal of Applied Bacteriology56, 465–477. L-forms of a small but varied selection of bacteria were found to enter viable plant cells and establish novel symbiotic relationships. Evidence is presented for the ability of the L-forms to spread and to express at least some of their metabolic functions within plant tissue.

of the staphylococci which have been reported suggest that they resemble those of the streptococci (Dienes and Sharp, 1956). Both alpha and beta hemolytic streptococci produce large bodies in appropriate mediums and the large bodies give rise to granules which develop into typical L colonies (Dienes, 1953a; Dienes and Sharp, 1956; Crawford et al, 1958). That L forms of staphylococci do develop in a manner similar to the

The bacteria and infection threads of the pea root nodule were examined by light and electron microscopy. The bacteria in the infection thread are enclosed in a microcapsule. This capsule disappears when the bacterium is released into the host cytoplasm. The membrane envelope which surrounds the bacteria in the host cell is shown to be derived from the plant cell membrane. The infection of the host cell is by means of a process akin to pinocytosis and the bacteria are confined to vacuoles in the host cytoplasm. As each membrane envelope contains only one bacterium, the envelopes must divide with the bacteria. The bacteria increase 40fold in volume from the infection thread to the stage of the mature bacteroid. The mature infected host cell contains few organelles. The mitochondria become confined to the periphery of the cell. Differences of membrane structure in gram negative bacteria found by other workers have been attributed to fixation artifacts.

r ady described, were being recovered from human blood specimens. Using the technique for blood culture described by Pickett and Nelson,12 one btains a confluent mass of growth on the primary plate which continues to develop over a period of several days and which increases in opacity and quantity but which has been, until recently, impossible of subculture. That is, subcultures of this material would not de

Previous s t u d i e s have a r t i f i c i a l l y i n d u c e d c e ll w a l l d e f i c i e n t forms of L i s t e r i a m o n o c y t o g e n e s w i th p e n i c i l l i n or s p e c i f i c a n t i s e r a ( 1 , 2 , 7 ) . C e l l w a ll d e f i c i e n t forms of L i s t e r i a monocytogenes have a l s o been d e m o n s t r a t e d in t i s s u e c u l t u r e s t u d i e s of i n f e c t ed a l v e o l a r macrophages ( 2 0 ) . Present s t u d i e s , u s i n g phase c o n t r a s t m i c r o s c o p y , i n d i c a t e t h a t c e l l w a l l d e f i c i e nt f o r m s of L i s t e r i a m o n o c y t o g e n e s a r e a n a t u r al p h e n o m e n o n . Samples t a k e n from v a r i o u s r e g i o n s of m o t i l i t y t e s t medium s t a b c u l t u r e s i n d i c a t e t h a t the o r g a n i s m s growing in the pathognomonic " u m b r e l l a " r e g i on a r e c e l l w a l l d e f i c i e n t forms. The c l a s s i c a l b a c i l l a ry f o r m s , s i m i l a r to t h o s e found in the s t a b r e g i o n , can be r e c o v e r e d from t h e " u m b r e l l a " only a f t e r 36-48 h o u r s. C h a n g e s in t h e o x y g e n a n d / o r c a r b o n d i o x i de c o n c e n t r a t i o n are i m p l i c a t e d as t h e primary cause of the d e f i c i e n c y in the c e l l w a l l. Previous studies have artificially induced cell Wall deficient forms of Listeria monocytogenes with Penicillin or specific antisera (1,2,7). Cell wall Deficient forms of Listeria monocytogenes have also been demonstrated in tissue culture studies of infected Alveolar macrophages (20). Present studies, using phase Contrast microscopy, indicate that cell wall deficient Forms of Listeria monocytogenes are a natural Phenomenon. Samples taken from various regions of Motility test medium stab cultures indicate that the Organisms growing in the pathognomonic “umbrella†region are cell wall deficient forms. The classical bacillary Forms, similar to those found in the stab region, can be recovered from the “umbrella†only after 36-48 hours. Changes in the oxygen and/or carbon dioxide concentration are implicated as the primary cause of the deficiency in the cell wall

A strain of Klebsiella pneumoniae virulent for mice was serially passaged in vitro as a penicillin-induced L-form. Periodically, the L-form was reverted to the bacterial form by removal of penicillin, and the revertant was tested for virulence. Virulence was gradually reduced, and after 109 passages the revertant was totally avirulent. Virulence was not restored by 40 passages of the revertant through mouse peritoneum. The revertant grew less vigorously and did not infect mice when inoculated subcutaneously; in contrast, the parental form was found in all tissues examined. After phagocytosis in vitro or in vivo, the revertant survived but did not increase in numbers, whereas the parental form did increase in numbers. The revertant had a much smaller capsule than did the parental form. Immunization with the live revertant resulted in type-specific protection against infection with the parental form.

Almost any bacterium can be converted into the so-called L-form by removal of the cell wall (1) and can be maintained indefinitely on nutritive media, supplemented with osmotic stabilisers. L-form bacteria differ from their parent cell walled form, in their size, vacuolation and pleomorphic nature (Fig. 1). Here we describe the longterm association of L-form bacteria with plants and show that bacterial secondary metabolites are produced by the L-form in association with the plant.

The adherence of Yersinia pseudotuberculosis to the surface of HeLa cells at 4 degrees C was studied. This temperature allows adhesion of bacteria but prevents engulfment. Adhesion between the bacteria and the cells was not dependent upon the presence of serum, Ca2+ or Mg2+ in the medium. Maximum adhesion was obtained at pH 6.5-7.9 and pretreatment of the cells with formaldehyde or glutaraldehyde inhibited the attachment of the bacteria. The interaction between the bacteria and the cell surface seems to involve cellular processes that are mostly microvilli. An intimate association between the bacteria and the cellular glycocalyx was found. Three virulent bacterial strains adhered more easily to the cell surface than five avirulent strains. Maximum adherence was obtained with bacteria from late logarithmic and early stationary phases of growth. The bacteria gradually lose their adhesive property when cultivated for several generations at 37 degrees C in nutrient broth but not when cultivated at 20 degrees C. Treatment of the bacteria with protease IV from Streptomyces caespitosus markedly reduced the efficiency of attachment.

Within the context of a Workshop concerned With aspects of the environmental releases of genetically engineered organisms it is appropriate to consider the implications of an alternative means of “engineering” Whereby desired expressions are obtained by creating stable somatic associations and hence avoiding actual genetic manipulation.

Bacteria lacking peptidoglycan come into four main categories: Mycoplasmas, bacterial L-forms, halobacteria and methanogenic bacteria. The last-named were only recently identified as falling in this class, when Kandier and Hippe [14] showed that the walls of Methanosarcina barken and some other species lacked muramic acid, glucosamine and D-glutamic acid. The structural component of the wall of M. barkeri consisted of galactosamine, uronic acids, glucose and a little galactose and thus resembled that of Halococcus morrhuae (see below) except that it was not sulphated. Further evidence has now indicated that many of the Methanobacteria have walls containing ‘pseudomurein’ (see Chapter 6) and that they may be considered as a group along with the Halobacteria, Thermosplasma and Sulfolobus to form a separate kingdom of prokaryotes designated Archaebacteria [66].

more susceptible than are parent bacterial forms to certain antibiotics [1], to killing action of serum [2], and to staphylococcal a-toxin or streptolysin S [3]. The present study was designed to compare the effect of bacteriocin from Streptococcus faecalis on bacterial and L-forms of strains of 5. faecalis and unrelated microbial species. A few studies were done with strains of Staphylococcus aureus and their L-forms, and several gram-negative bacteria and L-forms.

L-forms have been obtained from many different bacterial species by a wide variety of techniques [1, 2] since the L-form phenomenon was first described by Kleineberger-Nobel [3]. Because many reports have concentrated on the morphology of L-forms, it seemed important to study the quantitative aspects of L-form induction, the degree to which specific factors incorporated in various media favor or inhibit growth of L-forms, and the growth characteristics of L-forms established on agar and in broth culture. This report will concentrate on these aspects of the L-form phenomena, summarizing observations on an Lform derived from Streptococcus liquefaciens, a group D streptococcus (isolated by the Microbiology Laboratory, Memorial Sloan-Kettering Cancer Center, and typed by the Streptococcus Unit, Laboratory Division, National Communicable Disease Center).

The streptococcal cell wall mucopeptide when injected into mice either intraperitoneally or intravenously enhances the resitance to subsequent challenge with virulent Group A streptococci. Rabbits which are injected intravenously with solubilized mucopeptide develop a fever response which has a resemblance to that achieved with endotoxin. Mice which survive 6 to 7 weeks after challenge with virulent Group A streptococci yield at autopsy search Group A streptococci serologically identical to the challenge organisms. A preparative dose of cell walls injected into mice prior to challenge diminished this late recovery of streptococci. Group A-variant streptococci were recovered from mice which survived challenge and carried the organisms for several weeks. Filterable bacterial forms, which grew on L form media, were recovered from infected mice. The serologic type of the L forms was identical to that of the challenge organisms.