Six mutants of Salmonella typhimurium LT2 with defects in the heptose region of the lipopolysaccharide (LPS) ("rough" mutants) were more sensitive to the growth-inhibitory effects of erythromycin, bacitracin, vancomycin, novobiocin, kanamycin, and cloxacillin and of deoxycholate than smooth strains, but less sensitive to tetracycline and ampicillin. In general, growth of the three rough mutants of chemotype Rd2, which lack the distal but not the proximal heptose unit in the LPS, was less inhibited than the three mutants of chemotype Re, which are heptose-deficient. In addition, inhibition of uracil-1-14C incorporation in the presence of actinomycin D and spheroplast formation in the presence of lysozyme occurred in the rough mutants without ethylenediaminetetraacetate (EDTA) treatment of the cells, while actinomycin D and lysozyme were effective on smooth strains only after EDTA treatment. Since the major part of the LPS is in the outer membrane of the cell envelope, and since the target of the toxic agents used is located inside this layer, these data indicate that the carbohydrate part of the LPS component of the outer membrane is an essential part of a barrier layer preventing penetration of large molecules.

Small, acid-soluble spore proteins SASP-oa, SASP-,, and SASP--y as well as a SASP-j-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The aJ$-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-y was somewhat excluded from the nucleoid. These different locations of oda,-type and -y-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.Approximately 10% of the protein of dormant spores of Bacillus ,subtilis is composed of a group of small, acidsoluble spore proteins (SASP) (18). The SASP are synthesized in parallel during sporulation within the developing forespore and are rapidly degraded during spore germination, thus providing amino acids for protein synthesis during this period of development. Three proteins, termed SASP-a, SASP-1, and SASP-y, make up approximately 75% of the SASP pool. SASP-a and SASP-3 are almost identical in primary sequence, and there are a number of minor SASP with primary sequences similar to those of SASP-cx and SASP-P. In contrast, SASP-,y has a more different primary sequence, and there is only a single y-type SASP (18).Other than its role in providing amino acids for protein synthesis during spore germination, SASP--y appears to have no function in spores (6). However, the a/,B-type SASP also play a key role in the resistance of spores to UV light, as they are intimately involved in the modification of the UV photochemistry of spore DNA which is essential for spore UV resistance (11,12,14). While the. mechanism whereby a/p-type SASP affect spore DNA photochemistry is not known, it seems likely that it involves direct SASP-DNA interactions, and it is known that SASP are localiied within the spore core, the site of spore DNA (18). SASP can also bind to DNA in vitro (18). However, this binding is weak, and attempts to isolate spore DNA or spore nucleoids with significant associated SASP have failed (17). One study using intact Bacillus megaterium spores and UV-induced protein-DNA cross-linking did provide evidence that significant ao/-type SASP was associated with spore DNA in vivo (15). However, from this study it was not possible to determine what percentage of these SASP were DNA associated.Because of the limitations of these previous techniques, we decided to attempt to localize various SASP within B. subtilis spores and forespores by using immunoelectron microscopy. However, initial attempts to localize SASP in dormant spores by using this technique were unsuccessful, since the cross-linking agents used in fixation (paraformaldehyde with or without glutaraldehyde) did not penetrate dormant spores sufficiently to prevent SASP movement during subsequent steps. The lack of penetration of dormant spores by cross-linking agents is not surprising in light of * Corresponding author. what is known of dormant-spore permeability (5) as well as the resi...

Evidence is presented that the site of cell division in Salmonella typhimurium is flanked by two circumferential zones of cell envelope differentiation, the periseptal annuli, which separate the division site from the remainder of the cell envelope. Each annulus is composed of a continuous structure in which the membranous elements of the cell envelope are closely associated with, the murein cytoskeleton. The paired annuli appear early in the division process and the region between them defines a new cellular domain, the.periseptal compartment, within which the division septum is formed.The process of bacterial cell division occurs by ingrowth-of the cell envelope from a narrow circumferential zone at the midpoint of the cell. This leads to segregation of the cytoplasm into two compartments and finally to separation of the daughter cells. Ingrowth of the new septum at the proper location requires that the molecular organization of the division site differ from the organization of the envelope over-the remainder of the cell. The nature of this local differentiation and the cellular mechanisms that initiate and maintain it at the proper location are not known.In this paper we describe an organelle that is associated with early stages of the division process in Gram-negative bacteria and that is likely to play a role in these events. Fixation (1.5 hr) and subsequent washing and postfixation with 2.0% OS04, were carried out in the presence of the respective plasmolyzing solutions. In the experiment described in Fig. 3, the cells were subsequently washed twice more with distilled water and resuspended for 20 min in a saturated aqueous solution of thiocarbohydrazide (Sigma) to enhance contrast in the final sections; excess thiocarbohydrazide was removed by two washes with distilled water and the cells were again exposed to 2% OsO4 prior to embedding. Embedding, sectioning, and counterstaining with lead citrate and uranyl acetate were performed as described (2). Serial. sections approximately 60 nm thick were mounted on carbon-backed Formvar-coated slotted (2 X 1 mm) copper grids and examined in a Hitachi HUIIE electron microscope at 75 kV accelerating voltage. RESULTS The cell envelopes of Gram-negative bacteria contain two membranes which completely surround the cell. Between the inner (cytoplasmic) and outer membranes lies the continuous murein layer, a rigid crosslinked peptidoglycan that provides the only known cytoskeletal structure of these and other bacteria (3). During normal cell division the three layers invaginate coordinately to form the new septum (Fig. la). METHODSWhen cells are plasmolyzed by brief exposure to hypertonic solutions, the resulting decrease in cytoplasmic volume causes the inner membrane to shrink away from the rigid murein/ outer membrane layer. As originally shown by Bayer (4, 5), the plasmolysis procedure reveals the presence of small zones where the cytoplasmic membrane fails -to pull away from the murein/outer membrane layer. These sites of membrane adhesion are thought to re...

Some Streptococcus mutans strains change shape from bacillary to coccal or ellipsoid form in response to the ratio of bicarbonate to potassium or of borate to potassium in growth media. So that insight into determinants of shape of these streptococci could be gained, and future genetic studies facilitated, the shapes of a series of transformable and nontransformable strains of S. mutans were studied and attempts made to isolate a mutant of augmented transformability. Several strains were mutagenized by ethylmethane sulfonate and mutants with altered colonial and cellular morphologies isolated. Cell shapes were studied by Gram stain and Nomarski interference microscopy, and by scanning and transmission electron microscopy. Diverse shape-altered mutants were isolated from seven transformable and two nontransformable strains of S. mutans. Among these, length-to-width ratios ranged from > 10 to about 0.25. Regulation of timing of cell division, septum formation, or septum completion events may have been altered in these mutants. While most mutants substantially or completely lost transformability, mutant LT11 had transformation efficiency of 1.3 x 10(-4) to 2.3 x 10(-3), more than two to three orders of magnitude greater than its parental UA159 and the well-known transformable strain GS5(HK), respectively. There was no evidence of production of competence factor by LT11. Competence of LT11 was maintained for at least six months upon storage at -70 degrees C, facilitating its use for genetic studies. While the morphologies of several shape-altered mutants were no longer responsive to changes of the bicarbonate/potassium, unlike those of their parentals, the morphology of LT11 persisted in its response to this condition.(ABSTRACT TRUNCATED AT 250 WORDS)