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)

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.

Phenotypes were compared in two different classes of mutants with defects in murein-lipoprotein (IkyD mutants of Salmonella typhimurium and an lpo mutant of Escherichia coli). Both mutations are associated with the same triad of phenotypic abnormalities, consisting of defective formation ofthe division septum, leakage of periplasmic proteins during growth, and increased sensitivity to several unrelated external toxic agents. The abnormality in septum formation consists of a defect in invagination of the outer membrane during formation of the nascent septum. The results suggest that formation of the murein-lipoprotein link plays an important role in differentiation of the division septum and perhaps also in maintaining the normal barrier function of the outer membrane.

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in IkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983 [wt/vol]). After the cells were kept at room temperature for 3 min, they were fixed by the addition of glutaraldehyde to a final concentration of 2.5%. The suspension was allowed to stand for an additional 60 min at room temperature and centrifuged for 4 min at 14,000 x g, and the pellet was suspended in fresh medium (refractive index, 1.335).

Electron microscopy of plasmolyzed cells of Salmonella typhimurium revealed a continuous zone of membrane-murein attachment at the leading edge of the division septum at all stages of septal invagination. The membrane-murein attachment site had a characteristic ultrastructural appearance and remained as a bacterial birth scar at the new pole of each of the two daughter cells after cell separation. The continuous zone of membrane-murein attachment at the leading septal edge represents the second organelle based on a topologically ordered domain of membrane-murein adhesioh to be described at the site of cell division.Formation of the bacterial division septum occurs by the circumferential ingrowth of the cell envelope at the division site. In gram-negative bacteria, this formation requires the coordinate invagination of the three cell envelope layers, i.e., inner (cytoplasmic) membrane, murein, and outer membrane. The mechanism responsible for this topologically restricted differentiation is unknown.A new structure that appears at future division sites in gram-negative bacteria before the onset of septal invagination was recently described (4,8). The structure consists of two circumferential rings, the periseptal annuli, that flank the region of the cell envelope in which septal ingrowth will occur. Each annulus is a continuous zone where inner and outer membranes are closely apposed to the murein layer. It has been suggested (8) that the paired annuli define the periseptal domain and restrict essential elements of the division machinery to this location.In this paper, we describe a second division-related structure that is associated with ingrowth of the septum and that is also based on a topologically ordered domain of membrane-murein adhesion. The new structure consists of a continuous zone of membrane-murein attachment at the innermost edge of the nascent septum. After septal closure, the membrane-murein attachment site remains as a bacterial birth scar at the newly formed pole of each of the two daughter cells. MATERIALS AND METHODSSalmonella typhimurium SA534 (lkyD+), Rts34 [lkyD(Ts)] (5) and R71 (IkyD) (11) were grown in Proteose Peptone-beef extract medium (Difco Laboratories) containing 0.1 M NaCl to mid-exponential growth phase as previously described (5 30-min periods. The second method allows for a largely selective plasmolysis of the cell at the poles, while the first method results in general plasmolysis over the body of the cell and the poles (7,8,12).Unless otherwise noted, the final cell suspension was mixed with an equal volume of 4% glutaraldehyde in plasmolyzing buffer and immediately centrifuged, and the pellets were allowed to stand, without decanting of the supernatant solution, for 1.5 h at room temperature. The pellets were then washed three times with the plasmolyzing buffer, overlaid with 2% osmium tetroxide in the plasmolyzing buffer, and allowed to stand for an additional 1.5 h at room temperature. The cell pellets were then washed twice with distilled water, dehydrated through ethano...