Gram-negative cell walls are strong enough to withstand 3 atm of turgor pressure (40), tough enough to endure extreme temperatures and pHs (eg, Thiobacillus ferrooxidans grows at a pH of 1.5) and elastic enough to be capable of expanding several times their normal surface area (41). Strong, tough, and elastic... the gram-negative cell wall is a remarkable structure which protects the contents of the cell and which has stood the test of time for many, many years. Presumably, these three descriptive traits, have much to do with the tremendous success gram-negative bacteria have had as a life-form on our planet; members of the domain Bacteria inhabit almost all imaginable habitats except those excruciatingly extreme environments in which (some) members of the domain Archaea thrive. Molecular biological methods have not yet given scientists a precise historical record of the origin of gram-negative bacteria, but ancient stromatolites containing fossilized remains of cyanobacterium-like prokaryotes date back to the Archean eon. Over such extraordinary periods of time (much of it when no other life existed), we can imagine that random mutation, selection, and the slowly but ever-changing global environment gave rise to two fundamentally different cell wall formats in Bacteria; gram-positive and gram-negative varieties. Gram-positive cell walls, once thought to be relatively simple structural entities, can be quite different from one another, especially when cell wall turnover is taken into account (8, 9, 25, 29). The cell walls of gram-negative bacteria follow a more general structural format than that of gram-positive bacteria, which is strictly adhered to; gram-negative bacteria have an outer membrane situated above a thin peptidoglycan layer. Sandwiched between the outer membrane and the plasma membrane, a concentrated gel-like matrix (the periplasm) is found in the periplasmic space (7, 9). Because the periplasm exists above the plasma membrane, it is not part of the protoplast, and because the periplasm is differentiated from the external environment by the outer membrane, it is not part of the “outside.” It is in fact an integral compartment of the gram-negative cell wall (5). Together the plasma membrane and the cell wall (outer membrane, peptidoglycan layer, and periplasm) constitute the gram-negative envelope (5, 9). Our entire perception of gram-positive and gram-negative walls ultimately relies on the response of bacteria to Gram staining. Unwittingly, in 1884, Christian Gram developed a staining regimen for light microscopy which differentiated between these two types of bacteria because of the chemical composition and structural format of their cell walls. Because gram-negative bacteria possess a lipid-rich outer membrane (as well as a plasma membrane) and a thin peptidoglycan layer, the alcohol decolorizing step of Gram staining washes the primary stain (crystal violet) from the cells and the secondary stain (carbol fuchsin or saffranin) colors the bacteria red (57). Gram-positive bacteria are enshrouded in thicker, more resilient cell walls which do not allow the crystal violet to be removed and, accordingly, remain purple (57). Although the vast majority of bacteria adhere to the color differentiation of the Gram stain, to the chagrin of microbiological taxonomists, some bacteria refuse to obey; these are called gram-variable bacteria (6). Members of the Archaea cannot be easily differentiated by Gram staining (10). Interestingly, the staining response of gram-variable bacteria and archaea is also due to their cell wall composition and structure (6, 10). Advances in identifying gram-negative cell wall components, their cytoplasmic synthetic and plasma …