both sphingomyelin monolayers and monolayers of glycan-modified lipid A. Furthermore, both diplopterol and cholesterol could rescue pH-dependent phase transitions in glycan-modified lipid A monolayers. The role of hopanoids in membrane-mediated acid tolerance is further supported by observations of acid-inhibited growth and morphological abnormalities of the plasma membrane in hopanoid-deficient bacteria with mutant squalene-hopene cyclases. Hopanoids are produced in several nitrogen-fixing bacteria. In the actinomycete Frankia, hopanoids in the membranes of vesicles specialized for nitrogen fixation likely restrict the entry of oxygen by making the lipid bilayer more tight and compact. In Bradyrhizobium, hopanoids chemically bonded to lipid A increase membrane stability and rigidity, enhancing stress tolerance and intracellular survival in Aeschynomene legumes. In the cyanobacterium Nostoc punctiforme, large quantities of 2-methylhopanoids localize to the outer membranes of survival structures called akinetes. In another example of stress tolerance, hopanoids in the aerial hyphae (spore bearing structures) of the prokaryotic soil bacteria Streptomyces are thought to minimize water loss across the membrane to the air. Biosynthesis Squalene synthesis Since hopanoids are a C30 terpenoid, biosynthesis begins with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAP), which are combined to form longer chain isoprenoids. Synthesis of these smaller precursors proceeds either via the mevalonate pathway or the methylerythritol-4-phosphate pathway depending on the bacterial species, although the latter tends to be more common. DMAP condenses with one molecule of IPP to geranyl pyrophosphate, which in turn condenses with another IPP to generate farnesyl pyrophosphate (FPP). Squalene synthase, coded for by the gene sqs, then catalyzes the condensation of two FPP molecules to presqualene pyrophosphate (PSPP) before oxidizing NADPH to release squalene. However, some hopanoid-producing bacteria lack squalene synthase and instead use the three enzymes HpnC, HpnD and HpnE, which are encoded in the hpn operon with many other hopanoid biosynthesis genes. In this alternative yet seemingly more widespread squalene synthesis pathway, HpnD releases pyrophosphate as it condenses two molecules of FPP to PSPP, which HpnC converts to hydroxysqualene, consuming a water molecule and releasing another pyrophosphate. Then, hydroxysqualene is reduced to squalene in a dehydration reaction mediated by the FAD-dependent enzyme HpnE. Cyclization Next, a squalene-hopene cyclase catalyzes an elaborate cyclization reaction, engaging squalene in an energetically favorable all-chair conformation before creating 5 cycles, 6 covalent bonds, and 9 chiral centers on the molecule in a single step. This enzyme, coded for by the gene shc (also called hpnF in some bacteria), has a double