Structure of Compositionally Simple Lipopolysaccharide from Marine Synechococcus

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-d-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.Lipopolysaccharide (LPS) in the outer membrane layer is known to be the first line of defense against environmental factors in many gram-negative organisms, preventing lysis by complement, antimicrobial peptides and detergents (17, 21, 47). In proteobacteria, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and phosphate are key parts of the conserved inner core of the LPS which connects the less-well-conserved outer core and sometimes an attached polysaccharide to the lipid A anchor. Why heptose is so well conserved is a mystery, but the prevalence of Kdo and phosphate may be related to the charge which they impart to the outer membrane and to their ability to bind divalent cations. The Kdo-phosphate metal binding center is capable of binding calcium with a dissociation constant (K_d) of 12 to 13 μM (28). This high-affinity binding of divalent cations is known to be necessary for the low permeability of LPS bilayers to some antibiotics (32), and it has been hypothesized that divalent cation cross-bridges may link LPS molecules on the bacterial cell surfaces of enterobacteria into a giant complex with very low membrane permeability (16).Though the LPSs of many proteobacteria are well characterized, the LPSs from cyanobacteria are much less studied. The cell envelopes of cyanobacteria resemble those of gram-negative bacteria structurally, consisting of a cytoplasmic membrane, a peptidoglycan layer, an outer membrane containing LPS, and sometimes additional structures (9, 14). Previous chemical analyses have shown the LPS of some cyanobacteria to be devoid of phosphate, Kdo, and heptose (11, 12, 42, 43). Given the lack of Kdo in these organisms as well as the fact that the lability of the Kdo-glucosamine ketosidic linkage allows for the mild acid hydrolysis of LPS to lipid A, it is perhaps not surprising that many attempts at hydrolysis of cyanobacterial LPS to lipid A have failed (for an example, see reference 29).Within the cyanobacteria, the genus Synechococcus represents a polyphyletic group of unicellular morphotypes. Synechococcus cells are found in both freshwater and marine environments. Organisms from group A Synechococcus and its sister taxon Prochlorococcus are extremely important primary producers in marine environments, with multiple “clades” similar to “species” described for other bacteria, dominating in different environments (3, 22). Unlike enterobacteria, which must frequently contend with an onslaught of host factors, members of the Synechococcus face grazing by protists and bacteriophages as their primary survival challenges.The genome of Synechococcus sp. strain CC9311 has been shown to be devoid of the genes for Kdo biosynthesis, while strain WH8102 has several putative genes for Kdo biosynthesis (18, 20). This suggests that the LPS of cyanobacteria could be significantly different from that of enteric bacteria and could show species/strain variation as well. A comparison of the structures of LPS from cyanobacteria and enterobacteria would afford a unique opportunity to understand which elements of LPS structure are essential to bacterial survival and which are adaptations to the environment in which the bacteria live. To further this understanding, we present here an analysis of the LPS structure from two strains of marine Synechococcus: an open-ocean-dwelling strain having the putative genes for Kdo biosynthesis (strain WH8102; clade III) and a coastal strain lacking these genes (strain CC9311; clade I). We further present two novel methods for producing lipid A from bacteria lacking the labile Kdo ketosidic linkage.