Global and Targeted Lipid Analysis of Gemmata obscuriglobus Reveals the Presence of Lipopolysaccharide, a Signature of the Classical Gram-Negative Outer Membrane

Abstract

Planctomycete bacteria possess many unusual cellular properties, contributing to a cell plan long considered to be unique among the bacteria. However, data from recent studies are more consistent with a modified Gram-negative cell plan. A key feature of the Gram-negative plan is the presence of an outer membrane (OM), for which lipopolysaccharide (LPS) is a signature molecule. Despite genomic evidence for an OM in planctomycetes, no biochemical verification has been reported. We attempted to detect and characterize LPS in the planctomycete Gemmata obscuriglobus. We obtained direct evidence for LPS and lipid A using electrophoresis and differential staining. Gas chromatography-mass spectrometry (GC-MS) compositional analysis of LPS extracts identified eight different 3-hydroxy fatty acids (3-HOFAs), 2-keto 3-deoxy-D-manno-octulosonic acid (Kdo), glucosamine, and hexose and heptose sugars, a chemical profile unique to Gram-negative LPS. Combined with molecular/structural information collected from matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS analysis of putative intact lipid A, these data led us to propose a heterogeneous hexa-acylated lipid A structure (multiple-lipid A species). We also confirmed previous reports of G. obscuriglobus whole-cell fatty acid (FA) and sterol compositions and detected a novel polyunsaturated FA (PUFA). Our confirmation of LPS, and by implication an OM, in G. obscuriglobus raises the possibility that other planctomycetes possess an OM. The pursuit of this question, together with studies of the structural connections between planctomycete LPS and peptidoglycans, will shed more light on what appears to be a planctomycete variation on the Gram-negative cell plan. IMPORTANCEBacterial species are classified as Gram positive or negative based on their cell envelope structure. For 25 years, the envelope of planctomycete bacteria has been considered a unique exception, as it lacks peptidoglycan and an outer membrane (OM). However, the very recent detection of peptidoglycan in planctomycete species has provided evidence for a more conventional cell wall and raised questions about other elements of the cell envelope. Here, we report direct evidence of lipopolysaccharide in the planctomycete G. obscuriglobus, suggesting the presence of an OM and supporting the proposal that the planctomycete cell envelope is an extension of the canonical Gram-negative plan. This interpretation features a convoluted cytoplasmic membrane and expanded periplasmic space, the functions of which provide an intriguing avenue for future investigation.T he planctomycete bacterium Gemmata obscuriglobus (1) is of considerable interest to the fields of cell and evolutionary biology due to its uncommon cellular features and evolutionary placement within the Bacteria (2-5). G. obscuriglobus possesses an extensive endomembrane system (6) with superficial eukaryotelike properties, including the presence of membrane coat-like (MC-like) proteins (7), large quantities of sterols (8, 9), a propos...

Determining the bacterial cell biology of Planctomycetes

“…Both methods revealed proteins typical for the outer membrane of Gram-negative bacteria such as porins, as well as typical periplasmic proteins and proteins that are associated with the cytoplasmic membrane (Supplementary Table 2). Our findings are also in line with the recent report of lipid A in Planctomycetes24, a key component of lipopolysaccharides in outer membranes.…”

“…Peptidoglycan—the hallmark of free-living bacteria—has been found in several Planctomycetes2122. Recent bioinformatic and chemical analyses support a more typical Gram-negative cell plan than previously thought2324. However, to the best of our knowledge, endocytosis-like uptake has not been experimentally tested again after the initial report16.…”

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship

“…[112] Representative lipid A structures for V. fischeri and B. bacteriovorus are shown in Figure 4. In both cases, the structures represented are for the base peak in the single stage scan mass spectrum.…”

“…Planctomycetes are unique prokaryotes that contain both genotypic and phenotypic characteristics of eukaryotic cells. G. obscuriglobus is the first organism of its phylum to have a putative lipid A structure reported[112], as can be seen in Figure 5A. Most notably, the lipid A structure is mono-phosphorylated at C-1, with a GalA residue modification at the C-4’ position.…”

“…The ester-linked, β-hydroxyl-substituted myristate group at the C-2’ position is a unique finding among Gram-negative bacteria. [112] Synechococcus sp. CC9311 produces a rudimentary, tetra-acylated lipid A with no phosphate substitutions.…”