Manmilan Singh

The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

ABSTRACT When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then “boosted” after 2 days with additional acetate, the cells become “obese” after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of “sensitive” genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes—encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)—are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the “sensitive” genes are strongly responsive to the cells acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

Lactate metabolism is associated with mammalian mitochondria

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. Within the fermenting cell itself, however, it is generally assumed that lactate is produced to replenish NAD+ and then is secreted. Here we explored the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. With high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.

Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating Fibroblasts

Cellular proliferation requires the formation of new membranes. It is often assumed that the lipids needed for these membranes are synthesized mostly de novo. Here, we show that proliferating fibroblasts prefer to take up palmitate from the extracellular environment over synthesizing it de novo. Relative to quiescent fibroblasts, proliferating fibroblasts increase their uptake of palmitate, decrease fatty acid degradation, and instead direct more palmitate to membrane lipids. When exogenous palmitate is provided in the culture media at physiological concentrations, de novo synthesis accounts for only a minor fraction of intracellular palmitate in proliferating fibroblasts as well as proliferating HeLa and H460 cells. Blocking fatty acid uptake decreased the proliferation rate of fibroblasts, HeLa, and H460 cells, while supplementing media with exogenous palmitate resulted in decreased glucose uptake and rendered cells less sensitive to glycolytic inhibition. Our results suggest that cells scavenging exogenous lipids may be less susceptible to drugs targeting glycolysis and de novo lipid synthesis.

Staphylococcus aureus Peptidoglycan Tertiary Structure from Carbon-13 Spin Diffusion

The cell-wall peptidoglycan of Staphylococcus aureus is a heterogeneous, highly cross-linked polymer of unknown tertiary structure. We have partially characterized this structure by measuring spin diffusion from (13)C labels in pentaglycyl cross-linking segments to natural-abundance (13)C in the surrounding intact cell walls. The measurements were performed using a version of centerband-only detection of exchange (CODEX). The cell walls were isolated from S. aureus grown in media containing [1-(13)C]glycine. The CODEX spin diffusion rates established that the pentaglycyl bridge of one peptidoglycan repeat unit of S. aureus is within 5 A of the glycan chain of another repeat unit. This surprising proximity is interpreted in terms of a model for the peptidoglycan lattice in which all peptide stems in a plane perpendicular to the glycan mainchain are parallel to one another.

Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR.

Peptidoglycan is an essential component of cell wall in Gram-positive bacteria with unknown architecture. In this review, we summarize solid-state NMR approaches to address some of the unknowns in the Gram-positive bacteria peptidoglycan architecture: 1) peptidoglycan backbone conformation, 2) PG-lattice structure, 3) variations in the peptidoglycan architecture and composition, 4) the effects of peptidoglycan bridge-length on the peptidoglycan architecture in Fem mutants, 5) the orientation of glycan strands with respect to the membrane, and 6) the relationship between the peptidoglycan structure and the glycopeptide antibiotic mode of action. Solid-state NMR analyses of Staphylococcus aureus cell wall show that peptidoglycan chains are surprisingly ordered and densely packed. The peptidoglycan disaccharide backbone adopts 4-fold screw helical symmetry with the disaccharide unit periodicity of 40Å. Peptidoglycan lattice in the S. aureus cell wall is formed by cross-linked PG stems that have parallel orientations. The structural characterization of Fem-mutants of S. aureus with varying lengths of bridge structures suggests that the PG-bridge length is an important determining factor for the PG architecture.

Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Staphylococcus aureus peptidoglycan stem packing by rotational-echo double resonance NMR spectroscopy.

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine and l-[(15)N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between (13)C and (15)N of labeled amino acids incorporated in the peptidoglycan, and from those labels to (19)F of a glycopeptide drug specifically bound to the peptidoglycan. The observed (13)C-(15)N average distance of 4.1-4.4 Å between d- and l-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.

Cross-Link Formation and Peptidoglycan Lattice Assembly in the FemA Mutant of Staphylococcus aureus

Staphylococcus aureus FemA mutant grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-13C]alanine, l-[3-13C]alanine, [2-13C]glycine, and l-[5-19F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between 13C-labeled alanines and 19F-labeled lysine incorporated in the peptidoglycan. The alanyl 13C labels were preselected for REDOR measurement by their proximity to the glycine label using 13C–13C spin diffusion. The observed 13C–13C and 13C–19F distances are consistent with a tightly packed, hybrid architecture containing both parallel and perpendicular stems in a repeating structural motif within the peptidoglycan.

The Isotridecanyl Side Chain of Plusbacin-A3 Is Essential for the Transglycosylase Inhibition of Peptidoglycan Biosynthesis

Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm, and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer, thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [(2)H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [(2)H]pb-A3 has an isotopically (2)H-labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [(2)H]pb-A3 bound to [1-(13)C]glycine-labeled whole cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [(2)H]pb-A3 during the growth of S. aureus resulted in the accumulation of Parks nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.

REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant.

The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-¹³C]alanine, l-[3-¹³C]alanine, [2-¹³C]glycine, and l-[5-¹⁹F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between ¹³C-labeled alanines and ¹⁹F-labeled lysine incorporated in the peptidoglycan. The alanyl ¹³C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using ¹³C¹³C spin diffusion. The observed ¹³C¹³C and ¹³C³¹P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by ¹³C¹³C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-¹³C]alanine and l-[¹⁵N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an ¹⁹F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel-perpendicular mixed-geometry model for the FemA peptidoglycan lattice.