Bacillus subtilis Lipid Extract, A Branched-Chain Fatty Acid Model

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The Bacillus Subtilis Lipid Extract, A Branched-Chain Fatty Acid Model Membrane Jonathan D. Nickels, Sneha Chatterjee, Barmak Mostofian, Christopher B. Stanley, Michael Ohl, Piotr Zolnierczuk, Roland Schulz, Dean A. A. Myles, Robert F. Standaert, James Elkins, Xiaolin Cheng, and John Katsaras J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b01877 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

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The Journal of Physical Chemistry Letters

The Bacillus subtilis Lipid Extract, A Branched-Chain Fatty Acid Model Membrane Jonathan D. Nickels1,2,5,*, Sneha Chatterjee2, Barmak Mostofian3, Christopher B. Stanley2, Michael Ohl8, Piotr Zolnierczuk8, Roland Schulz9, Dean A.A. Myles2, Robert F. Standaert1,2,7, James G. Elkins7,8, Xiaolin Cheng3,7,*, John Katsaras1,2,5. 1Shull Wollan Center: A Joint Institute for Neutron Sciences, 2Biology and Soft Matter Division, 3Center for Molecular Biophysics, 4Biosciences Division, Oak Ridge National Laboratory, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America. 5Department of Physics & Astronomy, 6 Department of Biochemistry & Cellular and Molecular Biology, 7Department of Microbiology, University of Tennessee, Knoxville, Tennessee, United States of America. 8Jülich Center for Neutron Science, Forschungszentrum Juelich GmbH, Outstation at SNS, Oak Ridge, Tennessee, USA 9 Intel Corporation, Hillsboro, Oregon, United States of America.

ABSTRACT: Lipid extracts are an excellent choice of model biomembrane, however at present, there are no commercially available lipid extracts or computational models that mimic microbial membranes containing the branched-chain fatty acids found in many pathogenic and industrially relevant bacteria. We advance the extract of Bacillus subtilis as a standard model for these diverse systems, providing a detailed experimental description and equilibrated atomistic bilayer model included as supporting information to this manuscript and at (http://cmb.ornl.gov/members/cheng). The development and validation of this model represents an advance that enables more realistic simulations, and experiments, on bacterial membranes and reconstituted bacterial membrane proteins.

Natural lipid extracts are a popular choice as a membrane mimic for a wide range of structural, biochemical and biophysical studies. From determining the effects of small molecule additives in bilayers, to stabilizing membrane proteins, lipid extracts have wide-ranging utility. Lipid extracts pro1 vide the diverse composition of natural bilayers , though it is important to keep in mind that no extract fully captures the complexity found in living systems, such as lateral organiza2,3 4-6 tion and compositional asymmetry . A number of extracts are available commercially, including those from Escherichia coli, yeast, porcine brain, bovine heart and liver, chicken egg, and soybean. Unfortunately, none of these commercially

available extracts contain branched-chain fatty acids, such as those found in Bacillus subtilis and many pathogenic bacteria, including Clostridium difficile, Listeria monocytogenes, 7,8 Staphylococcus aureus, Legionella pneumophila and others . Branching is thought to regulate membrane fluidity in these organisms analogous to unsaturation in the membranes of animals, plants and other bacteria. Branched-chain fatty acids are also found in a wide number of organisms used in the bioprocessing of lignocellulose, such as Clostridium thermocellum, Bacillus macerans, and 9 certain strains of Pseudomonas ; as well as organisms utilized 10-12 in the industrial production of enzymes . This identifies a widespread role for the B. subtilis extract as a membrane mimic including branched-chain fatty acids. It is also worth noting is that B. subtilis is the dominant model system for Gram-positive bacteria, analogous to E. coli for Gramnegative bacteria. B. subtilis was also the first Gram-positive 13 bacterium to be genetically sequenced and is widely used as 14 a model organism to study biofilms , sporulation and mor15,16 3 phogenesis , and lipid bilayer organization . Here, we present a physio-chemical characterization of B. subtilis extract bilayers, then use these results to inform and validate an all-atom bilayer model of the B. subtilis lipid extract, the structure of which was equilibrated for over 1 μs. The extract can be produced using a total lipid extraction of 19 B. subtilis strain 168 cells using a modification of the Bligh 20 and Dyer method. The composition of the B. subtilis lipid extract contains phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL) and lysyl-PG lipids, in addition to approximately 30 percent neutral lipids (mostly di17,18,21,22 acylglycerol) (Figure 1). These lipids include seven fatty acid species identified from gas chromatography / mass spectroscopy analysis of the acid-catalyzed methanolysis of the extract (see Figure S1). The extract contained a distribution of saturated branched (iso- and anteiso-) and unbranched (normal-) fatty acids, consistent with prior charac7,17 terization (Figure 2). An atomistic bilayer model of the B. subtilis membrane with 420 lipids per leaflet was constructed according to the

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tions

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the

Figure 1. B. subtilis extract composition – Head Groups. (A) Structure of the four common lipid head groups found in the B. 17,18 subtilis extract . (B) The relative abundance of lipid head groups is shown, color coded with the MD snapshot in panel (C).

Figure 2. B. subtilis extract composition – fatty acids. (A) Structure of the seven main fatty acids found in the B. subtilis extract. The fatty acids are all saturated and predominantly have branched-chains, containing 14 to 17 carbon atoms. (B) The relative abundance of each fatty acid is shown, color coded with the MD snapshot in panel (C).


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Figure 3. Experimental and computational characterization of the B. subtilis extract. (A) Cross-section depicting the transverse structure of the extract from MD simulation at 37 ºC. The structure has been parsed into three categories, hydrophobic (CH3, CH2, and CH), head group (C=O, glycerol, etc.), and water (middle panel). In the lower panel, the scattering length density, ρ, has been computed from the averaged bilayer structure and is presented in comparison with the scattering length density profile extracted from analysis of the small angel neutron scattering measurements, shown in (B). In (B), the lines represent joint fits to the two independent datasets (details provided in the Supporting Information and Tables S1, S2 and S3). (C) Area per lipid (APL) and acyl thickness (Dc) are reported as a function of temperature, illustrating structural changes above 17 ºC, in agreement with the melting transition observed experimentally at 18.2 ºC. (D) and (E) The bending modulus, κ, was calculated from experimental observations of the intermediate scattering function, S(q,τ)/S(q,0). These data were fit using a decay 24-26 constant, Γ, describing the thermal fluctuations of the bilayer using the approach of Zilman and Granek, which are predicted 3 to be proportional to q . From this we obtained a value of κ=29.1 ± 5.3 kBT, consistent with a membrane in the fluid state. bilayer model, solvated in explicit water, were performed for over 1 μs at seven temperatures ranging from 2 °C to 37 °C, at 5 °C intervals. All simulations were performed using 23 GROMACS , as optimized for the Intel® Xeon Phi™ coprocessors at the National Energy Research Scientific Computing Center (NERSC). The details of the equilibrated MD extract bilayer are described in Figure 3, along with a characterization of the physical B. subtilis extract. The MD simulations were able to capture the temperature-dependent behavior of the physical extract, exhibiting the structural signatures of gel to fluid phase transitions (Figure 3D); namely, an increase in the area per lipid, APL, and decrease in the hydrophobic thickness, 2Dc, in close agreement with the experimentally observed melting transition (Tm = 18.2 ± 0.04 °C) (Figure S2). Focusing then on the fluid-phase bilayer at 37 °C, we present a detailed structural description, bringing together analysis of the MD simulations (Figure 3A) and the results of neutron scattering studies performed on the physical extract (Figure 3B). Small angle neutron scattering (SANS) is a structural technique sensitive to length scales from ~10 to 1000 Å, and is particularly useful in studies of hydrogen rich systems, like lipid bilayers, because of its sensitivity to the isotopes of hydrogen. The neutron scattering length density, ρ, (a function of atomic composition and molecular volume) can be directly calculated from the simulations and compared to

experimental results in Figure 3A, serving to validate the MD model (for details, see Supplementary Information and Tables S1 to S3). Subsequent analysis yields a number of physically meaningful parameters such as the area per lipid, APL, 2 which we estimated from the scattering results as 65.0 Å . This is in close agreement with the MD-derived value of 2 (67.0 Å ). The hydrophobic thickness, 2Dc, was estimated as 27.8 ± 1.0 Å from the scattering results, which matched well to the spacing between the carbonyl groups of the apposed leaflets rather than that the strict hydrophobic thickness defined between C2 carbons (23.9 ± 1.0 Å, see Figure S3). This difference emerges from the averaging in the z-axis that occurs in the slab model used to fit ρ from the experimental results. The head group region was found to contain 11.6 ± 1.7 water molecules per lipid from scattering studies, again in good agreement with the MD estimate of 11.4 molecules of water per lipid. The bending modulus, κ, of the extract bilayer was also measured using the neutron spin-echo technique which measures the thermal undulations of the bilayer (Figure 3D and 3E), yielding a bending modulus, κ = 29.1 ± 5.3 kBT. This compares favorably to an estimate from the simula27 tions based on the splay and tilt of the lipid tails which returns a value of 16 ± 1 kBT, slightly softer than the experimental result, though both values indicate a fluid phase bilayer, consistent with previously reported values from model 26,28 systems .



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The lipid extract of B. subtilis addresses the current need for a lipid extract model with branched-chain fatty acids. B. subtilis is straightforward to cultivate and process, making the lipid extract of B. subtilis a logical choice of model on the benchtop. We present a characterization and procedure for producing the physical extract, and use it to validate an equilibrated and publically available computational model of the extract (available as supporting information to this manuscript). Access to a more realistic bilayer will facilitate increasingly relevant investigations of cell membrane structure, as well as providing a crucial testbed for membrane integral and membrane associated proteins. We provide the quantitative tools needed to utilize the either the B. subtilis lipid extract or the associated computational model in a range of future in vitro and in silico studies where branchedchain fatty acid lipids are appropriate. Taken together, this is a significant advance in the toolkit for understanding the physical chemistry of bacterial lipid membranes and their embedded proteins.

ASSOCIATED CONTENT Supporting Information The Supporting Information contains the files needed to employ the computational model of the B. subtilis lipid extract; in addition to a document containing the Materials and Methods, three Supporting Figures, three Supporting Tables, and extended discussion of the experimental data treatment. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Jonathan D. Nickels Department of Chemical and Environmental Engineering University of Cincinnati, Cincinnati, OH 45221. [email protected] *Xiaolin Cheng Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy The Ohio State University, Columbus, OH 43210. [email protected]

ACKNOWLEDGMENT The authors would like to acknowledge M. Cochran and C. Gao for technical assistance. This research was sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), managed by UTBattelle, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC05-00OR22725, with support for J.K. provided by the DOE Office of Basic Energy Sciences, Scientific User Facilities Division. Small-angle neutron scattering was performed at ORNL using the EQ-SANS instrument at the Spallation Neutron Source, supported by the DOE Office of Basic Energy Sciences, Scientific User Facilities Division. This research was partially supported by an ASCR Leadership Computing Challenge (ALCC) award, and used resources of the Oak Ridge Leadership Computing Facility at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. This research also used resources

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of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

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(23) Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D. GROMACS 4.5: A High-Throughput and Highly Parallel Open Source Molecular Simulation Toolkit. Bioinformatics 2013, btt055. (24) Zilman, A.; Granek, R. Undulations and Dynamic Structure Factor of Membranes. Phys. Rev. Lett. 1996, 77, 4788. (25) Watson, M. C.; Brown, F. L. Interpreting Membrane Scattering Experiments at the Mesoscale: The Contribution of Dissipation Within the Bilayer. Biophys. J. 2010, 98, L9-L11.

(26) Woodka, A. C.; Butler, P. D.; Porcar, L.; Farago, B.; Nagao, M. Lipid Bilayers and Membrane Dynamics: Insight into Thickness Fluctuations. Phys. Rev. Lett. 2012, 109, 058102. (27) Khelashvili, G.; Kollmitzer, B.; Heftberger, P.; Pabst, G.; Harries, D. Calculating the Bending Modulus for Multicomponent Lipid Membranes in Different Thermodynamic Phases. J. Chem. Theory. Comput. 2013, 9, 3866. (28) Nickels, J. D.; Cheng, X.; Mostofian, B.; Stanley, C.; Lindner, B.; Heberle, F. A.; Perticaroli, S.; Feygenson, M.; Egami, T.; Standaert, R. F. et al. Mechanical Properties of Nanoscopic Lipid Domains. J. Am. Chem. Soc. 2015, 137, 15772.



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Figure 1. B. subtilis extract composition – Head Groups. (A) Structure of the four common lipid head groups found in the B. subtilis extract17,18. (B) The relative abundance of lipid head groups is shown, color coded with the MD snapshot in panel (C). 190x108mm (300 x 300 DPI)


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Figure 2. B. subtilis extract composition – fatty acids. (A) Structure of the seven main fatty acids found in the B. subtilis extract. The fatty acids are all saturated and predominantly have branched-chains, containing 14 to 17 carbon atoms. (B) The relative abundance of each fatty acid is shown, color coded with the MD snapshot in panel (C). 176x96mm (300 x 300 DPI)



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Figure 3. Experimental and computational characterization of the B. subtilis extract. (A) Crosssection depicting the transverse structure of the extract from MD simulation at 37 ºC. The structure has been parsed into three categories, hydrophobic (CH3, CH2, and CH), head group (C=O, glycerol, etc.), and water (middle panel). In the lower panel, the scattering length density, ρ, has been computed from the averaged bilayer structure and is presented in comparison with the scattering length density profile extracted from analysis of the small angel neutron scattering measurements, shown in (B). In (B), the lines represent joint fits to the two independent datasets (details provided in the Supporting Information and Tables S1, S2 and S3). (C) Area per lipid (APL) and acyl thickness (Dc) are reported as a function of temperature, illustrating structural changes above 17 ºC, in agreement with the melting transition observed experimentally at 18.2 ºC. (D) and (E) The bending modulus, κ, was calculated from experimental observations of the intermediate scattering function, S(q,τ)/S(q,0). These data were fit using a decay constant, Γ, describing the thermal fluctuations of the bilayer using the approach of Zilman and Granek,25-27 which are predicted to be proportional to q3. From this we obtained a value of κ=29.1 ± 5.3 kBT, consistent with a membrane in the fluid state. 169x85mm (300 x 300 DPI)


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Bacillus subtilis Lipid Extract, A Branched-Chain Fatty Acid Model

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