All samples were collected from Salt Pond, located near the northeastern end of San Salvador Island, Bahamas (24o05' N, 74o30' W). Intact mat samples (6 x 6 cm) were collected to a depth of approximately 2 cm and then returned to the Grice Research Center laboratory on San Salvador Island for further processing. For EPS analyses, the samples were sectioned into 1 mm thick slices under a dissecting microscope using sterile razor blades. The samples were transferred to 50 mL tubes and extracted (within 1 hour of collection) in the laboratory. The EPS sample was extracted with ethanol and dried at 35°C for several days.
Glass Formation
Solutions of 0.2 mg of glucose, trehalose, or dextran were prepared in sterile deionized water and incubated in open-top glass tubes at 50°C for 5 days on a heating block. Some
samples contained 0.1 mg N-hexanoyl-L-homoserine lactone (Cayman Chemical, Ann Arbor, MI) and/or 200 units acylase I from porcine kidney (Sigma-Aldrich). The first experiment was conducted at 50°C for five days to mimic the environment of a natural microbial mat. The second experiment was conducted at 60°C for five days followed by 15 minutes at 100°C to ensure the formation of a glass.
Solid State NMR Spectroscopy
Solid state NMR spectroscopy was conducted on samples of treated (glass) and untreated (control) trehalose and C6-AHL. Samples were incubated at 60°C for five days, followed by 15 minutes at 100°C. The pH of the samples was 7.5. Solid state 13C CP-MAS spectra were collected on a Bruker Avance III-HD 500 MHz spectrometer fitted with a 1.9 mm MAS probe. The spectra were collected at ambient temperature with sample rotation rate of 20 kHz. 1.5 msec contact time with linear ramping on the 1H channel and 62.5
kHz field on the 13C channel were used for cross polarization. 1H dipolar decoupling was performed with SPINAL64 modulation and 145 kHz field strength. Free induction decays were collected with a 27 msec acquisition time over a 300 ppm spectra width with a relaxation delay of 1.5s.
Quorum Sensing Signal Detection
Chromobacterium violaceum CV026 (provided by Professor Robert McLean of Texas
Tech University) was used to detect N-hexanoyl-L-homoserine lactone (C6-AHL). Glass samples were resuspended in 50 L sterile deionized water and allowed to rehydrate at room temperature for 10 minutes. 10 L of this solution was applied to a sterile paper
disk on Luria Bertram (30 g/L) agar plates inoculated with C. violaceum CV026. Plates were incubated at 30°C overnight. The production of a violet pigment by the bacteria indicated the presence of C6-AHL.
Temperature tolerance of C6-AHL
5 µM solutions of C6-AHL in water were subjected to 30 minutes of heat at 25, 35, 45, 55, and 65° C. Signal activity was determined by the biosensor C. violaceum CV026. The C6-AHL was dissolved in small volumes of Salt Pond water and dried over the course of three days at both 35 and 55° C. The initial salt concentrations were 280 ppt, 140 ppt, 70 ppt, and 35 ppt. Pure NaCl was used as the control in the same concentrations. The experiment was repeated with 1 mM trehalose added to the solutions.
Acylase I Enzymatic Assay
Acylase I was detected by spectrophotometric analysis according to Mitz & Schlueter (1958). Briefly, the decrease in absorbance at 238 nm was observed every minute for five minutes for solutions containing the treated (in glass) or pure (control) acylase I and 100 mM potassium phosphate and 14 nM N-acetyl-L-methionine (Sigma-Aldrich). The number of units/mL enzyme was determined by A238nm/min Test - A238nm/min
Blank)(60)(3)(dilution factor) / (0.019)(0.1).
Glass Experiment with Cations
Divalent cations were added to solutions of glucose, trehalose, or dextran at pH 7.5 or pH 11. Samples were comprised of 0.2 mg sugar, 200 units acylase I, and 0.1 mg dissolved
cation from either MnCl2⦁4H2O, CaCl2, NaCl, or MgCl2⦁6H2O. Samples were heated in
open-top glass tubes at 50°C for five days. After incubation, samples were resuspended in deionized water and acylase activity was determined spectrophotometrically with the aforementioned assay.
DSC, TGA, and ICP-MS
DSC was performed with a Q2000 Differential Scanning Calorimeter (TA Instruments, USA). Approximately 10 mg of sample was loaded into non-hermetic aluminum Tzero pans. An empty aluminum pan was used as a reference for all experiments. The heating rate was 10° C/min to 300° C under nitrogen flow. TGA characterization was performed with a Q500 Thermogravimetric Analyzer (TA Instruments, USA). Approximately 10 mg of sample was heated at a rate of 10 °C /min to 950 °C under nitrogen flow. Data were recorded by the instrument software. A qualitative trace metal analysis of water from Salt Pond was performed via inductively coupled plasma (ICP) – quadrupole mass spectrometry on a Thermo Finnigan Element XR ICP-MS.
Table 3.1Composition of sugar monomers within EPS extracted from natural mats. Carbohydrate monomer composition of EPS isolated from the upper layer of microbial mat as determined by GC/MS. Values represent mole percentage of total mass. N=2
Monomer Hydrated Desiccated
Arabinose 4.2 2.1 Rhamnose 7.6 11.8 Fucose 10.6 4.9 Xylose 16.3 13.3 Glucuronate 7.6 5.5 Galacturonate 4.7 2.5 Mannose 9.3 13.7 Galactose 8.9 17.5 Glucose 24.4 28.7 N-acetyl glucosamine 6.2 -- Unknown 0.2 --
Table 3.2 C6-AHL activity in trehalose as determined by pigmentation response of C. violaceum CV026. ‘Yes’ indicates the presence of a violet pigment during bacterial growth, which is produced in response to C6-AHL. ‘No’ indicates the absence of a response. Samples were incubated at 50°C for five days or 60°C for five days followed by 100°C for 15 minutes.
50°C 60°C/100°C
pH 7.5
C6-AHL Yes Yes
C6-AHL + Acylase I Yes Yes
pH 11
C6-AHL Yes No
C6-AHL + Acylase I Yes No
Table 3.3 Acylase activity in sugar after heat treatment at 50°C for five days at pH 7.5 and pH 11.
Glucose Trehalose Dextran
pH 7.5
Mn None None None
Mg None None None
Ca None Low Low
pH 11
Mn High High High
Mg None None None
Figure 3.1 Natural microbial mats collected from Salt Pond on San Salvador Island, Bahamasshowing wet- (A) and dry- (B, C) season sections of mat surface. Note underlying layer abundant in cyanobacteria during wet season (A). During early dry- season, evaporites, including gypsum and halite, begin to appear as salinity increases (B), and eventually encrust the dry surface mat (C).
B
A
Figure 3.2 SEM of dried surface layer of microbial mat.Sample consists of a continuous crystalline/amorphous structure of primarily halite, with lesser amounts of gypsum, anhydrite, and other salts.
Figure 3.3 TEM of dry, natural microbial mats showing intact cells surrounded by a dense capsular layer of EPS.
Figure 3.4 Vertical cross-section of microbial mat in Salt Pond showing distinct layering of microbial groups. The uppermost surface Layer (L1) is the layer containing abundant EPS, diatoms, archaea, as well as salt precipitates (during dehydration); Layer 2 (L2) is the dense green ‘cyanobacteria-rich’ layer; Layer 3 (L3) is a layer dominated by purple sulfur bacteria.
Figure 3.5Abundances of EPS isolated from surface layers of hypersaline mat in Salt Pond, San Salvador Island, Bahamas. Surface Layer (L1) is the layer containing the salt precipitate and polymers; Layer 2 (L2) is the dense green ‘cyanobacteria’ layer; Layer 3 (L3) is the layer dominated by purple sulfur bacteria.
EPS Abundance Across Hydration Gradient
SITE S1 S2 S3 S4 S5 ug E P S pe r m g C ell B iom ass 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Surface Layer (L1) Layer 2 (L2) Layer 3 (L3) L1 L2 L3
Figure 3.6 DSC thermogram recorded upon heating of trehalose dihydrate at rate of 10°C/minute. The sample was dried for five days at 35°C prior to analysis. The small shoulder at 85°C indicates a discontinuity in heat flow as the sample transitioned to the glassy state and absorbed heat (Tg). The large peak corresponds with the melting point of
trehalose dihydrate (97°C).
Figure 3.7 DSC thermogram recorded upon heating of trehalose anhydrous at rate of 10°C/minute. The sample was dried for five days at 35°C prior to analysis. The small shoulder at 85°C indicates a discontinuity in heat flow as the sample transitioned to the glassy state and absorbed heat (Tg). The larger peak corresponds with the melting point of
trehalose anhydrous (203°C).
Figure 3.8 DSC thermogram recorded upon heating of Salt Pond EPS at rate of
10°C/minute. The sample was dried for five days at 35°C prior to analysis. The multiple shoulders and peaks indicate the transition temperatures (Tg) of multiple compounds.
Specifically, the Tg of trehalose can be seen at 85°C.
Figure 3.9 TGA thermogram of trehalose dihydrate recorded upon heating at rate of 10°C/minute. The sample was dried for five days at 35°C prior to analysis. The green line represents weight loss occurred. The blue line represents the first derivative of the weight loss curve. The inflection point, the greatest rate of change on the weight loss curve (green), is indicated by the peak of the first derivative curve (blue).
Figure 3.10 TGA thermogram of Salt Pond EPS recorded upon heating at rate of 10°C/minute. The sample was dried for five days at 35°C prior to analysis. The small peak at 300°C corresponds with the peak expected for pure trehalose dihydrate. The blue line represents the first derivative of the weight loss curve. The inflection point, the greatest rate of change on the weight loss curve (green), is indicated by the peaks of the first derivative curve (blue).
Figure 3.11 Solid state 13C NMR spectra of (a) untreated C6-AHL, (b) heat-treated C6- AHL with trehalose, (c) heat-treated trehalose, and (d) untreated trehalose.
A
B
C
R
EFERENCES1. Feynman, R. P., Engineering and Science 1960, (23), 22-36.
2. Khanna, S. C.; Soliva, M.; Speiser, P., Epoxy resin beads as a pharmaceutical dosage form. II. Dissolution studies of epoxy-amine beads and release of drug. J Pharm Sci 1969, 58 (11), 1385-1388.
3. Khanna, S. C.; Jecklin, T.; Speiser, P., Bead polymerization technique for sustained-release dosage form. J Pharm Sci 1970, 59 (5), 614-618.
4. Merkle, H. P.; Speiser, P., Preparation and in vitro evaluation of cellulose acetate phthalate coacervate microcapsules. J Pharm Sci 1973, 62 (9), 1444-1448.
5. Birrenbach, G.; Speiser, P. P., Polymerized micelles and their use as adjuvants in immunology. J Pharm Sci 1976, 65 (12), 1763-1766.
6. Morimoto, Y.; Sugibayashi, K.; Kato, Y., Drug-carrier property of albumin
microspheres in chemotherapy. V. Antitumor effect of microsphere-entrapped adriamycin on liver metastasis of AH 7974 cells in rats. Chemical & Pharmaceutical Bulletin 1981,
29 (5), 1433-8.
7. Brasseur, F.; Couvreur, P.; Kante, B.; Deckers-Passau, L.; Roland, M.; Deckers, C.; Speiser, P., Actinomycin D absorbed on polymethylcyanoacrylate nanoparticles: increased efficiency against an experimental tumor. European Journal of Cancer 1980,
8. Chiannilkulchai, N.; Driouich, Z.; Benoit, J. P.; Parodi, A. L.; Couvreur, P., Doxorubicin-loaded nanoparticles: increased efficiency in murine hepatic metastases. Sel Cancer Ther 1989, 5 (1), 1-11.
9. Couvreur, P.; Kante, B.; Grislain, L.; Roland, M.; Speiser, P., Toxicity of
polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. J Pharm Sci
1982, 71 (7), 790-792.
10. Illum, L.; Davis, S. S.; Muller, R. H.; Mak, E.; West, P., The organ distribution and circulation time of intravenously injected colloidal carriers sterically stabilized with a block copolymer--poloxamine 908. Life Sci 1987, 40 (4), 367-374.
11. Borchard, G.; Audus, K. L.; Shi, F.; Kreuter, J., Uptake of surfactant-coated poly(methyl methacrylate)-nanoparticles by bovine brain microvessel endothelial cell monolayers. International Journal of Pharmaceutics 1994, 110 (1), 29-35.
12. Gulyaev, A. E.; Gelperina, S. E.; Skidan, I. N.; Antropov, A. S.; Kivman, G. Y.; Kreuter, J., Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles. Pharmaceutical Research 1999, 16 (10), 1564-9.
13. Ramge, P.; Unger, R. E.; Oltrogge, J. B.; Zenker, D.; Begley, D.; Kreuter, J.; Von Briesen, H., Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)- nanoparticles by human and bovine primary brain capillary endothelial cells. European Journal of Neuroscience 2000, 12 (6), 1931-40.
14. Steiniger, S. C.; Kreuter, J.; Khalansky, A. S.; Skidan, I. N.; Bobruskin, A. I.; Smirnova, Z. S.; Severin, S. E.; Uhl, R.; Kock, M.; Geiger, K. D.; Gelperina, S. E., Chemotherapy of glioblastoma in rats using doxorubicin-loaded nanoparticles.
15. Clardy, J.; Fischbach, M. A.; Currie, C. R., The natural history of antibiotics.
Current Biology : CB 2009, 19 (11), R437-41.
16. Demain, A. L.; Elander, R. P., The beta-lactam antibiotics: past, present, and future. Antonie Van Leeuwenhoek 1999, 75 (1-2), 5-19.
17. Berdy, J., Bioactive microbial metabolites. The Journal of Antibiotics 2005, 58
(1), 1-26.
18. Buxton, I. L. O. B., L.Z., Pharmacokinetics: The Dynamics of Drug Absorption, Distribution, Metabolism, and Elimination. McGraw Hill: New York, 2011.
19. OpenStax College, S. o. P. O. C. M., 2014 http://cnx.org/contents/9e7c7540- 5794-4c31-917d-fce7e50ea6dd@11@11.
20. Blumenthal, D. K. G., J.C., Pharmacodynamics: Molecular Mechanisms of Drug Action. McGraw Hill: New York, 2011.
21. Seral, C.; Van Bambeke, F.; Tulkens, P. M., Quantitative Analysis of Gentamicin, Azithromycin, Telithromycin, Ciprofloxacin, Moxifloxacin, and Oritavancin (LY333328) Activities against Intracellular Staphylococcus aureus in Mouse J774 Macrophages.
Antimicrobial Agents and Chemotherapy 2003, 47 (7), 2283-2292.
22. Lemaire, S.; Van Bambeke, F.; Mingeot-Leclercq, M. P.; Tulkens, P. M., Activity of three 1-lactams (ertapenem, meropenem and ampicillin) against intraphagocytic
Listeria monocytogenes and Staphylococcus aureus. The Journal of Antimicrobial Chemotherapy 2005, 55 (6), 897-904.
23. Barcia-Macay, M.; Seral, C.; Mingeot-Leclercq, M. P.; Tulkens, P. M.; Van Bambeke, F., Pharmacodynamic evaluation of the intracellular activities of antibiotics
against Staphylococcus aureus in a model of THP-1 macrophages. Antimicrob Agents Chemother 2006, 50 (3), 841-51.
24. Van Bambeke, F.; Barcia-Macay, M.; Lemaire, S.; Tulkens, P. M., Cellular pharmacodynamics and pharmacokinetics of antibiotics: current views and perspectives.
Current Opinion in Drug Discovery & Development 2006, 9 (2), 218-30.
25. van den Broek, P. J., Antimicrobial drugs, microorganisms, and phagocytes.
Reviews of Infectious Diseases 1989, 11 (2), 213-45.
26. Barza, M., Challenges to antibiotic activity in tissue. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 1994, 19 (5), 910-5. 27. Atalla, H.; Gyles, C.; Mallard, B., Persistence of a Staphylococcus aureus small colony variants (S. aureus SCV) within bovine mammary epithelial cells. Veterinary Microbiology 2010, 143 (2-4), 319-28.
28. Tuchscherr, L.; Medina, E.; Hussain, M.; Volker, W.; Heitmann, V.; Niemann, S.; Holzinger, D.; Roth, J.; Proctor, R. A.; Becker, K.; Peters, G.; Loffler, B.,
Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. EMBO Molecular Medicine 2011, 3
(3), 129-41.
29. Garcia, L. G.; Lemaire, S.; Kahl, B. C.; Becker, K.; Proctor, R. A.; Denis, O.; Tulkens, P. M.; Van Bambeke, F., Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of Staphylococcus aureus in models of extracellular (broth) and intracellular (THP-1 monocytes) infections. Antimicrob Agents Chemother 2012, 56 (7), 3700-11.
30. Wise, E. M.; Park, J. T., Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis. Proceedings of the National Academy of Sciences of the United States of America 1965, 54 (1), 75-81. 31. Tipper, D. J.; Strominger, J. L., Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine. Proceedings of the National Academy of Sciences of the United States of America 1965, 54 (4), 1133-1141. 32. Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.; Collins, J. J., A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007, 130
(5), 797-810.
33. Drawz, S. M.; Bonomo, R. A., Three decades of beta-lactamase inhibitors. Clin Microbiol Rev 2010, 23 (1), 160-201.
34. McDermott, P. F.; Walker, R. D.; White, D. G., Antimicrobials: Modes of Action and Mechanisms of Resistance. International Journal of Toxicology 2003, 22 (2), 135- 143.
35. Höltje, J.-V., Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiology and Molecular Biology Reviews 1998, 62 (1), 181-203. 36. Lee, W.; McDonough, M. A.; Kotra, L. P.; Li, Z.-H.; Silvaggi, N. R.; Takeda, Y.; Kelly, J. A.; Mobashery, S., A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis. Proceedings of the National Academy of Sciences 2001, 98 (4), 1427-1431. 37. Josephine, H. R.; Kumar, I.; Pratt, R., The perfect penicillin? Inhibition of a bacterial DD-peptidase by peptidoglycan-mimetic β-lactams. Journal of the American Chemical Society 2004, 126 (26), 8122-8123.
38. Waxman, D.; Strominger, J., Sequence of active site peptides from the penicillin- sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases. Journal of Biological Chemistry 1980, 255
(9), 3964-3976.
39. Carryn, S.; Chanteux, H.; Seral, C.; Mingeot-Leclercq, M.-P.; Van Bambeke, F.; Tulkens, P. M., Intracellular pharmacodynamics of antibiotics. Infectious Disease Clinics of North America 2003, 17 (3), 615-634.
40. Nikaido, H.; Pages, J.-M., Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiology Reviews 2012, 36
(2), 340-363.
41. Craig, W. A., Basic pharmacodynamics of antibacterials with clinical applications to the use of β-lactams, glycopeptides, and linezolid. Infectious Disease Clinics of North America 2003, 17 (3), 479-501.
42. Zapun, A.; Contreras-Martel, C.; Vernet, T., Penicillin-binding proteins and beta- lactam resistance. FEMS Microbiology Reviews 2008, 32 (2), 361-85.
43. Goffin, C.; Ghuysen, J.-M., Multimodular Penicillin-Binding Proteins: An Enigmatic Family of Orthologs and Paralogs. Microbiology and Molecular Biology Reviews 1998, 62 (4), 1079-1093.
44. Massova, I.; Mobashery, S., Kinship and Diversification of Bacterial Penicillin- Binding Proteins and β-Lactamases. Antimicrobial Agents and Chemotherapy 1998, 42
(1), 1-17.
45. Llarrull, L. I.; Testero, S. A.; Fisher, J. F.; Mobashery, S., The future of the beta- lactams. Current Opinion in Microbiology 2010, 13 (5), 551-7.
46. Bradford, P. A., Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001, 14 (4), 933-51, table of contents.
47. Therrien, C.; Levesque, R. C., Molecular basis of antibiotic resistance and β- lactamase inhibition by mechanism-based inactivators: perspectives and future directions.
FEMS Microbiology Reviews 2000, 24 (3), 251-262.
48. Fisher, J. F.; Meroueh, S. O.; Mobashery, S., Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chemical Reviews 2005,
105 (2), 395-424.
49. Chopra, I.; Roberts, M., Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews : MMBR 2001, 65 (2), 232-60 ; second page, table of contents.
50. Dax, S. L., Tetracycline Antibiotics. In Antibacterial Chemotherapeutic Agents, Springer: 1997; pp 159-205.
51. Mitscher, L. A., The Chemistry of the Tetracycline Antibiotics. M. Dekker: 1978; Vol. 9.
52. Rogalski, W., Chemical modification of the tetracyclines. In The Tetracyclines, Springer: 1985; pp 179-316.
53. Schnappinger, D.; Hillen, W., Tetracyclines: antibiotic action, uptake, and resistance mechanisms. Archives of Microbiology 1996, 165 (6), 359-369.
54. Chopra, I.; Hawkey, P.; Hinton, M., Tetracyclines, molecular and clinical aspects.
55. Becker, B.; Cooper, M. A., Aminoglycoside antibiotics in the 21st century. ACS Chemical Biology 2013, 8 (1), 105-15.
56. Kurland, C., Translational Accuracy and the Fitness of Bacteria. Annual Review of Genetics 1992, 26 (1), 29-50.
57. O'Connor, M.; Göringer, H. U.; Dahiberg, A. E., A ribosomal ambiguity mulation in the 530 loop of E. coli 16S rRNA. Nucleic Acids Research 1992, 20 (16), 4221-4227. 58. Bryan, L.; Kwan, S., Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin. Antimicrobial Agents and Chemotherapy 1983, 23 (6), 835-845.
59. Davis, B. D.; Chen, L.; Tai, P. C., Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides. Proceedings of the National Academy of Sciences 1986, 83 (16), 6164-6168.
60. Davis, B. D., Mechanism of bactericidal action of aminoglycosides.
Microbiological Reviews 1987, 51 (3), 341.
61. Hansen, J. L.; Ippolito, J. A.; Ban, N.; Nissen, P.; Moore, P. B.; Steitz, T. A., The structures of four macrolide antibiotics bound to the large ribosomal subunit. Molecular Cell 2002, 10 (1), 117-128.
62. Kannan, K.; Mankin, A. S., Macrolide antibiotics in the ribosome exit tunnel: species-specific binding and action. Annals of the New York Academy of Sciences 2011,
1241, 33-47.
63. Schlünzen, F.; Zarivach, R.; Harms, J.; Bashan, A.; Tocilj, A.; Albrecht, R.; Yonath, A.; Franceschi, F., Structural basis for the interaction of antibiotics with the