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Analysis of Mycolic Acids

Mycolic acids are extracted from wet biomass using minor modifications of the method described by Vilchèze and Jacobs (2007) for analysis of mycolic acids by HPLC. Dried extracts are reconstituted in chloroform:methanol and analyzed on an Agilent QTof mass spectrometer by direct infusion into the ESI source. Mycolic acids were identified based on comparison of exact masses from known mycolic acid structures.

Required material: 300 mg wet biomass (centrifugal pellet) send in frozen state on dry ice or suspended in isopropanol (send at ambient temperature). Lyophilized cells are suboptimal but can also be send corresponding to an amount of 60-80 mg. Alternatively, an actively growing culture (still in exponential growth phase upon arrival, without signs of sporulation or cell lysis) could be send. Subculturing (if required) will be charged separately. If subculturing is requested by the customer, detailed cultivation conditions (media, temperature, oxygen supply, cultivation duration) must be provided.

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References

  1. Vilchèze, C. and Jacobs, W.R. (2007), Isolation and Analysis of Mycobacterium tuberculosis Mycolic Acids. Current Protocols in Microbiology, 5: 10A.3.1-10A.2.11.
  2. Bouam, A., Armstrong, N., Levasseur, A., Drancourt, M. (2018) Mycobacterium terramassiliense, Mycobacterium rhizamassiliense and Mycobacterium numidiamassiliense sp. nov., three new Mycobacterium simiae complex species cultured from plant roots. Sci Rep 8, 9309.
  3. Fong-Fu Hsu, Kristina Soehl, John Turk, Albert Haas (2011) Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization. Analytical Biochemistry, 409, 112-122.

    

Analysis of Polyamines

Aliphatic linear polyamines are widely distributed in bacteria and archaea and can serve as a chemotaxonomic marker in bacterial classification. Polyamines are extracted from wet biomass according to the protocol for metabolite extraction described by Zech et al. (2009). Polyamines are derivatized and analyzed by GC-MS as described by Will et al. (2019).
The polyamine species and precursors that could be targeted with this method include: agmatine, cadaverine, homospermidine, norspermidine, 1,2- and 1,3-diaminopropane, putrescine, N-acetyl-putrescine, 2-hydroxyputrescine, spermidine and spermine.

Required material: 50-100 mg wet biomass send in frozen state on dry ice. Alternatively, an actively growing culture (still in exponential growth phase upon arrival, without signs of sporulation or cell lysis) could be send. Subculturing (if required) will be charged separately. If subculturing is requested by the customer, detailed cultivation conditions (media, temperature, oxygen supply, cultivation duration) must be provided.

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References

  1. Zech H, Thole S, Schreiber K, Kalhöfer D, Voget S, Brinkhoff T, Simon M, Schomburg D, Rabus R (2009) Growth phase-dependent global protein and metabolite profiles of Phaeobacter gallaeciensis strain DSM 17395, a member of the marine Roseobacter-clade. Proteomics 2009; 9:3677–3697.
  2. Will SE, Henke P, Boedeker C, Huang S, Brinkmann H, Rohde M, Jarek M, Friedl T, Seufert S, Schumacher M, Overmann J, Neumann-Schaal M, Petersen J (2019) Day and night: metabolic profiles and evolutionary relationships of six axenic non-marine cyanobacteria. Genome Biol Evol 2019; 11:270–294.

       

Analysis of Whole-Cell Sugars 

Diagnostic sugars in whole-cell hydrolysates (1N H2SO4, 100 °C, 2 hours) are analyzed by TLC on cellulose plates. The result supports the classification and identification of Actinobacteria.

Required material: For this method 15 mg freeze dried cells (send at ambient temperature) or 150 mg wet biomass (send frozen on dry ice) or suspended in isopropanol (send at ambient temperature) are needed. Alternatively, an actively growing culture (still in exponential growth phase upon arrival, without signs of sporulation or cell lysis) could be send. Subculturing (if required) will be charged separately. If subculturing is requested by the customer, detailed cultivation conditions (media, temperature, oxygen supply, cultivation duration) must be provided.

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References

  1. Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, 101-129.
  2. Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226-231.

    

Detection of isomers of 2,6-diaminopimelic acid (Dpm) and of 2,6-diamino-3-hydroxypimelic acid (OH-Dpm)

Whole-cell hydrolysates (4N HCl, 100°C, 16 hours) are examined by TLC on cellulose plates for the presence of Dpm isomers or OH-Dpm. The result supports the classification and identification of Actinobacteria.

Required material: For this method 50 mg wet biomass (send frozen on dry ice) or suspended in isopropanol (send at ambient temperature) are needed. Alternatively, an actively growing culture (still in exponential growth phase upon arrival, without signs of sporulation or cell lysis) could be send. Subculturing (if required) will be charged separately. If subculturing is requested by the customer, detailed cultivation conditions (media, temperature, oxygen supply, cultivation duration) must be provided. Freeze dried biomass (min 5 mg) might give suboptimal results.

Download the service order form here.

Team Services

References 

  1. Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, 101-129.
  2. Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407-77.
  3. Rhuland, L. E., Work, E., Denman, R. F. & Hoare, D. S. (1955). The behaviour of the isomers of 2,6-diaminopimelic acid on paper chromatograms. J Am Chem Soc 77, 4844-4846.