Research on peptidoglycan

Research on bacterial cell wall material has focused on abundance of peptidoglycan in various environments, degradation of peptidoglycan, and on charactetrization of specific microorganisms that can degrade peptidoglycan.

General information on peptidoglycan and on methods used in our laboratory are presented below. Recent articles on peptidoglycan include:


1.  Jorgensen NOG, KK Brandt, O Nybroe & M Hansen (2009) Delftia lacustris sp. nov., a peptidoglycan-degrading bacterium from freshwater and emended description of D. tsuruhatensis as a peptidoglycan-degrading bacterium. International Journal of Systematic and Evolutionaty Microbiology (in press)

2. Jorgensen NOG, P Engel, R Jellison & JT Hollibaugh (2008) Contribution of bacterial cell wall components to DOM in alkaline, hypersaline Mono Lake, California. Geomicrobiology Journal 25: 38-55

3. Jorgensen NOG & M. Middelboe (2006) Occurrence and bacterial cycling of D amino acid isomers in an estuarine environment. Biogeochemistry 81: 77-94

4. Middelboe M & NOG Jorgensen (2006) Viral lysis of bacteria: an important source of dissolved amino acids and cell wall compounds. Journal of The Marine Biological Association of the United Kingdom 86: 605-612

Facts about peptidoglycan

he cell wall of Gram-positive (G+) bacteria consists of a thick and uniform peptidoglycan layer, making up 90% of the cell wall. In contrast, Gram-negative (G-) bacteria have a complex, multilayered cell wall structure with a relatively thin inner peptidoglycan layer (constitutes 10% of the cell wall) and an outer membrane of lipopolysaccharides and proteins. Peptidoglycan consists of strands of alternating N-acetylglucosamine and N-acetylmuramic acid units, cross-linked by short peptides. Amino acids (AA) in these peptides include D-isomers of alanine, aspartate, glutamate, and serine, which have been used as markers of peptidoglycan in environmental studies.

Degradation of peptidoglycan

acterial cell walls are believed to be more resistant to degradation than the remaining part of the cell. In marine waters and sediments, D-AA have been found to make up 10 to 30% of the hydrolyzable AA, indicating that peptidoglycan may comprise a relatively large fraction of the dissolved organic matter (DOM). Considering that <10% of all AA in living marine bacteria are D-isomers (own unpublished results), and that bacterial biomass in pelagic waters and sediments makes up <5% of the total organic carbon pool, a proportion of D-isomers >10% in this organic matter indicates a 20- to 60-fold enrichment with D-AA. Most likely, this enrichment is caused by a selective biological degradation of L-AA relative to D-AA. Despite the observed abundance of D-AA in aquatic DOM, mechanisms of bacterial cell wall preservation or degradation in natural environments are largely unknown.

Peptidoglycan may reside for several thousands years, at least in marine environments. Thus, in marine samples peptidoglycan has been found to account for the major and refractory part of 4,000-6,000 years old high molecular dissolved organic matter.

Natural racemization of amino acids in peptidoglycan

Natural racemization of amino acids in protozoa, invertebrates and skeletons of vertebrates has been used for chronological study. The method is based on the fact that amino acids change their configuration from the natural L into D forms during aging, although at a low rate. The time for conversion of L amino acids to a equilibrium of L and D amino acids is estimated to ~10⁴ to ~10⁶ years. This low rate of racemization suggests that D amino acids in most environments originate from bacteria rather than racemization.

Fig. 1

2. Application of isotopic approaches for measuring peptidoglycan degradation

Various isotopes (³H, ¹⁴C and ¹⁵N) were attempted incorporated into peptidoglycan of different estuarine bacteria. The preliminary results indicate a variable labeling. Using ³H leucine as a food source, a relatively high specific activity of D alanine in peptidoglycan was detected, while other peptidoglycan components had a low ³H content. When the labeled peptidoglycan was offered to growing bacteria, most of its ³H activity was respired during a 20 day period.

According to literature, most bacterial enzymes used for hydrolysis of peptidoglycan are cell-associated. Then, how can a bacterium utilize extracellular peptidoglycan? To test if bacteria produce extracellular, peptidoglycan-degrading enzymes, zymogram technique is used (polyacrylamide gels with peptidoglycan) are used. If enzymes in the “supernatant” of bacterial cultures are capable of degrading peptidoglycan, presence of peptidoglycan-degradaed bands will appear in the zymograms. The technique appears promising although some adjustments are required.