The biodegradation of bacterial lipopolysaccharides by marine invertebrates and marine bacteria

• Main Author: Jamieson, Thomas
• Format: Thesis
• Language: English
• Published: ProQuest Dissertations & Theses 1986
• Subjects: Modiolus modiolus
• Online Access: http://theses.gla.ac.uk/72286/; http://theses.gla.ac.uk/72286/1/10646103.pdf; https://eleanor.lib.gla.ac.uk/record=b1279041

Gram-negative bacteria are abundant in marine environments and also act as a food source for filter-feeding animals such as bivalve molluscs. The question posed in this investigation was : are lipopolysaccharides (LPSs) - the cell-wall polymers unique to Gram-negative bacteria - subject to biodegradation by either molluscan gut extracts or by heterotrophic bacteria in marine sediments? Extracts of digestive gland from 5 species of marine molluscs were prepared : Cerastoderma edule (common cockle), Modiolus modiolus (horse mussel), Mya arenaria (sand gaper), Mytilus edulis (common mussel) and Pecten maximus (great scallop) . Purification was not taken beyond crude ammonium sulphate (AS) fractionation. To explore their possible hydrolytic action on LPSs, each was incubated for up to 24h with purified LPS from Salmonella minnesota, Escherichia coli Olll:B4 and Methylophilus methylotrophus. Degradation of the polysaccharide moieties of all three LPSs by all molluscan extracts was readily detected by the combined applications of descending paper chromatography, thin layer chromatography (TLC), colorimetry and gel filtration chromatography. S. minnesota LPS was degraded most by all extracts and evidence for the breakdown of the O-chain repeating unit was detected, with glucose, N-acetylglucosamine, N-acetylgalactosamine and/or galactose being released. The core-oligosaccharides of the other two LPSs were degraded : E. coli 0111:B4 LPS yielded glucose and N-acetylglucosamine upon hydrolysis by the extracts, Meth. methylotrophus LPS hydrolysis released only glucose. Degradation of all three LPSs resulted in the release of other unidentified monosaccharides. Most extracts had optimum glycosidase activity at pH 4.5 against all three LPSs tested although Modiolus was most potent at pH 5.0. Of all the extracts tested, only one fraction, from Modiolus, had demonstrable endoglycolytic activity towards LPS, releasing oligosaccharides from S. minnesota and E. coli 0111:B4 LPSs. All extracts released large quantities of inorganic phosphate from LPSs, as determined colorimetrically, but no free ethanolamine was detected by TLC. Action on the lipid A component of the LPS molecule was investigated by the release of free fatty acids, detectable colorimetrically or by TLC. Most digestive gland preparations failed to release significant amounts of free fatty acids from LPS, despite having lipase activity towards tributyrin (the pH optimum of which was characterised for each AS fraction). However, one preparation from Cerastoderma released non-hydroxylated fatty acids from LPSs, as did Helix pomatia (common snail) gut juice, although a commercially available lipase preparation from the yeast Candida cylandracea, had very little activity against LPSs. Marine sediments and sands from different environments were examined for both aerobic and anaerobic LPS-degrading bacteria. Numerous bacterial strains were isolated by ability to degrade tributyrin, whole Gram-negative bacterial cells, LPS or lipid A. One lipolytic bacterial isolate (P8) released fatty acids when the latter was present in nutrient culture medium. A total of nine bacterial isolates was able to grow on a medium containing LPS from Meth, methylotrophus as the sole organic nutrient and produced visible zones of degradation. These isolates were also degradative towards protein, lipids, DNA and starch. Cell-free extracts of one of these bacterial strains (Ml ) liberated reducing material from LPS when incubated for 24h,which suggested a saccharolytic mode of attack. In numerous tests, culture supernatant preparations from all of the other bacterial isolates (including LPS-degrading bacterial strains isolated by earlier investigators) had no detectable effect on LPS. Anaerobic LPS-degrading bacteria were sought, but not isolated, although LPS degradation was observed in anaerobic mixed liquid cultures. In conclusion, whereas molluscan digestive gland extracts readily degraded the polysaccharide moieties of LPSs, breakdown of the lipid A component was less easily detected. Although bacterial isolates had readily demonstrable degradative activity towards LPS and other compounds on solid growth media, enzymatic breakdown of LPS was more difficult to detect and thus characterise.