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Estimations of Uronic Acids As Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples

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Estimations of Uronic Acids As Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1982, p. 1151-1159 Vol. 43, No. 5 0099-2240/82/051151-09$02.00/0 Estimations of Uronic Acids as Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples STEVEN A. FAZIO, DAVID J. UHLINGER, JEFFREY H. PARKER, AND DAVID C. WHITE* Departments ofBiological Science and Oceanography, Florida State University, Tallahassee, Florida 32306 Received 31 August 1981/Accepted 1 December 1981 The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrat- ed in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideu- terated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater. Extracellular polymers are becoming increas- lar polymers may be particularly important in ingly important in microbial ecology. These heat transfer resistance by the microfouling polymers are important in conferring virulence community that is formed when metal surfaces on pathogenic bacteria by inhibiting phagocyto- are exposed to rapidly flowing seawater (33, 34). sis and the action of serum antimicrobial factors. Since these extracellular polymers are ofgreat They protect organisms from predation and im- importance, the means to quantitatively mea- pede desiccation in soil microorganisms. Exo- sure their formation and catabolism needed to be polysaccharides regulate the ionic traffic at the developed. For this type of analysis, a compo- cell surface, particularly the traffic of magne- nent found outside the cytoplasmic membrane sium ions. They concentrate nutrients such as but not found in intercellular polymers, such as amino acids, phosphates, and silicon and protect the hydroxyproline in collagen or the desmosine microbes from toxic heavy metals or antibacteri- in elastin, would be ideal. From an examination al agents often used in antifouling treatments. In of compendia of extracellular and cellular poly- nitrogen-fixing organisms, these polymers act as mer compositions (2, 18, 30, 35, 42, 43), it is carbon storage materials (15). Surfaces are par- clear that uronic acids are almost unique to the ticularly important in microbial ecology (29), polymers found outside the cytoplasmic mem- and all known marine periphytic microbes attach brane of the cells. Uronic acids are also found in irreversibly to surfaces containing acid polysac- the polysaccharide polymers of higher plant cell charides (10-14, 17). Indirect effects of these walls, in gram-positive microbes grown with polymers include the stabilization of sediments phosphate limitation (16), and in some gram- and soils by the presence of invertebrate feeding negative microbial lipopolysaccharides. Amino- tubes and microbial extracellular polysaccharide uronic acids found in the walls of micrococci polymers (19, 20, 36, 49). Recent evidence has (45) cannot be assayed by the methods described indicated that microbially produced extracellu- in this study. However, the electron micro- 1151 1152 FAZIO ET AL. APPL. ENVIRON. MICROBIOL. graphic examination of environmental samples extracellular polymers, as employed by Smith shows extensive extracellular polymers stained (41), was adapted for the analysis of environ- by ruthenium red (polymers containing uronic mental samples. The method involves the reduc- acid) surrounding the bacteria (13, 14). tion of the uronic acids to primary alcohols while Uronic acids are often estimated by their acid- they are still a part of the polysaccharide poly- catalyzed decarboxylation under controlled con- mers. Uronic acids as monomers are very diffi- ditions (24). Although this method is very sensi- cult to quantitatively reduce to the correspond- tive, it is not specific and does not differentiate ing neutral carbohydrate, even with repeated between the various uronic acids. The carbazole treatments with NaBH4 (39). However, in the reaction has been used to measure the uronic polymer, the uronic acids can be quantitatively acids in hydrolysates (4). This is a relatively reduced with one treatment with NaB2H4, pro- insensitive colorimetric reaction, and the extinc- vided the methyl ester is formed first (41). tion coefficients are different for different uronic After much frustration with methods that acids and depend on other components in the were designed to reproducibly hydrolyze, sepa- polymers (38). This method, like the decarboxyl- rate, and assay uronic acids, a method in which ation method, does not differentiate between the the uronic acids are reduced to dideuterated different uronic acids. neutral sugars while still in the polymer and then Consequently, a method that allowed quanti- are assayed quantitatively was adapted. tative recovery of each uronic acid was sought. The known microbial exopolysaccharides con- MATERIALS AND METHODS tain D-glucuronic acid, D-galacturonic acid, D- Materials. Glass-distilled solvents (Burdick and mannuronic acid, and L-gulonic acid (15). Possi- Jackson, Muskegon, Mich.), freshly distilled chloro- bly, other components, such as those isolated form, and derivatizing reagents (Pierce Chemical Co., from marine sediments by Mopper and Larsson Rockford, Ill.; Aldrich Chemical Co., Milwaukee, (31), could be measured in such an analysis. If Wis.; and PCR Research Chemicals, Inc., Gainesville, each component were isolated in the analysis, it Fla.) were used. Standard sugars and uronic acid- would be possible to use isotopes to monitor containing gums were purchased from Sigma Chemical rates of formation and catabolism. Co., St. Louis, Mo. Sodium borodeuteride (98 atom% Polymers of deuterium) was acquired from Merck and Co., Inc./ containing uronic acids resist acid hydrolysis Isotopes, St. Louis, Mo. because the carboxylic acid moiety stabilizes the Bacterial cultures. Pseudomonas marina (ATCC glycosidic linkage (25). This stabilization causes 27129) and Pseudomonas atlantica strain T6C (the gift very low yields of galacturonic acid when poly- of W. A. Corpe, Columbia University) were main- galacturonic acid polymers are acid hydrolyzed. tained on Difco marine agar (3). Bacteria that were Once hydrolyzed, the uronic acids become ex- forming mucoid colonies were isolated from marine tensively lactonized. The extent ofthe lactoniza- sediments near the Florida State University marine tion in our experiments and as reported previ- laboratory on sterilized glass slides that had been left ously (5) is irreproducible. Since in the sediment for 3 days (11). These organisms were quantitative maintained on agar containing 1% (wt/vol) glucose and analysis requires the separation of the uronic 0.5% (wt/vol) peptone in sea salts (Instant Ocean, acids from the neutral aldoses (6), anion-ex- Aquarium Systems Inc., East Lake, Ohio) with a change columns are usually used. Uronic acids salinity of 25 ,ug/liter or were frozen in medium react with and thus are not quantitatively recov- containing 15% glycerol at -70°C. The extracellular erable from the weak anion-exchange columns polymers for analysis were recovered by the modified necessary to separate the acids from the neutral Bligh and Dyer chloroform-methanol-water extraction sugars (32). The lactones will not sorb to strong (47) after precipitation. anion-exchange columns in the acetate form and Collection of invertebrates. Maldanid polychaete can cause degradation of the aldoses and uronic worms of Clymenella spp. were collected by the sieving of subtidal mud flat sediments. The worm acids in the hydroxide form (6). The conversion tubes were washed extensively, frozen, and lyophi- of the lactones to the free acids or to salts by lized before the analysis for uronic acids. using weak alkali causes the formation of col- Fresh fecal mounds formed by the enteropneust ored degradation products unless very carefully Ptychodera bahamensis were identified on the sedi- titrated. If the anomeric carbons of the aldoses ment surface and carefully collected by divers with and uronic acids are reduced, the resulting aldi- SCUBA equipment as previously described (44). Con- tols and aldonic acids can be separated on anion- trol sediments were collected at least 25
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