O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770

The O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770 was obtained by mild acid degradation of the corresponding lipopolysaccharide and found to contain D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine residues in equimolar ratio. Based on methylation analysis, periodate oxidation, and 13C-NMR spectroscopy data of native and modified polysaccharides, the following structure of the trisaccharide repeating unit of the O-specific polysaccharide was established: [structure: see text]     

Development and validation of an NMR-based identity assay for bacterial polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

Monitoring the polysaccharide quality of Agaricus blazei in submerged culture by examining molecular weight distribution and TNF-α release capability of macrophage cell line RAW 264.7

A method for monitoring the biological activity of broth polysaccharides of Agaricus blazei (AB-BP) in a submerged culture is described. The TNF-alpha releasing capability of AB-BP on the macrophage cell line, RAW 264.7, correlated with the molecular weight of AB-BP. The quality of polysaccharide in a submerged culture of Agaricus blazei was indirectly monitored by analyzing the distribution of its molecular weight within 1 h. The harvest time of the maximum polysaccharide production did not coincide with that of the maximum biological characteristics in the batch culture.     

Purification of an alcohol dehydrogenase involved in the conversion of methional to methionol in <Emphasis Type="Italic">Oenococcus oeni</Emphasis> IOEB 8406

Oenococcus oeni, the major lactic acid bacteria involved in malolactic fermentation (MLF) in wine, is able to produce volatile sulfur compounds from methionine. Methional reduction is the last enzymatic step of methionol synthesis in methionine catabolism. Alcohol dehydrogenase (ADH) activity was found to be present in the soluble fraction of O. oeni IOEB 8406. An NAD(P)H-dependent ADH involved in the reduction of methional was then purified to homogeneity. Sequencing of the purified enzyme and amino acid sequence comparison with the database revealed the presence of a conserved sequence motif specific to the medium-chain zinc-containing NAD(P)H-dependent ADHs. Despite the great importance of ADH activities in wine flavor modification, this is the first report of the purification of an ADH isolated from O. oeni. The purified ADH does not seem to be involved in the modification of buttery and lactic notes or to be involved in the specific formation of volatile alcohols during MLF. The enzyme was not strictly specific of methional reduction and the highest reducing activity was obtained with acetaldehyde as substrate. The function of the purified ADH remains unclear, although the role of the sulfur atom in methional molecules in the interaction between enzyme and substrate was evidenced.     

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.     

Comparative study of the cell wall composition of broccoli, carrot, and tomato: structural characterization of the extractable pectins and hemicelluloses

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.     

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.     

Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.     

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72?h conversion assay (CONV) with in vitro rumen microflora incubation for 72?h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r?=?0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.     

Immunocytochemical localization of cell wall polysaccharides in the root tip ofAvena sativa

Summary Two polyclonal antisera, anti-xyloglucan (anti-XG) and anti-polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), which recognize, respectively, noncellulosic -(14)-D-glucan containing polysaccharides and the unesterified forms of the acidic pectic polysaccharide polygalacturonic acid/rhamnogalacturonan I, were used to localize epitopes recognized by the two antisera in the root tip of oat (Avena sativa). Immunoblot analysis shows that epitopes recognized by the anti-XG antibodies are present in both the mixed linkage -(13)-(14)-D-glucans (MG) and in xyloglucan (XG). Immunogold electron microscopy shows that the cell walls of meristematic, cortical, epidermal, columella, and peripheral cells contain significant amounts of such epitopes. In contrast, the molecules that carry these MG/XG epitopes appear to be sparse in the expanded middle lamella of meristematic cells, but dense in the expanded middle lamella of peripheral root cap cells. This finding suggests that the porosity of the middle lamella is altered in peripheral root cap cells to facilitate mucilage secretion. In contrast, few PGA/RG-I epitopes were detected in any cell walls of any of the cell types examined. Double immunogold labeling experiments revealed an intriguing localization pattern of MG/XG and of PGA/RG-I epitopes in the peripheral mucilage-secreting cells of the root cap. Whereas MG/XG epitopes were abundant in the cell wall, they were sparse in both the secreted mucilage and in intracellular secretory vesicles. In marked contrast, PGA/RG-I epitopes were detected at high density in intracellular secretory vesicles, but unexpectedly, were quite sparse in both the cell wall and in the mucilage. These immunolabeling patterns are consistent with the hypotheses that the synthesis and secretion of particular -D-glucans is subject to both activation and down-regulation during cell development and differentiation and that post-secretory alterations of pectic polysaccharides, such as enzymatic release of RG-I-type mucilage molecules from PGA/RG-I precursors, may occur in the peripheral cell walls of the oat root cap.Abbreviations MG mixed linkage -(13)-(14)-D-glucan - PGA/RG-I polygalacturonic acid/rhamnogalacturonan I - SEPS sycamore extracellular polysaccharides - TGN trans Golgi network - XG xyloglucan     

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.     

ALCIAN BLUE: A QUANTITATIVE AQUEOUS ASSAY FOR ALGAL ACID AND SULFATED POLYSACCHARIDES1

Alcian Blue, a cationic copper phthalocyanine dye, complexes with the anionic carboxyl and half-ester sulfate groups of acidic algal polysaccharide in aqueous solution to form an insoluble precipitate. The quantity of dye removed from solution is proportional to the quantity of polyanion in solution, and this principle forms the basis for the quantitative determination of acid and/or sulfated algal polysaccharides. The assay is linear between 0 and 100 μg/ml agar, alginic acid, carrageenan, pectin and Porphyridium aerugineum Geit. polysaccharide. In addition, the technique is used to determine the anion density of acid polysaccharides on a molar or weight equivalency basis.     

The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell

The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.     

Soil saccharide extraction and detection

Extraction of soil saccharides involves the use of reagents effective in breaking hydrogen and covalent bonds between soil constituents and the saccharides. Of the many extractants proposed for saccharide determination, water is commonly used for extraction of water-soluble mono- and polysaccharides in soil. Analysis of these water extracts by colorimetric assays (anthrone-sulfuric acid and phenol-sulfuric acid methods) often show color development indicating that saccharides are present. However, high performance liquid chromatography (HPLC) and gas chromatography analyses have indicated that these colorimetric assays are prone to errors due to interferences from inorganic soil constituents such as Cl⁻, NO₃ ⁻ and Fe⁺³. When water extracts (25° or 80°C) are put through deionization resins to remove interferences little to no saccharides are present when assayed by the phenol-sulfuric acid analysis. The inability of water to extract saccharides from soil or microbial polymers was confirmed by HPLC analysis. The phenol-sulfuric acid assay was found to be acceptable for saccharide analysis of soil extracts only after being subjected to resin deionization for interference removal. The anthrone-sulfuric acid method is not considered acceptable for determining saccharides in soil.     

Evidence for the adaptive significance of enzyme activity levels: Interspecific variation in α-GPDH and ADH in Drosophila

The activity levels of alcohol dehydrogenase and -glycerophosphate dehydrogenase were compared among nine species of Drosophila representing three phylogenetic groups. For any given life stage, interspecific variability in activity level was much greater for ADH than for -GPDH. Patterns of ontogenetic expression of enzyme activity were also much more variable among species for ADH than for -GPDH. These results are consistent with the interpretation that -GPDH is involved with a relatively uniform adaptive function among species, whereas ADH levels may reflect variable adaptive capabilities. There is a significant correlation between ADH activities and survivorship on alcohol-treated media for these nine species.This research was supported by Contract AT(04-3)-34 200 with ERDA. The authors are supported by an NIH training grant in genetics.     

Osmotically-regulated trehalose accumulation and cyclic β-(1,2)-glucan excretion by Rhizobium leguminosarum biovar trifolii TA-1

Rhizobium leguminosarum biovar trifolii TA-1 produced high molecular weight extracellular (EPS) and capsular polysaccharides (CPS) as the main carbohydrate products in a medium (10 g of mannitol and 1 g of glutamic acid per liter) with low osmotic pressure of 0.20 MPa. By increasing the osmotic pressure of the medium with the addition of NaCl or other osmolytes up to 1.44 MPa, the synthesis of EPS and CPS was suppressed. Cyclic -(1,2)-glucans were excreted instead. Concentrations of over 1500 mg of glucans/l medium were produced by a biomass of 520 mg protein at 200 mM NaCl (1.20 MPa). Intracellular cyclic -(1,2)-glucan concentrations remained at 45 to 100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells as osmo-protectant. Concentrations of up to 130 mg/g protein were reached. Strain TA-1 could tolerate 350 mM NaCl.Abbreviations CPS capsular polysaccharide - EPS extracellular polysaccharide - LM_r low molecular weight - HM_r high molecular weight     

Antibody-based assay for N-deacetylase activity of heparan sulfate/heparin N-deacetylase/N-sulfotransferase (NDST): novel characteristics of NDST-1 and -2

A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs), which are key enzymes in sulfation of heparan sulfate (HS)/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in HSs by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stably transfected with mouse NDST-1 or -2. We found HSs to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta ( approximately 0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 microM (calculation based on [HexA]) being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 microM, respectively). Comparison of various HS preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (>or=6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated HS, and heparin. Our primary findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, whereas other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.     

Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis

Within the capsule gene complex (cps) of Neisseria meningitidis two functional regions B and C are involved in surface translocation of the cytoplasmically synthesized capsular polysaccharide, which is a homopolymer of α-2,8 polyneuraminic acid. The region-C gene products share characteristics with transporter proteins of the ABC (ATP-binding cassette) superfamily of active transporters. For analysis of the role of region B in surface translocation of the capsular polysaccharide we purified the polysaccharides of region B- and region C-defective Escherichia coli clones by affinity chromatography. The molecular weights of the polysaccharides were determined by gel filtration and the polysaccharides were analysed for phospholipid substitution by polyacrylamide gel electrophoresis and immunoblotting. The results indicate that the full-size capsular polysaccharide with a phospholipid anchor is synthesized intracellularly and that lipid modification is a strong requirement for translocation of the poly saccharide to the cell surface. Proteins encoded by region B are involved in phospholipid substitution of the capsular polysaccharide. Nucleotide sequence analysis of region B revealed two open reading frames, which encode proteins with molecular masses of 45.1 and 48.7 kDa.     

Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light

The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We describe the characteristics of C. neoformans biofilm development using a microtiter plate model, microscopic examinations, and a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of cryptococci within a biofilm. A strong correlation between XTT and CFU assays was demonstrated. Chemical analysis of the exopolymeric material revealed sugar composition consisting predominantly of xylose, mannose, and glucose, indicating the presence of other polysaccharides in addition to glucurunoxylomannan. Biofilm formation was affected by surface support differences, conditioning films on the surface, characteristics of the medium, and properties of the microbial cell. A specific antibody to the capsular polysaccharide of this fungus was used to stain the extracellular polysaccharide matrix of the fungal biofilms using light and confocal microscopy. Additionally, the susceptibility of C. neoformans biofilms and planktonic cells to environmental stress was investigated using XTT reduction and CFU assays. Biofilms were less susceptible to heat, cold, and UV light exposition than their planktonic counterparts. Our findings demonstrate that fungal biofilm formation is dependent on support surface characteristics and that growth in the biofilm state makes fungal cells less susceptible to potential environmental stresses.