Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose Synthase in the Plant Pathogenic Bacterium Ralstonia solanacearum Strain GMI1000, a Distinct Member of the 4,6-Dehydratase and Decarboxylase Family

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-β-l-threo-pentopyranosyl-4″-ulose in the presence of NAD⁺. The second activity converts UDP-β-l-threo-pentopyranosyl-4″-ulose and NADH to UDP-xylose and NAD⁺, albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.     

Biosynthesis of UDP-glucuronic acid and UDP-galacturonic acid in Bacillus cereus subsp. cytotoxis NVH 391-98

The food borne pathogen Bacillus cereus produces uronic acid-containing glycans that are secreted in a shielding biofilm environment, and certain alkaliphilic Bacillus deposit uronate-glycan polymers in the cell wall when adapting to alkaline environments. The source of these acidic sugars is unknown and, in the present study, we describe the functional identification of an operon in Bacillus cerues subsp. cytotoxis NVH 391-98 that comprises genes involved in the synthesis of UDP-uronic acids in Bacillus spp. Within the operon, a UDP-glucose 6-dehydrogenase converts UDP-glucose in the presence of NAD(+) to UDP-glucuronic acid and NADH, and a UDP-GlcA 4-epimerase (UGlcAE) converts UDP-glucuronic acid to UDP-galacturonic acid. Interestingly, in vitro, both enzymes can utilize the TDP-sugar forms as well, albeit at lower catalytic efficiency. Unlike most of the very few bacterial 4-epimerases that have been characterized, which are promiscuous, the B. cereus UGlcAE enzyme is very specific and cannot use UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylglucosaminuronic acid or UDP-xylose as substrates. Size exclusion chromatography suggests that UGlcAE is active as a monomer, unlike the dimeric form of plant enzymes; the Bacillus UDP-glucose 6-dehydrogenase is also found as a monomer. Phylogenic analysis further suggests that the Bacillus UGlcAE may have evolved separately from other bacterial and plant epimerases. Our results provide insight into the formation and function of uronic acid-containing glycans in the lifecycle of B. cereus and related species containing homologous operons, as well as a basis for determining the importance of these acidic glycans. We also discuss the ability to target UGlcAE as a drug candidate.     

Synthesis of Flavonoid O-Pentosides by Escherichia coli through Engineering of Nucleotide Sugar Pathways and Glycosyltransferase

Plants produce two flavonoid O-pentoses, flavonoid O-xyloside and flavonoid O-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study, Escherichia coli was used to synthesize the plant-specific flavonoid O-pentosides quercetin 3-O-xyloside and quercetin 3-O-arabinoside. Two strategies were used. First, E. coli was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes, UXS (UDP-xylose synthase) from Arabidopsis thaliana and ugd (UDP-glucose dehydrogenase) from E. coli, were overexpressed. In addition, the gene encoding ArnA (UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis, UXE (UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The E. coli strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-O-xyloside and quercetin 3-O-arabinoside.     

Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family,UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms

UDP-xylose (Xyl) is an important sugar donor for the synthesis of glycoproteins, polysaccharides, various metabolites, and oligosaccharides in animals, plants, fungi, and bacteria. UDP-Xyl also feedback inhibits upstream enzymes (UDP-glucose [Glc] dehydrogenase, UDP-Glc pyrophosphorylase, and UDP-GlcA decarboxylase) and is involved in its own synthesis and the synthesis of UDP-arabinose. In plants, biosynthesis of UDP-Xyl is catalyzed by different membrane-bound and soluble UDP-GlcA decarboxylase (UDP-GlcA-DC) isozymes, all of which convert UDP-GlcA to UDP-Xyl. Because synthesis of UDP-Xyl occurs both in the cytosol and in membranes, it is not known which source of UDP-Xyl the different Golgi-localized xylosyltransferases are utilizing. Here, we describe the identification of several distinct Arabidopsis genes (named AtUXS for UDP-Xyl synthase) that encode functional UDP-GlcA-DC isoforms. The Arabidopsis genome contains five UXS genes and their protein products can be subdivided into three isozyme classes (A-C), one soluble and two distinct putative membrane bound. AtUxs from each class, when expressed in Escherichia coli, generate active UDP-GlcA-DC that converts UDP-GlcA to UDP-Xyl. Members of this gene family have a large conserved C-terminal catalytic domain (approximately 300 amino acids long) and an N-terminal variable domain differing in sequence and size (30-120 amino acids long). Isoforms of class A and B appear to encode putative type II membrane proteins with their catalytic domains facing the lumen (like Golgi-glycosyltransferases) and their N-terminal variable domain facing the cytosol. Uxs class C is likely a cytosolic isoform. The characteristics of the plant Uxs support the hypothesis that unique UDP-GlcA-DCs with distinct subcellular localizations are required for specific xylosylation events.     

Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance

The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 A conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors.     

The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases

Glutamate decarboxylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II. Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.     

Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases

The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.      

Stereo-electronic control of reaction selectivity in short-chain dehydrogenases: Decarboxylation,epimerization, and dehydration

Sugar nucleotide–modifying enzymes of the short-chain dehydrogenase/reductase type use transient oxidation–reduction by a tightly bound nicotinamide cofactor as a common strategy of catalysis to promote a diverse set of reactions, including decarboxylation, single- or double-site epimerization, and dehydration. Although the basic mechanistic principles have been worked out decades ago, the finely tuned control of reactivity and selectivity in several of these enzymes remains enigmatic. Recent evidence on uridine 5'-diphosphate (UDP)-glucuronic acid decarboxylases (UDP-xylose synthase, UDP-apiose/UDP-xylose synthase) and UDP-glucuronic acid-4-epimerase suggests that stereo-electronic constraints established at the enzyme's active site control the selectivity, and the timing of the catalytic reaction steps, in the conversion of the common substrate toward different products. The mechanistic idea of stereo-electronic control is extended to epimerases and dehydratases that deprotonate the Cα of the transient keto-hexose intermediate. The human guanosine 5'-diphosphate (GDP)-mannose 4,6-dehydratase was recently shown to use a minimal catalytic machinery, exactly as predicted earlier from theoretical considerations, for the β-elimination of water from the keto-hexose species.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance

Gram-negative bacteria have evolved mechanisms to resist the bactericidal action of cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. The strategy involves the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A in their outer membrane. ArnA is a key enzyme in the Ara4N-lipid A modification pathway. It is a bifunctional enzyme catalyzing (1) the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) to the UDP-4' '-ketopentose [UDP-beta-(l-threo-pentapyranosyl-4' '-ulose] and (2) the N-10-formyltetrahydrofolate-dependent formylation of UDP-Ara4N. Here we demonstrate that the transformylase activity of the Escherichia coli ArnA is contained in its 300 N-terminal residues. We designate it the ArnA transformylase domain and describe its crystal structure solved to 1.7 A resolution. The enzyme adopts a bilobal structure with an N-terminal Rossmann fold domain containing the N-10-formyltetrahydrofolate binding site and a C-terminal subdomain resembling an OB fold. Sequence and structure conservation around the active site of ArnA transformylase and other N-10-formyltetrahydrofolate-utilizing enzymes suggests that the HxSLLPxxxG motif can be used to identify enzymes that belong to this family. Binding of an N-10-formyltetrahydrofolate analogue was modeled into the structure of ArnA based on its similarity with glycinamide ribonucleotide formyltransferase. We also propose a mechanism for the transformylation reaction catalyzed by ArnA involving residues N(102), H(104), and D(140). Supporting this hypothesis, point mutation of any of these residues abolishes activity.     

Bacteroides fragilis synthesizes a DNA invertase affecting both a local and a distant region

The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of Bacteroides fragilis was characterized. Its gene, tsr19, is adjacent to mpi, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of B. fragilis, Tsr19 brings about inversion of two DNA regions, one local and one located distantly.     

Identification of Bacteroides fragilisby a modified immuno polymerase chain reaction (mIPCR), using a specific monoclonal antibody

Bacteroides fragilis is the anaerobic species most commonly isolated from human clinical specimens, and is resistant to many antimicrobial agents. A monoclonal antibody, mAb4H8 (IgG3), reacting with a specific epitope in the lipopolysaccharide (LPS) isolated from most of the B. fragilis strains, was produced and employed with modified Immuno Polymerase Chain Reaction (mIPCR) for identification of B. fragilis with a detection limit of 10(4) cfu/mL bacterial suspension. A number of bacterial strains were examined, including B. fragilis, Bacteroides spp. other than B. fragilis and other genera. All the B. fragilis strains with the immunodominant (beta1,6-linked D-galactosyl chain) epitope were positive. None of the other strains showed the positive reaction. The results indicate that mIPCR assay with mAb4H8 has a high specificity and high sensitivity.     

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography–mass spectrometry.     

Structural motifs for pyridoxal-5'-phosphate binding in decarboxylases: an analysis based on the crystal structure of the Lactobacillus 30a ornithine decarboxylase.

Two of the five domains in the structure of the ornithine decarboxylase (OrnDC) from Lactobacillus 30a share similar structural folds around the pyridoxal-5''-phosphate (PLP)-binding pocket with the aspartate aminotransferases (AspATs). Sequence comparisons focusing on conserved residues of the aligned structures reveal that this structural motif is also present in a number of other PLP-dependent enzymes including the histidine, dopa, tryptophan, glutamate, and glycine decarboxylases as well as tryptophanase and serine-hydroxymethyl transferase. However, this motif is not present in eukaryotic OrnDCs, the diaminopimelate decarboxylases, nor the Escherichia coli or oat arginine decarboxylases. The identification and comparison of residues involved in defining the different classes are discussed.     

Bacteroides and appendicitis (author's transl)]

Bacteroides fragilis is frequently recovered from cases of appendicitis with perforation and from infections developing secondary to appendicitis. In order to assess the part played by B. fragilis in the aetiology of appendicitis, quantitative aerobic and anaerobic culture studies of the contents of 49 inflammated appendices were performed. Anaerobic gram-negative non-sporing rods were cultivated from 43 appendices in the range 10(3)-10(9)/g. A total of 1,473 isolates was differentiated by biochemical methods, and 1,374 cultures were found to belong to the saccharolytic species of the genus Bacteroides (B. fragilis, B. thetaiotaomicron, B. vulgatus, B. distasonis etc.). B fragilis was detected in 31 appendices; the species predominated in 18 samples. B theraiotamicron, recovered from 27 samples, was prevalent in 4 appendices. In one sample, B. fragilis and B. thetaiotaomicron outnumbered the other appendicular bacterial. B. vulgatus was cultivated from 12 appendices, but did once constitute the prevalent group. It has been previously shown that B. vulgatus (43% of intestinal isolates) and B. thetaiomicron predominate in the normal narge bowel flora. On the other hand, approximately 80% of pyrogenic Bacteroides strains belong to B. fragilis, B. thetaiotaomicron accounting for 19% and B. vulgatus being virtually absent. From these striking differences in species distribution the conclusion was drawn that B. fragilis possesses the highest virulence for man. Species distribution within the 1,374 appendicular isolates of saccharolytic Bacteroides (percentages of 62, 19 and 4.3 for B. fragilis, B. thetaiotaomicron, and B. vulgatus, respectively) was very similar to that encountered in clinical specimens. From the results obtained it becomes evident that pyrogenic Bacteroides, in particular B. fragilis, plays an important role in nearly 50% of cases of appendicitis.     

Distribution of the human faecal bacterium Bacteroides fragilis, its bacteriophages and their relationship to current sewage pollution indicators in bathing water

Although several bacteria are currently used as possible indicators of human pathogens in sewage-polluted sea water, they are often viewed as inadequate and especially inadequate as indicators of viral pathogens. This study investigates the distribution of Bacteroides fragilis and closely related Bacteroides spp. and their associated bacteriophages in sea water frequently used for recreational purposes. These organisms may provide a potentially more appropriate indicator. Bacteroides fragilis is one of about 10 species which are loosely placed together in the 'B. fragilis' group. Samples down-current from a sewage outfall were examined for the presence of B. fragilis group organisms and associated bacteriophages. Numbers were correlated with current bacterial and possible viral indicators at these sites. These B. fragilis group isolates were used as hosts to successfully isolate bacteriophages. The host range of these bacteriophages was investigated. It is hoped to expand this study by using these B. fragilis group hosts and their bacteriophages to identify a more suitable, European-wide, indicator of bacterial pathogens which can also be used to detect bacteriophages which are suitable as viral indicators.     

Crystal structure of Escherichia coli ArnA (PmrI) decarboxylase domain. A key enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance

Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. ArnA is a key enzyme in the lipid A modification pathway, and its deletion abolishes both the Ara4N-lipid A modification and polymyxin resistance. ArnA is a bifunctional enzyme. It can catalyze (i) the NAD(+)-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the N-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose. We show that the NAD(+)-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of ArnA. This domain is separable from the N-terminal fragment, and its activity is identical to that of the full-length enzyme. The crystal structure of the ArnA decarboxylase domain from E. coli is presented here. The structure confirms that the enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family. On the basis of sequence and structure comparisons of the ArnA decarboxylase domain with other members of the short-chain dehydrogenase/reductase (SDR) family, we propose a binding model for NAD(+) and UDP-glucuronic acid and the involvement of residues T(432), Y(463), K(467), R(619), and S(433) in the mechanism of NAD(+)-dependent oxidation of the 4'-OH of the UDP-glucuronic acid and decarboxylation of the UDP-4-keto-glucuronic acid intermediate.     

Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch

The extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, and elongation with N-acetylglucosamine, catalyzed by members of the Fringe family, modulates Notch activity. Only recently, genes encoding enzymes involved in the O-glucosylation pathway have been cloned. In the Drosophila mutant rumi, characterized by a mutation in the protein O-glucosyltransferase, Notch signaling is impaired in a temperature-dependent manner, and a mouse knock-out leads to embryonic lethality. We have previously identified two human genes, GXYLT1 and GXYLT2, encoding glucoside xylosyltransferases responsible for the transfer of xylose to O-linked glucose. The identity of the enzyme further elongating the glycan to generate the final trisaccharide xylose-xylose-glucose, however, remained unknown. Here, we describe that the human gene C3ORF21 encodes a UDP-xylose:α-xyloside α1,3-xylosyltransferase, acting on xylose-α1,3-glucoseβ1-containing acceptor structures. We have, therefore, renamed it XXYLT1 (xyloside xylosyltransferase 1). XXYLT1 cannot act on a synthetic acceptor containing an α-linked xylose alone, but requires the presence of the underlying glucose. Activity on Notch EGF repeats was proven by in vitro xylosylation of a mouse Notch1 fragment recombinantly produced in Sf9 insect cells, a bacterially expressed EGF repeat from mouse Notch2 modified in vitro by Rumi and Gxylt2 and in vivo by co-expression of the enzyme with the Notch1 fragment. The enzyme was shown to be a typical type II membrane-bound glycosyltransferase localized in the endoplasmic reticulum.     

Evidence that polygalacturonic acid may not be a major source of carbon and energy for some colonic Bacteroides species.

Five Bacteroides species that are found in the human colon can utilize polygalacturonic acid (PGA) when they are grown in laboratory media: Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis subsp. a, and Bacteroides sp. strain 3452A (an unnamed DNA-DNA homology group). PGA-degrading enzymes from B. thetaiotaomicron have been isolated and characterized previously. To determine whether a PGA lyase activity in human feces could be attributed to any of these species, we first determined the properties of PGA lyases from the other four Bacteroides species. PGA lyases from all the Bacteroides species were soluble, cell associated, and inducible by PGA. All had similar pH optima (8.4 to 8.8) and similar molecular weights (50,000). All activities were enhanced by calcium. The PGA lyases from the five species differed with respect to isoelectric point: B. thetaiotaomicron (pI 7.5), B. vulgatus (pI 7.7), B. ovatus (pI 5.8, 7.2), B. fragilis subsp. a (pI 6.1), and Bacteroides sp. strain 3452A (pI 7.7). The PGA lyase activity in human feces resembled those of the Bacteroides PGA lyases in that it had a pH optimum of 8.4 to 8.8 and was enhanced by calcium. However, it differed from the Bacteroides PGA lyases both with respect to isoelectric point (pI 4.2 to 4.4) and molecular weight (100,000). On the basis of these findings, it appears that the PGA lyase activity in human feces is not produced by any of the Bacteroides species surveyed in this survey. Moreover, there was no detectable PGA lyase activity in feces that had the same properties as the Bacteroides enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)