Assessment of microbial communities by graph partitioning in a study of soil fungi in two Alpine meadows.

Understanding how microbial community structure and diversity respond to environmental conditions is one of the main challenges in environmental microbiology. However, there is often confusion between determining the phylogenetic structure of microbial communities and assessing the distribution and diversity of molecular operational taxonomic units (MOTUs) in these communities. This has led to the use of sequence analysis tools such as multiple alignments and hierarchical clustering that are not adapted to the analysis of large and diverse data sets and not always justified for characterization of MOTUs. Here, we developed an approach combining a pairwise alignment algorithm and graph partitioning by using MCL (Markov clustering) in order to generate discrete groups for nuclear large-subunit rRNA gene and internal transcript spacer 1 sequence data sets obtained from a yearly monitoring study of two spatially close but ecologically contrasting alpine soils (namely, early and late snowmelt locations). We compared MCL with a classical single-linkage method (Ccomps) and showed that MCL reduced bias such as the chaining effect. Using MCL, we characterized fungal communities in early and late snowmelt locations. We found contrasting distributions of MOTUs in the two soils, suggesting that there is a high level of habitat filtering in the assembly of alpine soil fungal communities. However, few MOTUs were specific to one location.

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene.

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.

Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases.

Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.

Production of O-acetylated and sulfated chitooligosaccharides by recombinant Escherichia coli strains harboring different combinations of nod genes.

High cell density cultivation of recombinant Escherichia coli strains harboring the nodBC genes (encoding chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose and tetra-N-acetylchitopentaose (Samain, E., Drouillard, S., Heyraud, A., Driguez, H., Geremia, R.A., 1997. Carbohydr. Res. 30, 235-242). We have now extended this method to the production of sulfated and O-acetylated derivatives of these two compounds by coexpressing nodC or nodBC with nodH and/or nodL that encode chitooligosaccharide sulfotransferase and chitooligosaccharide O-acetyltransferase, respectively. In addition, these substituted chitooligosaccharides were also obtained as tetramers by using nodC from Rhizobium meliloti instead of nodC from A. caulinodans. These compounds should be useful precursors for the preparation of Nod factor analogues by chemical modification.

A (1----4)-alpha-D-glucan-protein involved in liver glycogen biosynthesis.

A macromolecular (1----4)-alpha-D-[14C]glucan-protein complex was synthesized with a rat liver preparation and uridine diphosphate D-[14C]glucose. The size of the complex is contributed by both the protein and the (1----4)-alpha-D-glucosyl-oligomer components. Iodoacetamide treatment did not change the migration properties on Bio-Gel A-50m. Therefore, disulfide bonds linking glucan-protein subunits seem not to be involved. The [14C]glucan-protein, precipitated by diluted trichloroacetic acid, was digested by alpha-amylase, phosphorylase a, and proteases. The extent of proteolysis was greater for a complex having fewer D-glucose units incorporated. After proteolytic digestion of that complex, the labeled fragments behaved on electrophoresis, and ion-exchange and gel chromatography as [14C]glucosylated peptides. These findings support previous conclusions that the primer for liver glycogen synthesis is a protein on which glycogen is built up by covalent attachment.

Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli.

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.

Microbes on the cliff: alpine cushion plants structure bacterial and fungal communities.

Plants affect the spatial distribution of soil microorganisms, but the influence of the local abiotic context is poorly documented. We investigated the effect of a single plant species, the cushion plant Silene acaulis, on habitat conditions, and microbial community. We collected soil from inside (In) and outside (Out) of the cushions on calcareous and siliceous cliffs in the French Alps along an elevation gradient (2,000-3,000 masl). The composition of the microbial communities was assessed by Capillary-Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP). Univariate and multivariate analyses were conducted to characterize the response of the microbial beta-diversity to soil parameters (total C, total N, soil water content, [Formula: see text], and pH). Cushions affected the microbial communities, modifying soil properties. The fungal and bacterial communities did not respond to the same abiotic factors. Outside the cushions, the bacterial communities were strongly influenced by bedrock. Inside the cushions, the bacterial communities from both types of bedrock were highly similar, due to the smaller pH differences than in open areas. By contrast, the fungal communities were equally variable inside and outside of the cushions. Outside the cushions, the fungal communities responded weakly to soil pH. Inside the cushions, the fungal communities varied strongly with bedrock and elevation as well as increases in soil nutrients and water content. Furthermore, the dissimilarities in the microbial communities between the In and Out habitats increased with increasing habitat modification and environmental stress. Our results indicate that cushions act as a selective force that counteracts the influence of the bedrock and the resource limitations on the bacterial and fungal communities by buffering soil pH and enhancing soil nutrients. Cushion plants structure microbial communities, and this effect increases in stressful, acidic and nutrient-limited environments.

Expression and study of recombinant ExoM, a beta1-4 glucosyltransferase involved in succinoglycan biosynthesis in Sinorhizobium meliloti.

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative beta1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti. The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter. Escherichia coli BL21(DE3)/pLysS containing exoM expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in E. coli as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region. E. coli membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an S. meliloti mutant strain which accumulates the ExoM substrate (Glcbeta1-4Glcbeta1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a beta1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcbeta1-4Glcbeta1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.

Identification of essential amino acids in the Azorhizobium caulinodans fucosyltransferase NodZ.

The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an alpha 1-6 linkage to the reducing N-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors. Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity. Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides. These peptide motifs are thought to play important roles in catalysis. NodZ was expressed as an active and soluble form in Escherichia coli and was subjected to site-directed mutagenesis to investigate the role of the most conserved residues. Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity. A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form. The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily. Secondary structure predictions and threading analyses suggest the presence of a "Rossmann-type" nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases. Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases.

Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM.

ExoM is a beta(1-4)-glucosyltransferase involved in the assembly of the repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti. By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic acid residues of ExoM were identified. Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitro activity but could restore succinoglycan production in vivo. However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro and in vivo assays. These results indicate that Asp-44, Asp-96, and Asp-187 are essential for the activity of ExoM. Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycosyltransferase reaction.

A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan.

A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form beta-(1----2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in beta-(1----2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type.

Osmotic regulation of beta(1-2) glucan synthesis in members of the family Rhizobiaceae.

High osmolarity in the culture medium of growing Agrobacterium tumefaciens strongly inhibited the accumulation of cellular beta(1-2) glucan. However, the enzymatic system required for the synthesis of this polysaccharide from UDP-glucose was not repressed by high osmolarity. Mutants of A. tumefaciens and Rhizobium meliloti affected in beta(1-2) glucan synthesis were unable to grow normally in low-osmolarity media.

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Identification of two essential glutamic acid residues in glycogen synthase.

The detailed catalytic mechanism by which glycosyltransferases catalyze the transfer of a glycosyl residue from a donor sugar to an acceptor is not known. Through the multiple alignment of all known eukaryotic glycogen synthases we have found an invariant 17-amino acid stretch enclosed within the most conserved region of the members of this family. This peptide includes an E-X(7)-E motif, which is highly conserved in four families of retaining glycosyltransferases. Site-directed mutagenesis was performed in human muscle glycogen synthase to analyze the roles of the two conserved Glu residues (Glu-510 and Glu-518) of the motif. Proteins were transiently expressed in COS-1 cells as fusions to green fluorescence protein. The E510A and E518A mutant proteins retained the ability to translocate from the nucleus to the cytosol in response to glucose and to bind to intracellular glycogen. Although the E518A variant had approximately 6% of the catalytic activity shown by the green fluorescence protein-human muscle glycogen synthase fusion protein, the E510A mutation inactivated the enzyme. These results led us to conclude that the E-X(7)-E motif is part of the active site of eukaryotic glycogen synthases and that both conserved Glu residues are involved in catalysis. We propose that Glu-510 may function as the nucleophile and Glu-518 as the general acid/base catalyst.

Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA.

The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.

The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase.

Nod factors are signal molecules produced by Azorhizobium, Bradyrhizobium, and Rhizobium species that trigger nodule formation in leguminous host plants. The backbone of Nod factors consists of a beta-1,4-N-acetylglucosamine oligosaccharide from which the N-acetyl group at the nonreducing end is replaced by a fatty acid. The nodABC gene products are necessary for backbone biosynthesis. By incubation of cell extracts from Azorhizobium caulinodans with radioactive uridine diphosphate-N-acetylglucosamine, Nod factor precursors were identified and characterized as beta-1,4-N-acetylglucosamine oligosaccharides. By analysis of different nod gene mutants and by expression of nodC in Escherichia coli, the N-acetylglucosaminyltransferase activity was ascribed to the NodC protein. The results suggest that the first step in biosynthesis of Nod factors is the assembly of the oligosaccharide chain.

Phytanyl-pyrophosphate-linked substrate for a bacterial alpha-mannosyltransferase.

The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate. In order to find a suitable synthetic substrate for the alpha1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared. In the presence of GDP-[14C]mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a 14C-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate. These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.

Towards a classification of glycosyltransferases based on amino acid sequence similarities: prokaryotic alpha-mannosyltransferases.

A number of genes encoding bacterial glycosyltransferases have been sequenced during the last few years, but their low sequence similarity has prevented a straightforward grouping of these enzymes into families. The sequences of several bacterial alpha-mannosyltransferases have been compared using current alignment algorithms as well as hydrophobic cluster analysis (HCA). These sequences show a similarity which is significant but too low to be reliably aligned using automatic alignment methods. However, a region spanning approx. 270 residues in these proteins could be aligned by HCA, and several invariant amino acid residues were identified. These features were also found in several other glycosyltransferases, as well as in proteins of unknown function present in sequence databases. This similarity most probably reflects the existence of a family of proteins with conserved structural and mechanistic features. It is argued that the present IUBMB classification of glycosyltransferases could be complemented by a classification of these enzymes based on sequence similarities analogous to that which we proposed for glycosyl hydrolases [Henrissat, B. (1991) Biochem. J. 280, 309-316].

Identification of nodSUIJ genes in Nod locus 1 of Azorhizobium caulinodans: evidence that nodS encodes a methyltransferase involved in Nod factor modification.

The Azorhizobium caulinodans strain ORS571 nodulation genes nodSUIJ were located downstream from nodABC. Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon. Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp. strain NGR234, contain a consensus with similarity to S-adenosylmethionine (SAM)-utilizing methyltransferases. A naringenin-inducible nodS-dependent protein of approximately 25 kDa could be cross-linked to radiolabelled SAM. By applying L-[methyl-3H]-methionine in vivo, Nod factors of ORS571, known to be N-methylated, could be labelled in wild type and nodU mutants but not in nodS mutants. Therefore, we propose that NodS is a SAM-utilizing methyltransferase involved in Nod factor synthesis.

Electrophoretic karyotype and gene assignment to resolved chromosomes of Trichoderma spp.

A molecular karyotype for three different Trichoderma species (T. harzianum, T. viride, and T. reesei) was determined by using two different systems: countour-clamped electric-field and rotating-electrode electrophoresis. Six chromosomal DNA bands were observed in T. harzianum and T. reesei and five in T. viride. The sizes of these molecules were estimated by their mobility relative to the Schizosaccharomyces pombe chromosomes and ranged between 2.2 and 7.4 megabase pairs (mbp). The estimated genome sizes range from 31 to 39 mbp. A number of genes were located in the different chromosomes by means of Southern analysis. The implications of these findings are discussed.