The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of L-guluronic acid, D-glucose, and D-glycero-D-mannoheptose.13 C-labeled EPS and 13 C-labeled cellulose were purified from bacterial cultures grown on [ 13 C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from 13 C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo-or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species. Some major phyla of the domain Bacteria, like the Proteobacteria and Actinobacteria, are well-known from cultivation studies. Others, like Acidobacteria, Verrucomicrobia, and Planctomycetes, have few cultured species, although they make up large proportions of the bacterial 16S rRNA genes detected in environments such as soil (1). One theory proposed to explain the difficulty in culturing bacteria from these phyla is that most are K selected as opposed to r selected. Typical r-selected bacteria grow rapidly on simple monomeric substrates in nutrient-rich environments, conditions presented by most microbiological media (2, 3). On the other hand, K-selected bacteria have a more efficient cell metabolism and strong competitive ability and grow slowly on recalcitrant, complex substances (3-5). Several studies have demonstrated that members of difficult-to-culture phyla like Acidobacteria, Verrucomicrobia, and Planctomycetes can be cultivated using polysaccharides, such as xylan, xanthan, and pectin, as energy sources (e.g., see references 6 to 8). The heteropolysaccharide gellan, which is often used as a gelling agent as an alternative to agar, can also act as an energy source for some soil bacteria (9), in part explaining the observation that it improves cultu...
Many archaeal proteins undergo posttranslational modifications. S-layer proteins and flagellins have been used successfully to study a variety of these modifications, including N-linked glycosylation, signal peptide removal and lipid modification. Use of these well-characterized reporter proteins in the genetically tractable model organisms, Haloferax volcanii, Methanococcus voltae and Methanococcus maripaludis, has allowed dissection of the pathways and characterization of many of the enzymes responsible for these modifications. Such studies have identified archaeal-specific variations in signal peptidase activity not found in the other domains of life, as well as the enzymes responsible for assembly and biosynthesis of novel N-linked glycans. In vitro assays for some of these enzymes have already been developed. N-linked glycosylation is not essential for either Hfx. volcanii or the Methanococcus species, an observation that allowed researchers to analyze the role played by glycosylation in the function of both S-layers and flagellins, by generating mutants possessing these reporters with only partial attached glycans or lacking glycan altogether. In future studies, it will be possible to consider questions related to the heterogeneity associated with given modifications, such as differential or modulated glycosylation.
The genetics and biochemistry of the N-linked glycosylation system of Archaea have been investigated over the past 5 years using flagellins and S layers as reporter proteins in the model organisms, Methanococcus voltae, Methanococcus maripaludis, and Haloferax volcanii. Structures of archaeal N-linked glycans have indicated a variety of linking sugars as well as unique sugar components. In M. voltae, M. maripaludis, and H. volcanii, a number of archaeal glycosylation genes (agl) have been identified by deletion and complementation studies. These include many of the glycosyltransferases and the oligosaccharyltransferase needed to assemble the glycans as well as some of the genes encoding enzymes required for the biosynthesis of the sugars themselves. The N-linked glycosylation system is not essential for any of M. voltae, M. maripaludis, or H. volcanii, as demonstrated by the successful isolation of mutants carrying deletions in the oligosaccharyltransferase gene aglB (a homologue of the eukaryotic Stt3 subunit of the oligosaccharyltransferase complex). However, mutations that affect the glycan structure have serious effects on both flagellation and S layer function.
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