Abstract

We used a comparative genomics approach implemented in the SEED annotation environment to reconstruct the chitin and GlcNAc utilization subsystem and regulatory network in most proteobacteria, including 11 species of Shewanella with completely sequenced genomes. Comparative analysis of candidate regulatory sites allowed us to characterize three different GlcNAc-specific regulons, NagC, NagR, and NagQ, in various proteobacteria and to tentatively assign a number of novel genes with specific functional roles, in particular new GlcNAc-related transport systems, to this subsystem. Genes SO3506 and SO3507, originally annotated as hypothetical in Shewanella oneidensis MR-1, were suggested to encode novel variants of GlcN-6-P deaminase and GlcNAc kinase, respectively. Reconstitution of the GlcNAc catabolic pathway in vitro using these purified recombinant proteins and GlcNAc-6-P deacetylase (SO3505) validated the entire pathway. Kinetic characterization of GlcN-6-P deaminase demonstrated that it is the subject of allosteric activation by GlcNAc-6-P. Consistent with genomic data, all tested Shewanella strains except S. frigidimarina, which lacked representative genes for the GlcNAc metabolism, were capable of utilizing GlcNAc as the sole source of carbon and energy. This study expands the range of carbon substrates utilized by Shewanella spp., unambiguously identifies several genes involved in chitin metabolism, and describes a novel variant of the classical three-step biochemical conversion of GlcNAc to fructose 6-phosphate first described in Escherichia coli.

Highlights

  • The genus Shewanella is composed of a number of species and strains, all of which are Gram-negative ␥-proteobacteria

  • This study expands the range of carbon substrates utilized by Shewanella spp., unambiguously identifies several genes involved in chitin metabolism, and describes a novel variant of the classical three-step biochemical conversion of GlcNAc to fructose 6-phosphate first described in Escherichia coli

  • Shewanella oneidensis MR-1 was originally isolated from freshwater sediments of Oneida Lake, NY [1], whereas other species have been isolated from marine sediments, marine waters, and a variety of other environments [2]

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Summary

The abbreviations used are

Fru-6-P, fructose 6-phosphate; GlcNAc-6-P, N-acetylglucosamine-6-phosphate; NAG pathway, N-acetylglucosamine catabolic pathway; PTS, phosphoenolpyruvate-carbohydrate phosphotransferase system; GlcN-6-P, glucosamine 6-phosphate; ManNAc-6-P, N-acetylmannosamine 6-phosphate; Ni-NTA, nickel-nitrilotriacetic acid; PIPES, 1,4-piperazinediethanesulfonic acid. Characterized in this study) provides the first strong evidence of an elaborate chitin utilization machinery, which is likely to be conserved in many Shewanella spp

EXPERIMENTAL PROCEDURES
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