Profile of secreted hydrolases, associated proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the degradation of hemicellulose

• Published in: Applied and environmental microbiology 2014-08, Vol.80 (16), p.5001-5011
• Main Authors: Currie, D H, Guss, A M, Herring, C D, Giannone, R J, Johnson, C M, Lankford, P K, Brown, S D, Hettich, R L, Lynd, L R
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biodegradation, Environmental; Chromatography; Enzymes; Gram-positive bacteria; Hydrolases - genetics; Hydrolases - metabolism; Mass spectrometry; Physiology; Polysaccharides - metabolism; Protein Transport; Proteins; Thermoanaerobacterium - enzymology; Thermoanaerobacterium - genetics; Thermoanaerobacterium - metabolism
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00998-14

Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars.