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  • Depending upon the varied habitats e g soil


    Depending upon the varied habitats (e.g. soil, water, animal digestive tracts, etc.), microbes possess a diverse range of carbohydrate ABC transporters and metabolic pathways (Rodionov et al., 2013; Bräsen et al., 2014). Thermus thermophilus HB8, a thermophilic gram-negative bacterium dwelling in hot springs, utilizes a limited number of SU5416 as carbon and energy source. Unlike several other bacteria, it harbors only major facilitator superfamily (MFS, a class of secondary transporters) and ABC transporter superfamily (a class of primary active transporters) for carbohydrate uptake (Elbourne et al., 2017). The bacterium T. thermophilus HB8 consists of 127 open reading frames (ORFs) encoding ABC transporter subunits. Out of which, a total of 35 ORFs have been annotated to encode 11 carbohydrate ABC transport systems including 11 SBP, 19 TMD and 5 NBD subunits. Quite interestingly, 9 out of 11 carbohydrate ABC transport systems lack their corresponding NBD subunits (Elbourne et al., 2017). In many gram-positive bacteria, the operons encoding ABC transporters of the CUT1 family are known to be devoid of NBD subunits. In addition, the genes encoding NBD components of carbohydrate ABC transporters are observed to be located at a distant position on the genome (Ferreira and Sa-Nogueira, 2010; Marion et al., 2011; Wuttge et al., 2012). Earlier studies have suggested a sharing mechanism of the NBD subunits among several carbohydrate ABC transporters to compensate for their low number (Eitinger et al., 2011). Despite having identified the subunits of carbohydrate ABC transporters in T. thermophilus HB8, their function remains underexplored. The usual method of sequence-similarity-based characterization of ABC transporters to assign their cognate ligands is often inadequate. Thus, in addition to the sequence-based analysis, integrating the information of genetic contents, their organization and regulation as well as metabolomic analysis are known to significantly enhance the preciseness of gene annotation (Rodionov et al., 2013). Detailed information regarding carbohydrate transport and metabolism has been made available from many model organisms such as Thermotoga maritima (Conners et al., 2005). However, projection of these information for the study of carbohydrate transport in distantly related bacteria is difficult due to different habitats. Thus, it becomes essential to perform a functional annotation of carbohydrate transporters considering the bacterium-specific carbohydrate metabolism. In this study, we have attempted to assign the cognate substrate to each carbohydrate ABC transporter as well as correlate their function with the carbohydrate metabolic network in T. thermophilus HB8. To fulfill this purpose, a total of 35 ORFs encoding the 11 putative carbohydrate ABC transport systems and 106 ORFs encoding proteins involved in carbohydrate metabolism have been investigated. Since the UgpABCE (uptake glycerol phosphate) transporters are the members of the CUT1 family and are also the closest homolog of the sugar ABC transporters, they often get misannotated as sugar ABC transporters. Thus, we have also performed functional annotation of the UgpABCE transporters. In summary, we present the functional characterization of six sugar, four Ugp and one purine ABC transporter(s) utilizing the approaches of sequence-, structure-, genetic- and metabolic-based analysis.
    Materials and methods
    Results and discussion
    Conclusion This study demonstrates an integrated approach for the functional annotation of carbohydrate ABC transporters, where cognate ligand for each transporter is verified by their metabolic profiling. Through this study, a total of 11 putative carbohydrate ABC transporters from T. thermophilus HB8 are identified and their cognate ligands are assigned. Out of these, six are characterized as sugar, four as phospholipid (viz. UgpABCE) and the remaining one as purine ABC importer. This study also supports the relationship between microbial habitats and the carbohydrate diversity. Functional annotation of carbohydrate ABC transporters and its metabolic profiling suggest the transport and synthesis of two major osmolytes viz. trehalose and mannosylglycerate. In addition, the bacterium T. thermophilus HB8 seems to transport starch- and cellulose-degraded products as carbon and energy source. Apart from cognate ligand identification, this study also suggests the hypothesis of the sharing phenomenon of ABC transporter subunits. Further, a thorough analysis of ABC transporter subunits reveals that the NBDs are not coexistent with the carbohydrate ABC transporter operons and thus are shared among multiple carbohydrate ABC transporters. Interestingly, UgpABCE transporters lack both the NDB as well as TMD components and hence seem to borrow from other sugar ABC transport systems. In summary, the predicted carbohydrate metabolism network along with the carbohydrate ABC transport systems suggest that most of the carbohydrate ABC transporters are interlinked by carbohydrate metabolism which, in turn, may help the bacterium to survive even in the scarcity of some carbohydrates.