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Influence of in situ progressive N-terminal is still controversial truncation of glycogen branching enzyme in Escherichia coli DH5? on glycogen structure, accumulation, and bacterial viability.

ABSTRACT: Glycogen average chain length (ACL) has been linked with bacterial durability, but this was on the basis of observations across different species. We therefore wished to investigate the relationship between bacterial durability and glycogen ACL by varying glycogen average chain length in a single species. It has been shown that progressive shortening of the N-terminus of glycogen branching enzyme (GBE) leads to a lengthening of oligosaccharide inter-?-1,6-glycosidic chain lengths, so we sought to harness this to create a set of Escherichia coli DH5? strains with a range of glycogen average chain lengths, and assess these strains for durability related attributes, such as starvation, cold and desiccation stress resistance, and biofilm formation.A series of Escherichia coli DH5? mutants were created with glgB genes that were in situ progressively N-terminus truncated. N-terminal truncation shifted the distribution of glycogen chain lengths from 5-11 DP toward 13-50 DP, but the relationship between glgB length and glycogen ACL was not linear. Surprisingly, removal of the first 270 nucleotides of glgB (glgB?270) resulted in comparatively high glycogen accumulation, with the glycogen having short ACL. Complete knockout of glgB led to the formation of amylose-like glycogen containing long, linear ?1,4-glucan chains with significantly reduced branching frequency. Physiologically, the set of mutant strains had reduced bacterial starvation resistance, while minimally increasing bacterial desiccation resistance. Finally, although there were no obvious changes in cold stress resistance or biofilm forming ability, one strain (glgB?180) had significantly increased biofilm formation in favourable media.Despite glgB being the first gene of an operon, it is clear that in situ mutation is a viable means to create more biologically relevant mutant strains. Secondly, there was the suggestion in the data that impairments of starvation, cold and desiccation resistance were worse for the strain lacking glgB, though the first of these was not statistically significant. The results provide prima facie evidence linking abiotic stress tolerance with shorter glycogen ACL. However, further work needs to be done, perhaps in a less labile species. Further work is also required to tease out the complex relationship between glycogen abundance and glycogen structure.

SUBMITTER: Wang L

PROVIDER: S-EPMC4433092 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Influence of in situ progressive N-terminal is still controversial truncation of glycogen branching enzyme in Escherichia coli DH5α on glycogen structure, accumulation, and bacterial viability.

Wang Liang L Regina Ahmed A Butardo Vito M VM Kosar-Hashemi Behjat B Larroque Oscar O Kahler Charlene M CM Wise Michael J MJ

BMC microbiology 20150507

<h4>Background</h4>Glycogen average chain length (ACL) has been linked with bacterial durability, but this was on the basis of observations across different species. We therefore wished to investigate the relationship between bacterial durability and glycogen ACL by varying glycogen average chain length in a single species. It has been shown that progressive shortening of the N-terminus of glycogen branching enzyme (GBE) leads to a lengthening of oligosaccharide inter-α-1,6-glycosidic chain leng ...[more]

PMID: 25947105

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Project description:BACKGROUND:Branched polymers of glucose are universally used for energy storage in cells, taking the form of glycogen in animals, fungi, Bacteria, and Archaea, and of amylopectin in plants. Some enzymes involved in glycogen and amylopectin metabolism are similarly conserved in all forms of life, but some, interestingly, are not. In this paper we focus on the phylogeny of glycogen branching and debranching enzymes, respectively involved in introducing and removing of the ?(1-6) bonds in glucose polymers, bonds that provide the unique branching structure to glucose polymers. RESULTS:We performed a large-scale phylogenomic analysis of branching and debranching enzymes in over 400 completely sequenced genomes, including more than 200 from eukaryotes. We show that branching and debranching enzymes can be found in all kingdoms of life, including all major groups of eukaryotes, and thus were likely to have been present in the last universal common ancestor (LUCA) but have been lost in seemingly random fashion in numerous single-celled eukaryotes. We also show how animal branching and debranching enzymes evolved from their LUCA ancestors by acquiring additional domains. Furthermore, we show that enzymes commonly perceived as orthologous, such as human branching enzyme GBE1 and E. coli branching enzyme GlgB, are in fact related by a gene duplication and consequently paralogous. CONCLUSIONS:Despite being usually associated with animal liver glycogen and plant starch, energy storage in the form of branched glucose polymers is clearly an ancient process and has probably been present in the last universal common ancestor of all present life. The evolution of the enzymes enabling this form of energy storage is more complex than previously thought and illustrates the need for explicit phylogenomic analysis in the study of even seemingly "simple" metabolic enzymes. Patterns of conservation in the evolution of the glycogen/starch branching and debranching enzymes hint at some as yet unknown mechanisms, as mutations disrupting these patterns lead to a variety of genetic diseases in humans and other mammals.

Project description:In bacteria, glycogen plays important roles in carbon and energy storage. Its structure has recently been linked with bacterial environmental durability. Among the essential genes for bacterial glycogen metabolism, the glgB-encoded branching enzyme GBE plays an essential role in forming α-1,6-glycosidic branching points, and determines the unique branching patterns in glycogen. Previously, evolutionary analysis of a small sets of GBEs based on their N-terminal domain organization revealed that two types of GBEs might exist: (1) Type 1 GBE with both N1 and N2 (also known as CBM48) domains and (2) Type 2 GBE with only the N2 domain. In this study, we initially analyzed N-terminal domains of 169 manually reviewed bacterial GBEs based on hidden Markov models. A previously unreported group of GBEs (Type 3) with around 100 amino acids ahead of the N1 domains was identified. Phylogenetic analysis found clustered patterns of GBE types in certain bacterial phyla, with the shorter, Type 2 GBEs predominantly found in Gram-positive species, while the longer Type 1 GBEs are found in Gram-negative species. Several in vitro studies have linked N1 domain with transfer of short oligosaccharide chains during glycogen formation, which could lead to small and compact glycogen structures. Compact glycogen degrades more slowly and, as a result, may serve as a durable energy reserve, contributing to the enhanced environmental persistence for bacteria. We were therefore interested in classifying GBEs based on their N-terminal domain via large-scale sequence analysis. In addition, we set to understand the evolutionary patterns of different GBEs through phylogenetic analysis at species and sequence levels. Three-dimensional modeling of GBE N-termini was also performed for structural comparisons. A further study of 9,387 GBE sequences identified 147 GBEs that might belong to a possibly novel group of Type 3 GBE, most of which fall into the phylum of Actinobacteria. We also attempted to correlate glycogen average chain length (ACL) with GBE types. However, no significant conclusions were drawn due to limited data availability. In sum, our study systematically investigated bacterial GBEs in terms of domain organizations from evolutionary point of view, which provides guidance for further experimental study of GBE N-terminal functions in glycogen structure and bacterial physiology.

Dataset Information

Influence of in situ progressive N-terminal is still controversial truncation of glycogen branching enzyme in Escherichia coli DH5? on glycogen structure, accumulation, and bacterial viability.

Publications

Influence of in situ progressive N-terminal is still controversial truncation of glycogen branching enzyme in Escherichia coli DH5α on glycogen structure, accumulation, and bacterial viability.

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