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

Abstract

Background: 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 lengths, so we sought to harness this to create a set of Escherichia coli DH5α 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.

Results: A series of Escherichia coli DH5α 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 (glgBΔ270) 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 α1,4-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 (glgBΔ180) had significantly increased biofilm formation in favourable media.

Conclusions: 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.

Figure 1

GlgB and glgB-mutated strains in their E. coli DH5α genomic context, and the scar sequence left following in situ mutation. A. The five essential glycogen metabolism genes are organized into a single transcriptional unit as glgBXCAP in E. coli DH5α (Montero et al. [44]). For GlgB in E. coli DH5α, four domains have been identified, which are N-terminus, CBM48, α-amylase, and C-terminus. B. P1 and P2 (italic letters) were primers for amplifying linear PCR products from plasmid pKD4. Underlined sequence was FRT site with a palindromic sequence (blue sequences), separated by 12 asymmetric nucleotides. Red italic sequence was a ribosome-binding site and the green italic letters represented start codon for downstream gene expression.

Figure 2

Growth of E. coli DH5α strains in 1 × M9 minimal media (T/G = 1:2) and corresponding glycogen accumulation. A. Cell density is plotted against time averaged over four independent OD₆₀₀ readings. B. Glycogen accumulation is expressed as ratio of glucose to protein amount over time. Three independent replicates were performed. All data were presented as means ± standard error.

Figure 3

Chain length distributions of oligosaccharides in glycogen extracted from the six E. coli DH5α strains. A. Chain length distributions of isoamylase-debranched glycogen, which are expressed as molar percentage (%) in terms of oligosaccharide chain length. B. Difference plot generated by subtracting the molar percentage of the respective WT oligosaccharide DP from the corresponding glgBΔ90, glgBΔ180, glgBΔ270, and glgBΔ369 molar percentages. The experiment was performed twice independently with two repeats for each replicate.

Figure 4

Starvation survival assay for E. coli DH5α strains in PBS buffer for 15 days. For each strain, two independent replicates were performed. Each replicate includes. four repeats. Viable cells of the six strains drop sharply for the first six days. E. coli DH5α survived better, especially at day 3, than E. coli DH5α ΔglgB from day 0 to 9. The other four strains behaved similar with no obvious difference. After day 6, cells died at a very slow rate. At day 15, the number of colony-forming units (CFU) for the six E. coli DH5α strains converged together.

Figure 5

Desiccation survival abilities of E. coli DH5α strains. Counts of colony-forming units (CFU) are plotted for each strain at time point 0 h, 3 h, and 6 h. Two independent biological replicates were performed for each strain at each time point. For each replicate, four technical repeats were included.

Figure 6

Quantification of biofilm formation abilities of E. coli DH5α strains on 96-well polystyrene plates in LB. Crystal violet staining method is used. Findings are expressed as fold changes versus WT based on based on average of three independent replicates. Bacteria in LB broth had enhanced biofilm formation abilities compared to bacteria growing in M9 (T/G = 1:2), where biofilm formation abilities were uniform (including Δ180), but a quarter of the level of that seen for the strains cultured in LB broth (Data not shown).