A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Abstract

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.

Fig 1

A. Organization of the glycogen metabolism gene cluster (glg-amy-pgm) encoded in the L. acidophilus NCFM genome. Predicted rho-independent transcriptional terminators are indicated in hairpin loops with overall confidence score (ranges from 0 to 100).B. Comparative structural organization of glycogen metabolism gene clusters in other microorganisms. The gene cluster is present in a human microbiome reference strain of L. bulgaricus (strain PB2003/044-T3-4) and a probiotic strain of L. helveticus (strain R0052) but not in other sequenced dairy-associated L. bulgaricus and L. helveticus strains. Putative glycogen synthesis genes are indicated in light grey arrows, whereas glycogen degradation genes are indicated in black arrows. amyA or pulA, α-amylase/pullulanase/glycogen-debranching enzyme, glgX, glycogen-debranching enzyme.

Fig 2

Phylogenetic tree of GlgA orthologues among lactobacilli and other microorganisms. The protein sequences were aligned and the phylogenetic tree was constructed using clustalx v2.1 and visualized using MEGA 5.1 software. The species names followed by the GenBank accession number of the corresponding GlgA proteins are indicated. Abbreviated genus: B., Bifidobacterium; C., Corynebacterium; E., Escherichia; Ent., Enterococcus; L., Lactobacillus; Lc., Lactococcus; M., Mycobacterium; Strep., Streptococcus. The scale bar indicates an evolutionary distance of 0.05 amino acid substitutions per position.

Fig 3

Transcript levels of the glg operon (A) and intracellular glycogen content (B) in L. acidophilus grown in SDM supplemented with 2% (w/v) of various carbohydrates or no carbohydrate (no CHO). Intracellular glycogen content was expressed as mg of glucose (released from glycogen by amyloglucosidase) per g of cell wet weight. The data are the mean ± standard deviation for two independent biological replicates.

Fig 4

A. Growth and glycogen accumulation profiles of L. acidophilus in SDM containing 2% trehalose or raffinose. The intracellular glycogen contents under both carbohydrate conditions at various growth phases (indicated by OD₆₀₀) from (A) were compiled in (B) and plotted against the transcript levels of the glg operon. The data represent the mean ± standard deviation for two independent biological replicates.

Fig 5

A. Iodine staining of L. acidophilus NCK1909 parent strain and glycogen metabolism mutant colonies grown on solid SDM medium containing 2% trehalose. Both ΔglgA and ΔglgB mutant cells appeared as yellow/colourless indicative of glycogen-deficient phenotype. Like the parent cells, the ΔglgP and Δamy mutants were stained brown, indicating that their ability to synthesize intracellular glycogen was unaffected.B. Intracellular glycogen content of mid-log-phase cells cultivated in SDM containing 2% trehalose. Glycogen was not detected from both ΔglgA and ΔglgB cultures. Data shown represent the mean ± standard deviation for two independent biological replicates.

Fig 6

A. Growth of L. acidophilus glycogen metabolism mutants compared with the parent strain in SDM containing 1% raffinose.B. Survival of the strains in simulated small intestinal juice (SIJ; contains 0.3% Oxgall bile). Percentage of survival represents viable cells (cfu ml⁻¹) at various time points (1, 2, 3 and 4 h) versus at time zero.C. Growth in MRS broth supplemented with 0.5% Oxgall bile. The data shown are representation of at least three independent biological replicates.