A LacI-family regulator activates maltodextrin metabolism of Enterococcus faecium

Abstract

Enterococcus faecium is a gut commensal of humans and animals. In the intestinal tract, E. faecium will have access to a wide variety of carbohydrates, including maltodextrins and maltose, which are the sugars that result from the enzymatic digestion of starch by host-derived and microbial amylases. In this study, we identified the genetic determinants for maltodextrin utilization of E. faecium E1162. We generated a deletion mutant of the mdxABCD-pulA gene cluster that is homologous to maltodextrin uptake genes in other Gram-positive bacteria, and a deletion mutant of the mdxR gene, which is predicted to encode a LacI family regulator of mdxABCD-pulA. Both mutations impaired growth on maltodextrins but had no effect on the growth on maltose and glucose. Comparative transcriptome analysis showed that eight genes (including mdxABCD-pulA) were expressed at significantly lower levels in the isogenic ΔmdxR mutant strain compared to the parental strain when grown on maltose. Quantitative real-time RT-PCR confirmed the results of transcriptome analysis and showed that the transcription of a putative maltose utilization gene cluster is induced in a semi-defined medium supplemented with maltose but is not regulated by MdxR. Understanding the maltodextrin metabolism of E. faecium could yield novel insights into the underlying mechanisms that contribute to the gut commensal lifestyle of E. faecium.

Figure 1. Schematic representation of the gene clusters involved in maltodextrin and maltose utilization of E. faecium E1162.

Genes are represented by arrows (drawn to scale). Genes putatively encoding proteins involved in maltodextrin transport and/or metabolism are indicated in blue or in red. Genes predicted to be involved in the uptake and/or metabolism of maltose are indicated in green or in purple. The genes that encode putative transcriptional regulators are indicated in red or purple. The grey arrows represent the adjacent genes that are not predicted to be involved in maltodextrin or maltose utilization. Gene names, without the EfmE1162-prefix (omitted for reasons of space), are indicated in the arrows and the gene locus tags are indicated above or below the arrows. The homologs in L. monocytogenes EGD-e or in E. faecalis V583 are shown with corresponding colors above or below the gene clusters of E. faecium. The gene locus tags of the homologs are indicated in the arrows. Lines link the homologous genes with corresponding genes in E. faecium and amino acid identities are indicated.

Figure 2. Growth of E. faecium on starch, maltotetraose, maltose and glucose.

Growth curves of E. faecium E1162 wild-type (black), its isogenic mutants ΔmdxR (red) and ΔmdxABCD-pulA (blue), and the in trans complemented strain ΔmdxR+mdxR (green) on starch (panel A), maltotetraose (panel B), maltose (panel C) and glucose (panel D) are shown. The growth curve of E1162 wild-type in M1 was shown in grey as a negative control. Overnight cultures were inoculated at an initial OD₆₆₀ of 0.0025 into 300 µl semi-defined minimal medium M1 [9], M1 supplemented with 2.5 g/l of starch, maltotetraoise, maltose or glucose as sole carbon source, respectively, and then incubated in the Bioscreen C system at 37° C with continuous shaking. The absorbance of 600nm (A₆₀₀) was recorded every 15 min for 12 hours. Growth curves are mean data of three independent experiments. Note that A₆₀₀ at the start of the experiment is higher in M1 + starch than in the other conditions, due to increased turbidity of the medium containing starch.

Figure 3. qRT-PCR analysis of mdxB, malT and malP expression ratios in wild-type E1162 and its isogenic mutant ΔmdxR grown in BHI and maltose.

The data from the qRT-PCR were normalized using tufA as an internal standard [28]. The differences in gene expression (log₂-transformed data) relative to tufA are shown. Error bars represent the standard deviation of the mean of four biological replicates. Asterisks represent significant differences (*: P<0.01, **: P<0.001, ***: P<0.0001, as determined by an unpaired two-tailed Student’s t-test) between the indicated samples.