Ethanolamine Utilization and Bacterial Microcompartment Formation Are Subject to Carbon Catabolite Repression

Abstract

Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR).IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.

Keywords: bacterial microcompartments; carbon catabolite repression; enterococcus; ethanolamine utilization.

FIG 1

Model depicting the regulation of eut gene expression. As previously established, the TCS, EutV and EutW, is activated by EA and positively regulates eut gene expression by preventing termination at the nascent transcripts generated by the eutP, eutG, and eutS promoters (shown in gray). However, the sRNA, EutX, also contains a EutV binding site (depicted as two gray loops) and will act as a “sponge,” preventing positive regulation by EutV. AdoCbl disrupts this negative regulation by binding to the riboswitch (red loop) when EutX is being transcribed, causing an early termination event resulting in a shortened form of EutX that can no longer bind EutV. By this manner, AdoCbl positively regulates eut gene expression. As shown in this current work and circled in red, glucose negatively regulates eut gene expression by activating CcpA, which binds the cre site within the eutS promoter, directly blocking transcription and preventing expression of the TCS. Expression at the eutP and eutG promoters is thereby indirectly blocked by CCR due to the lack of the TCS. +, positive effect on eut gene expression; −, negative effect.

FIG 2

E. faecalis utilizes EA and forms BMCs in CDM under aerobic conditions. (A) Graphical representation of the growth of E. faecalis OG1RF (WT) strain when grown in CDM. Growth curves were initiated at an OD₆₀₀ of ∼0.1, and cells were allowed to grow for 12 h in air with shaking (aerobic; left graph) (A) or covered with no air (microaerobic; right graph) in CDM containing either no carbon source, 33 mM EA, 0.2% ribose, or both 33 mM EA and 0.2% ribose. All cultures were supplemented with 40 μg/ml AdoCbl. (B) Representative transmission electron micrographs showing the formation of BMCs (arrows) in E. faecalis OG1RF (WT) strain. BMC formation was induced by addition of 33 mM EA (bottom panels) compared to the control condition where no EA was added (top panels). Left panels, aerobic conditions; right panels, microaerobic conditions.

FIG 3

Bacterial microcompartment numbers and their effect on the growth of E. faecalis mutants. (A) Representative transmission electron micrographs displaying the formation of BMCs (arrows) in E. faecalis strains OG1RF (WT) and the ΔeutX mutant when induced with 33 mM EA (bottom) versus no EA control condition (top). (B) Graphical representation of the number of BMCs per cell calculated for E. faecalis OG1RF (WT) and ΔeutX mutant grown under inducing conditions. BMCs were counted in 30 cells per strain. An unpaired t test with Welch’s correction for the number of BMCs per cell observed in the ΔeutX mutant versus that in the WT was used to calculate the P values. (C) Graphical plot of the growth of E. faecalis strains OG1RF (WT) and ΔeutV and ΔeutX mutants with or without 33 mM EA. All the cultures were supplemented with 0.2% ribose and 40 μg/ml AdoCbl and were grown under aerobic conditions for 12 h.

FIG 4

Glucose-associated carbon catabolite repression of ethanolamine utilization in E. faecalis. Graphical representation of E. faecalis OG1RF (WT) strain growth characteristics when grown in CDM under aerobic conditions for 12 h. Cultures utilizing 0.2% ribose as the carbon source with or without the addition of 33 mM EA were compared to cultures containing 0.2% glucose as an alternative carbon source, similarly supplemented with 33 mM EA; 40 μg/ml AdoCbl was added to all the cultures.

FIG 5

CcpA negatively regulates expression of the eut locus in E. faecalis. qRT-PCR analysis of transcript levels of eutP, eutG, and eutS relative to that of the control, gyrA, for the E. faecalis strains OG1RF (WT), the ΔccpA mutant, and the ΔccpA::ccpA complement. Cultures for each strain grown using 0.2% ribose, with or without the addition of 33 mM EA, were compared to those cultured with 0.2% glucose with or without 33 mM EA.

FIG 6

The cre site substitution and its influence on eut gene expression in E. faecalis. (A) Illustration showing the intergenic region between eutG and eutS, bearing the modified cre site (AATCGA was substituted for CCATGG to bring in an NcoI restriction site). The EutV regulatory region corresponds to the terminator loops in the 5′ untranslated region (UTR) region of the nascent eutS transcripts to which EutV binds to facilitates antitermination. SD, ribosomal binding site upstream of eutS open reading frame. (B) Transcript levels of eutP, eutG, and eutS normalized to the control, gyrA, for the E. faecalis strains OG1RF (WT) and the P_eutS::cre* mutant. Each strain was grown aerobically in 0.2% ribose with or without 33 mM EA, and its transcript levels were compared to those grown in 0.2% glucose with or without 33 mM EA.

FIG 7

Identification of cre motifs in bacterial microcompartment loci. (A) Frequencies of cre motifs found in intergenic regions of BMC loci in Firmicutes (CcpA containing) and non-Firmicutes. (B) Frequencies of cre motifs found in intergenic regions of different microcompartment subtype loci in Firmicutes genomes. Unknown, metabolosome of unknown function; PDU, 1,2-propanediol utilization; PVM, Planctomycetes and Verrucomicrobia type; BUF, BMC of unknown function; GRM, glycyl radical enzyme containing; EUT, ethanolamine utilization; MIC, incomplete core locus; ETU, ethanol utilization. Schematics of eut or pdu-eut loci from four bacterial genomes as identified in reference . (C) Enterococcus faecalis OG1RF. (D) Listeria monocytogenes EGD-e. (E) Streptococcus sanguinis SK36. (F) Peptoclostridium difficile 630. Genes are colored by association with different components of the BMC loci. Green, eut-associated genes; blue, pdu-associated genes; brown, cobalamin biosynthesis or transport genes; orange, regulatory genes; gray, other/unrelated genes. Regulatory RNAs, eutX and aspocR, are show in light green and light orange, respectively. Only putative promoter sequences associated with cre motif sequences are identified by directional arrows.