Figure 1.
The agmatine catabolic pathway in E. faecalis.
A) Schematic representation of agmatine metabolism and B) genetic organization of the agu locus in E. faecalis.
Table 1.
Strains, plasmids and oligonucleotides used in this study.
Figure 2.
Growth curves of E. faecalis strains.
Wild type strain JH2-2 with empty vector pBM02 (A), aguR-deficient strain (B) and aguR−-complemented strain (C) were grown in LB Gal with (○) or without 10 mM agmatine (•).
Table 2.
Ammonium production and final pH values of E. faecalis strains grown in the presence or absence of agmatine.
Figure 3.
Transcriptional analysis of agu genes in E. faecalis.
A) Schematic representation of agu operons. PaguR and PaguB indicate promoter regions. Secondary structures TaguR and TaguB represent putative Rho-independent transcriptional terminators. B) Northern blot analysis. E. faecalis wild-type (lanes 1 and 2) or aguR (lane 3) cells were grown in LB Gal with or without agmatine (Ag). Total RNA (extracted after 6 h of growth) was hybridized against specific probes I or II. Transcript size was determined by comparison to RNA markers. C) Primer extension experiments for the determination of aguR and aguB transcriptional start sites (lanes +1). Lanes G, A, C and T show homologous sequence ladders. D) Nucleotide sequence of aguR-aguBDAC intergenic region. Positions of transcriptional start sites are indicated (+1). Putative Shine-Dalgarno (SD), −10 and −35 regions are shown underlined. Translational start sites are shown in bold. The predicted cre site is highlighted in purple.
Table 3.
β-galactosidase activity (MU) of strains carrying PaguB- or PaguR-lacZ fusions grown in the presence or absence of agmatine.
Table 4.
β-galactosidase activity (MU) of JH2-2 PaguB-lacZ grown under different conditions, pH values and in the presence or absence of agmatine.
Figure 4.
Analysis of CcpA and PTSMan effects on expression of the aguBDAC operon.
β-galactosidase activity of PaguB-lacZ transcriptional fusion in wild type (JH2-2), ccpA− (CL14), mpt− (JH98) and ccpA− mpt− (CL98) strains. Cells were grown in LBA with or without 30 mM glucose (Glu), lactose (Lac), maltose (Mal) or fructose (Fru). Error bars represent standard deviation of at least triplicate measurements.
Figure 5.
CcpA interaction with the aguR-aguBDAC intergenic region.
For band shift assays, agu or agumut amplicons (2.69 nM each) were incubated with increasing concentrations of CcpA (0.025–0.7 mM), 5 mM of P-Ser-HPr and 20 mM FBP. The arrow indicates position of the retarded complex (C). Consensus, wild type and mutated sequence of cre sites are indicated.
Figure 6.
Gene context analysis of the AgDI system from different sources.