Article Figures & Data
Figures
- FIG. 1.
(A) Sequence of the ace promoter region. Potential −35 and −10 region and ribosome binding site (RBS) sequences are underlined. The transcriptional start site (+1) is in boldface, and the Ers box is boxed. (B) EMSA with the promoter region of the ace gene and different concentrations (2 to 0.2 μg) of His6-ErsHTH protein. (C) EMSA of His6-ErsHTH binding to the D4-labeled DNA fragment containing the ace regulatory region. Amplifications were performed with D4-Pu and D4-Pr (Table 2). Crude cell extracts prepared from an E. faecalis Δers mutant strain (0.1 μg protein) were added to all of the reaction mixtures. Shown are the labeled ace promoter without protein (lane 1), with His6-ErsHTH (1 μg protein) (lane 2), with His6-ErsHTH and an unlabeled competitor (lane 3), and with His6-ErsHTH and a nonspecific DNA fragment (internal fragment of the ef1843 gene) (lane 4).
- FIG. 2.
Expression of ers and ace determined by Western blot or RT-qPCR analysis. (A) Western blot analysis (with antiserum against His-tagged Ers) of E. faecalis protein extracts (40 μg) from strain JH2-2 grown at 37°C (lane 2), at 46°C (lane 3), or in the presence of bile salts (lane 4) and from strain JH2-2/p3535ers (lane 5). A 0.1-μg sample of purified His6-Ers protein was loaded into lane 1. (B) RT-qPCRs experiments with the ers and ace genes and primers described in Table 2. RNAs were extracted from cells cultured under the same conditions used for protein extracts. Values that are significantly different are >2 (P < 0.05).
- FIG. 3.
Effect of ace inactivation on virulence. Percent survival of G. mellonella larvae at 16, 20, and 24 h after infection with L. lactis IL1403 (black bar), E. faecalis JH2-2 (gray bar), the JH2-2 Δace mutant (hatched bar), and the complemented Δace mutant strain (white bar). We used 6 × 106 CFU counted on an agar plate per injection. Experiments were repeated at least three times, and the results represent the mean ± standard deviation of live larvae.
- FIG. 4.
Time course of intracellular survival of E. faecalis JH2-2 (squares) and the Δace mutant (triangles) within murine peritoneal macrophages. The results shown represent the mean number ± the standard deviation of viable intracellular bacteria per 105 macrophages of three independent experiments with three wells.
- FIG. 5.
Infection with 104 cells of wild-type E. faecalis JH2-2 (•) and its isogenic Δace mutant strain (▪). Kidney pair homogenates were obtained from groups of 15 mice that were sacrificed and necropsied 48 h after a transurethral challenge. Results are expressed as log10 CFU per gram of tissue. A value of 0 was assigned to uninfected kidneys. Horizontal bars represent the geometric means.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Reference(s) or source E. faecalis JH2-2 Fusr Rifr; plasmid-free wild-type strain 9, 35 Δers JH2-2 isogenic Δers mutant 25 Δace JH2-2 isogenic Δace mutant This study Complemented Δace ace insertion in the JH2-2 Δace mutant This study JH2-2/p3535ers Strain JH2-2 harboring plasmid p3535ers This study E. coli XL1Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F′ proAB lacIqZΔM15] Tn10 (Tetr) Stratagene M15/pQE30ersHTH Strain harboring pQE30ersHTH This study L. lactis IL 1403 4 Plasmids pMAD oripE194ts Emr AmprbgaB 1 pMSP3535 3 pGEM-T easy f1 ori lacZ Ampr Promega pMad-Δace Construction for the ace deletion mutant This study pMad-ace Construction for the complemented ace mutant strain This study pMSP3535ers ers cloned into overexpression vector p3535 This study pGEM-T-Pace Promoter of ace cloned into pGEM-T This study - TABLE 2.
Primers used in this study
Locusa or primer name Sequence (5′-3′)a Use Forward Reverse ers CGCGGATCCATAATAGATAATTGAGG TTGCTGCATCTTTTTCATGG RT-qPCR ef0082 TTGACGTCAGCACCTTCTTC CGTAGCGTTCACCTTTGACA RT-qPCR ef0049 CAGCTTTTCCGACACGTCTT TTCCCCCACGTACTTTAGCA RT-qPCR ef0782 TTAGAGCGCGCAGTGAATC ATGGCCGATCTGCTTCTAAA RT-qPCR ace ATGAAGGAAGCCCACAGTTG GTTGTGCCTGTTTTGGGAAG RT-qPCR kat GCTGTTTGGGATTTTTGGTC CATGCATATGACGGAACGAC RT-qPCR ef1656 TTTATTTGTCACCCAACCAACA CAATTGAAACGGTTGATCAGAA RT-qPCR ef1664 TGGCGACCTAATTACCTTGG CGTTTGCATCGTACGTGAGT RT-qPCR ef1816 CGTCAAGACGATACCCGATT TTTCCATGTTGTGCAGGAAG RT-qPCR ef2185 GCGATTGCTCCAACTGAATC GCCGTCCGTAATAAAAAGCA RT-qPCR ef3146 CCAGAATTCTCAATTCCTCGTT CAAATTTTTGTACACCGACAGG RT-qPCR aceRace1 ATTCCAACTAGTGCGTCTGG 5′RACE PCR aceRace2 TTGAGGTCATCTCTCCTTG 5′RACE PCR aceRace3 TCAATTCGGCGCCAAACGAA 5′RACE PCR Prom-ace TCATTCTTTTCCTCCTTATCT CCGCAATTGGTAGAATCATTA Cloning in pGEM-T Prom-ers TAAGTTTGGTTCTGTCATTA CGACTCGAAAGGAATGTTCA Cloning in pGEM-T aceDC-Ub CCGGAATTCAGCTCAACTATGCCTGTCGA CGCGGATCCGTCATTCCAACTAGTGCGTC Cloning in pMAD aceDC-Db CGCGGATCCCAGCCATCCACAGAAACAAC GAAGATCTTTCCTCTGCCATTAACGCGT Cloning in pMAD p3535ers CGCGGATCCATAATAGATAATTGAGGAGTTA CGCGCGCTGCAGTTAAACAATAATGTTATCTCTAATC Cloning in pMSP35535 pQE30-ersHTH GCGGATCCAAAAAGATGCAGCAAATGTTGAT CGCGCGCTGCAGTTAAACAATAATGTTATCTCTAATC Cloning in pQE30 mad1F TCTAGCTAATGTTACGTTACAC Cloning verification mad2R TCATAATGGGGAAGGCCATC Cloning verification Pu GTTGTAAAACGACGGCCAGT Cloning verification Pr CACAGGAAACAGATATGACC Cloning verification ↵ a From the annotated sequence available at http://www.tigr.org .
↵ b Primers used to clone upstream (U) and downstream (D) sequences of ace in order to construct a deletion mutant and complemented strain by a double-crossover (DC) event.
- TABLE 3.
Identification and expression of genes harboring a putative Ers box in the promoter region
ORFd Gene name Putative Ers box Expressiona (P value) with reference to that of: 23S rRNA gene gyrA ef0049 AACAAATGTTC-N75-ATG NDb ND ef0782 AACAAATGTTG-N272-ATG +1.01 +1.1 ef1099 ace AACAAATGTTA-N176-ATG +5.11 (0.02) +7.4 (0.016) ef1656 AACATATGTTA-N77-ATG −1.17 −1.72 ef1664 AACAATTGTTA-N132-ATG +1.12 −1.01 ef1816 AACAAATGTTT-N81-ATG +1.36 +1.27 ef2185 AACATATGTTT-N129-ATG —c — ef3146 AACAATTGTTT-N424-ATG +1.99 +2.4 (>0.05)
