Article Figures & Data
Figures
- Fig. 1.
Construction of transcriptional reporter vector pLCR. Plasmids pTV53 and pTV30, which provided the promoterless B. pumilus cat-86 gene and the E. coli lacZ gene with an upstream spoVG ribosome binding site (rbs) and aBamHI site, respectively, were described previously (28, 37). Plasmid pTV1OK (13) provided the kanamycin resistance gene (kan), a type III aminoglycoside phosphotransferase derived from Streptococcus faecalis(35), and the pWVO1ts origin of replication, a temperature-sensitive derivative of the origin of replication fromLactococcus lactis plasmid pWVO1 (18). Plasmid pTV30cat was isolated from B. subtilis by selection for chloramphenicol resistance at 30°C following recombination between plasmids pTV30 and pTV53. Similarly, pTV30catts was isolated by selection for kanamycin resistance at 28°C following recombination between plasmids pTV30cat and pTV1OK. Details of plasmid construction by in vivo recombination in B. subtilis are described in Materials and Methods. pLCR was generated in two conventional cloning steps by partial XbaI digestion and religation of pTV30catts followed by insertion of a PCR-generated fragment containing the trpA terminator (7) into theNcoI and BamHI sites. X, XbaI; E,EcoRI; K, KpnI; P, PstI; N,NcoI; B, BamHI. E, N, and B are unique restriction sites in pLCR.
- Fig. 2.
Schematic of pLCR fusion constructs integrated into theL. monocytogenes chromosome. (A) Integration of pLCR by homologous recombination between the cloned hlypromoter-containing fragment (hly′) and the corresponding region of the L. monocytogenes chromosome, yielding strain DP-L2986. (B and C) Integration structures which resulted from recombination between the actA promoter-containing fragment (actA′) cloned in the forward (B) and reverse (C) orientations with respect to the transcription of lacZ andcat and the corresponding region of the L. monocytogenes chromosome, yielding strains DP-L2989 and DP-L2812, respectively. Broken lines represent plasmid sequences, and solid lines represent flanking chromosomal regions. Arrows depict promoter positions and the direction of transcription for each of the indicated genes. Stem and loop structures represent transcriptional terminators.
- Fig. 3.
Promoter activity in LB broth as measured by chloramphenicol resistance. L. monocytogenes DP-L2986, DP-L2989, and DP-L2812 carrying the hly, actA, and control reverse-orientation actA promoter fusions, respectively, were cultured in buffered LB broth in the absence (light bars) or presence (dark bars) of 10 μg of chloramphenicol per ml. After 5 h, bacterial growth was assessed by measuring the OD600. The data represent the mean ± standard deviation for three individual experiments. The addition of increasing doses of chloramphenicol at the onset of L. monocytogenes10403S culturing in LB broth and measurement of the OD600at various time points demonstrated an MIC of 5 μg/ml (data not shown).
- Fig. 4.
Promoter activity in J774 cells as measured by chloramphenicol resistance. Monolayers of J774 cells grown on glass coverslips were infected with DP-L2986, DP-L2989, or DP-L2812. Chloramphenicol (10 μg/ml) (cm) was added at 2.5 h postinfection. Monolayers were lysed at the indicated time points, and the number of bacteria per coverslip was determined. Results are expressed as the mean number of bacteria ± the standard deviation for three coverslips per time point. One of three experiments with similar results is shown. The MIC of chloramphenicol for L. monocytogenes 10403S grown in J774 cells was determined to be 5 μg/ml by measuring the number of CFU per monolayer in the presence and absence of increasing doses of chloramphenicol at various time points after infection (data not shown).
- Fig. 5.
Promoter activity in LB broth as measured by a β-galactosidase assay. L. monocytogenes DP-L2986 (dark bars), DP-L2989 (hatched bars), and DP-L2812 (see below) carrying thehly, actA, and control reverse-orientationactA promoter fusions, respectively, were cultured in buffered LB broth, and β-galactosidase activity was measured at the indicated time points. Results are expressed as units of β-galactosidase activity per 107 CFU per minute. DP-L2812 background activity, which in no case exceeded 0.08 U, was subtracted from each value. The circles indicate OD600 values for DP-L2986 and DP-L2989, which were identical over 8 h of growth. The data represent the mean ± standard deviation for three individual experiments.
- Fig. 6.
SDS-PAGE of L. monocytogenes secreted and membrane-anchored proteins produced in LB broth. Wild-type L. monocytogenes 10403S was grown in buffered LB broth for 5.5 h, and secreted proteins (lanes 1 and 2) from 10 ml of culture were precipitated with trichloroacetic acid and resuspended in SDS-PAGE sample buffer. Membrane-anchored proteins (lanes 3 to 6) were extracted from bacterial pellets by boiling for 5 min in SDS-PAGE sample buffer. Eighty percent of each sample was subjected to SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Lane 1, 10403S; lane 2, DP-L2161 (10403SΔhly) (14); lane 3, 10403S; lane 4, DP-L1942 (10403SΔactA) (3); lane 5, SLCC-5764 (5); lane 6, DP-L1955 (SLCC-5764ΔactA) (20). Arrows indicate the positions of LLO and ActA. Lanes M show molecular mass standards (kilodaltons [kd]).
- Fig. 7.
Immunoprecipitation of LLO and ActA from L. monocytogenes-infected J774 cells. Bacterial proteins were metabolically labeled during growth in J774 cells or in vitro and immunoprecipitated with monoclonal anti-LLO antibody B3-19 (lanes 1 to 3) or polyclonal anti-ActA antibody 2553 (lanes 4 and 5). Lanes 1 and 4, 10403S-infected J774 cells; lane 2, DP-L2817 (10403S::pfoH438Y)-infected J774 cells; lane 3, supernatant from 10403S cultured in vitro as described in Materials and Methods; lane 5, DP-L1942 (10403SΔactA)-infected J774 cells. An autoradiograph depicting one of two experiments with similar results is shown. The exposure times for the visualization of ActA and LLO by autoradiography were 18 h and 7 days, respectively. For quantitation of ActA and LLO as described in Results, the gel was scanned with a Molecular Dynamics PhosphorImager, and the resulting images were analyzed with ImageQuant software (Molecular Dynamics). Numbers at left are in kilodaltons.
Tables
- Table 1.
Promoter activity during bacterial growth in J774 cells
Expt DP-L2986 DP-L2989 β-Galactosidase activity (U/107CFU/min)a 107CFU/dishb β-Galactosidase activity (U/107 CFU/min) 107 CFU/dish 1 13.6 2.4 31.1 1.5 2 7.1 2.6 21.2 1.6 3 15.4 1.8 41.8 1.0 4 7.1 2.6 32.9 1.4 Mean ± SDc 10.8 ± 4.3 31.7 ± 8.4 ↵a Background activity, derived from DP-L2812-infected monolayers, was subtracted from each value. β-Galactosidase activity derived from DP-L2812-infected monolayers never exceeded the endogenous β-galactosidase activity produced by uninfected J774 cells.
↵b The number of CFU per dish was determined by lysing infected J774 cells grown on coverslips in duplicate dishes and plating a portion of the lysate on LB agar. The total number of CFU per dish was extrapolated by multiplication by a factor which corrected for the area of the coverslip relative to that of the 60-mm dish.
↵c Derived from four individual experiments. The P value, determined by Student’s t test, for a comparison of the values shown was <0.01.
