Article Figures & Data
Figures
- FIG. 1.
Chromosomal actA and mpl deletion mutants in the presence of actA-gus-plcB transcriptional reporter gene fusions in L. monocytogenes. The mpl and actA promoters are shown as thin arrows with associated transcripts (wavy lines). WT, wild type. ΔP actA contains a complete deletion of the actA proximal promoter but maintains the ribosome binding site immediately upstream of the actA coding sequences. Δmpl274-510 contains a frameshift mutation at codon 274 of mpl resulting in the loss of the protease active site and approximately 50% of the protein. Δmpl131-361 contains an in-frame deletion of amino acids 131 through 361 of Mpl resulting in the loss of the protease active site. Δmpl contains a complete deletion of the mpl promoter and coding regions. A promoterless copy of gus was inserted between the actA and plcB coding regions in the chromosomes of all mutants via homologous recombination.
- FIG.2.
Examination of actA expression in L. monocytogenes mpl and actA mutant strains grown in broth media. GUS activity was measured following 5 h of growth in BHI broth or BHI broth treated with 0.2% charcoal. Units of GUS were normalized for optical density at 595 nm as described by Youngman (61) for the measurement of β-galactosidase activity but with the appropriate substrate substitution of 4-methylumbilliferyl-β-d-glucuronide. Units are expressed as a percentage of NF-L476 (wild-type [WT]) activity in BHI broth or BHI broth treated with 0.2% charcoal. Each assay was done in triplicate, and the data represent the mean and SE for at least three individual experiments.
- FIG. 3.
Intracellular actA expression in mpl and actA mutant strains. Tissue culture dishes containing monolayers of J774 cells were infected with the indicated L. monocytogenes strains for 5 h as described in Materials and Methods. GUS activity was determined following lysis of the monolayers. 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 lysates on LB agar. The total number of CFU per dish was extrapolated by multiplication by a factor that corrected for the area of the coverslip relative to that of the 60-mm dish. Units of GUS are as described by Youngman (61) for the measurement of β-galactosidase activity but with the appropriate substrate substitution of 4-methylumbilliferyl-β-d-glucuronide. Units are expressed as a percentage of NF-L476 (wild-type [WT]) activity. Each assay was done in triplicate, and the data represent the mean and SE for at least three individual experiments. Background activities from 10403S-infected monolayers and from uninfected cells were equivalent and never represented greater than 2% of the activity detected for NF-L476.
- FIG. 4.
Intracellular expression of ActA by L. monocytogenes in J774 cells. J774 mouse macrophage-like cells were infected with L. monocytogenes; at 2.5 h postinfection, the infected cells were starved for methionine and cysteine, host protein synthesis was blocked, and cell-to-cell spread was inhibited as described in Materials and Methods. Infected cells were pulse-labeled with 35S-methionine prior to host cell lysis, and SDS-extractable bacterial proteins were resolved on an SDS-8% polyacrylamide gel. The amount of sample loaded per lane was normalized for the number of bacteria. The three species of ActA, representing different phosphorylation states of the protein, are the major protein bands observed by using this approach (6). WT and WT*, proteins derived from 10403S- and NF-L476-infected cells, respectively.
- FIG. 5.
Examination of actA expression in P(hly)-actA and PWT-actA L. monocytogenes strains grown in various broth media. GUS activity was measured following 5 h of growth in BHI broth (B), MEM (M), BHI broth treated with 0.2% charcoal (BC), and LB. Units of GUS are as described by Youngman (61) for the measurement of β-galactosidase activity but with the appropriate substrate substitution of 4-methylumbilliferyl-β-d-glucuronide. Each assay was done in triplicate, and the data represent the mean and SE for at least three individual experiments.
- FIG. 6.
Comparison of PWT-actA and P(hly)-actA promoter activation in L. monocytogenes strains containing PrfA or PrfA*. GUS activity was measured following 5 h of growth in BHI broth or BHI broth treated with 0.2% charcoal or following bacterial growth in J774 cells. Units of GUS are as described by Youngman (61) for the measurement of β-galactosidase activity but with the appropriate substrate substitution of 4-methylumbilliferyl-β-d-glucuronide. Each assay was done in triplicate, and the data represent the mean and SE for at least three individual experiments. (A) Measurement of actA-gus expression in cultures following growth in either BHI broth (B) or BHI broth treated with 0.2% charcoal (BC). (B) Comparison of the levels of actA-gus expression achieved under optimal extracellular conditions versus the level of activity observed for L. monocytogenes located within the host cell cytosol (intracellular). The intracellular value shown is for NF-L476. Background activities from 10403S-infected monolayers and from uninfected cells were equivalent and never represented greater than 2% of the activity detected for NF-L476.
Tables
- TABLE 1.
Bacterial strains and relevant characteristics
Straina Relevant characteristic(s) Reference or source 10403S Wild type 23 DP-L1942 ΔactA164-465b 6 DP-L1465 Δmpl274-510c Dan Portnoy DP-L2296 Δmpl131-361d 39 DP-L2343 Δmple Dan Portnoy NF-L559 P(hly)-actA This work NF-L476 actA-gus-plcB This work NF-L767 ΔPactA-gus-plcB This work NF-L707 Δmpl274-510 actA-gus-plcB This work NF-L861 Δmpl131-361 actA-gus-plcB This work NF-L862 Δmpl actA-gus-plcB This work NF-L572 P(hly)-actA-gus-plcB This work NF-L753 actA-gus-plcB prfA* This work NF-L754 P(hly)-actA-gus-plcB prfA* This work NF-L904 ΔPactA-gus-plcB comK::actA This work
