Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence

Abstract

We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L. monocytogenes in the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L. monocytogenes for biofilm growth and pathogenesis.

FIG. 1.

Adhesion and growth of L. monocytogenes in microtiter plates. Wild-type strain C52 (⧫), Sag-1 (▪), and Sag-2 (▴) were grown statically at 37°C in TSB in the wells of a microtiter plate. At each time point irreversibly attached bacteria were quantified by crystal violet staining as described in Materials and Methods. No change in crystal violet staining was observed in wells containing only TSB (•). The error bars represent the SEM (n = 6).

FIG. 2.

Growth of the adhered bacteria in a microtiter plate. Wild-type strain C52 (⧫), Sag-1 (▪), and Sag-2 (▴) were grown statically at 37°C in TSB in the wells of a microtiter plate for 1 h. At this point (depicted by the arrow) the planktonic bacteria were gently removed from the well and replaced by an equal volume of fresh TSB. Subsequently, at each time point attached bacteria were quantified by crystal violet staining. The error bars represent the SEM (n = 6).

FIG. 3.

(A) Hypothetical chromosomal insertion of Tn917-LTV3. In this case a transcriptional fusion between the lacZ gene and a chromosomal promoter is shown. Only unique sites which can be used to clone sequence flanking the lacZ end are shown. Restriction enzyme abbreviations: X, XbaI; A, Asp7I8; Sm, SmaI; Xh, XhoI; H, HindIII. Antibiotic resistance genes: cat, chloramphenicol; neo, kanamycin-neomycin; ble, bleomycin; erm, erythromycin-lincomycin. (B) Site of Tn917-LTV3 insertion in mutant Sag-1. Nucleotide positions given are based on similarity with the relA gene of B. subtilis. (C) Site of Tn917-LTV3 insertion in mutant Sag-2. In panels B and C, the black arrows indicate the orientation of the lacZ gene of Tn917-LTV3.

FIG. 4.

Complementation of the mutations in Sag-1 and Sag-2. Sag-1 was complemented with the relA gene of B. subtilis on plasmid pDG148relA. Sag-2 was complemented with plasmid pCThpt containing the wild-type hpt gene with a constitutive XylA promoter. The graph shows the growth of adhered bacteria quantified after 6 h as described in Materials and Methods. The error bars represent the SEM (n = 6).

FIG. 5.

Accumulation of (p)ppGpp after amino acid starvation. Bacteria were labeled with [³²P]H₃PO₄ in MOPS starvation medium lacking phosphate and amino acids, but containing serine hydroxamate (1 mg ml⁻¹) and ℒ-valine (0.5 mg ml⁻¹), for 30 min at 37°C. An equal volume of 13 M formic acid was added and the samples were subjected to freeze (dry ice)-thawing twice. Acid extracts were centrifuged briefly, and the supernatants were spotted onto polyethyleneimine cellulose for thin-layer chromatography in 1.5 M K₂HPO₄ (pH 3.4). Strains: CF1943, E. coli W3110 parental strain; CF1944, E. coli CF1943 (ΔrelA::kan); CF1946, E. coli CF1943 (ΔrelA::kan ΔspoT::cm).