The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions - PubMed (original) (raw)

The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions

Laura D Wonderling et al. Appl Environ Microbiol. 2004 Apr.

Abstract

This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L. monocytogenes encounters in some ready-to-eat foods. A library of L. monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments. One of the L. monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced. A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria. An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress. Additionally, strain LDW1 was tested for its response to temperature and H(2)O(2) stresses. The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44 degrees C but at a rate similar to that of the wild-type strain when incubated at 4 degrees C. In addition, strain LDW1 was significantly more sensitive to a 52 degrees C heat shock than the wild-type strain. Strain LDW1 was also defective in its response to H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g of H(2)O(2) was more inhibitory for the growth of strain LDW1 than for that of the parent strain. The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA(-) organisms, which suggests that L. monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions.

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Figures

FIG. 1.

FIG. 1.

Effects of NaCl on growth of L. monocytogenes strains 10403S and OSM1. Strains 10403S and OSM1 were grown at 37°C in Pine's defined broth with varying concentrations of NaCl. Growth was measured by optical density at 600 nm. Growth curves represent the average optical density from two or more individual growth assays. Error bars, standard deviations among the growth assays. Solid symbols, 10403S; open symbols, OSM1. Squares, 1.8% NaCl; triangles, 3% NaCl; circles, 4% NaCl.

FIG. 2.

FIG. 2.

Effects of glycine betaine and NaCl on growth of L. monocytogenes strains 10403S and OSM1. Strains were grown at 37°C in Pine's defined broth with 4% NaCl in either the presence or the absence of 1 mM glycine betaine. Growth was measured by optical density at 600 nm. Growth curves represent the average optical density from two or more individual growth assays. Error bars, standard deviations among the growth assays. Symbols: ▪, 10403S; □, OSM1. Dashed lines represent cultures where 1 mM glycine betaine was added to the growth medium.

FIG. 3.

FIG. 3.

Location and arrangement of the htrA gene on the chromosomes of L. monocytogenes strains EGD-e and F2365 and L. innocua strain CLIP 11262. lmo0289, lmo0290, lmo0291, and lmo0293 correspond to the ORF names obtained from the annotated genome sequence of strain EGD-e. Horizontal arrows show the 5′-to-3′ orientation of each ORF. Solid rectangles, predicted ribosomal binding sites. The vertical arrow indicates the position of the predicted transcriptional terminator sequence for the htrA gene. The ranges of nucleotide distances between the htrA gene and the adjacent ORFs represent the distances obtained from the completed genome sequences of L. monocytogenes strains EGD-e and F2365 and L. innocua strain CLIP 11262.

FIG. 4.

FIG. 4.

Alignment of the L. monocytogenes (Lm) HtrA protein with HtrA homologs from B. subtilis (Bs), L. lactis (Ll), L. helveticus (Lh), Y. enterocolitica (Ye), and E. coli (Ec). Symbols below residues represent amino acid identity (*) and strong similarity (:). Arrows indicate the H-D-S catalytic residues. Boxed region, putative PDZ domain.

FIG. 5.

FIG. 5.

Growth of strain LDW1 in BHI with 5% added NaCl and complementation of the salt-sensitive phenotype with plasmid pLW1. Growth curves represent the average optical density from two or more individual growth assays. Error bars, standard deviations among the growth assays. (a) Growth of strains 10403S and LDW1 at 30°C in BHI broth with or without 5% added NaCl. Symbols: ▪, 10403S in BHI; □, LDW1 in BHI; ▴, 10403S in BHI plus 5% added NaCl; ▵, LDW1 in BHI plus 5% added NaCl. (b) Complementation of the NaCl-sensitive phenotype with a plasmid carrying the wild-type htrA gene. All strains were grown at 30°C in BHI broth plus chloramphenicol and 5% added NaCl. Symbols: ▪, LDW2 (10403S plus pCON-1); □, LDW4 (LDW1 plus pCON-1); ▴, LDW3 (10403S plus pLW1); ▵, LDW5 (LDW1 plus pLW1).

FIG. 6.

FIG. 6.

Effect of elevated temperature on growth of L. monocytogenes strains 10403S and LDW1. Cultures were grown in BHI with aeration at 44°C, and optical densities at 600 nm were measured hourly. Growth curves represent the average optical density from two or more individual growth assays. Error bars, standard deviations among the growth assays. Symbols: ▪, 10403S; □, LDW1.

FIG. 7.

FIG. 7.

Effect of heat shock on survival of L. monocytogenes strains 10403S and LDW1. Cultures were grown at 30°C in BHI to mid-log phase and then shifted to 52°C. Samples were taken during the 4 h of heat shock and plated on BHI agar to enumerate survivors. Inactivation data were obtained from four individual cultures for each strain, and each experiment was run in duplicate. Error bars, standard deviations. Symbols: ▪, 10403S; □, LDW1.

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References

    1. Bayles, D. O., and B. J. Wilkinson. 2000. Osmoprotectants and cryoprotectants for Listeria monocytogenes. Lett. Appl. Microbiol. 30:23-27. - PubMed
    1. Begley, M., C. Hill, and C. G. M. Gahan. 2003. Identification and disruption of btlA, a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes. FEMS Microbiol. Lett. 218:31-38. - PubMed
    1. Behari, J., and P. Youngman. 1998. Regulation of hly expression in Listeria monocytogenes by carbon sources and pH occurs through separate mechanisms mediated by PrfA. Infect. Immun. 66:3635-3642. - PMC - PubMed
    1. Bianchi, A. A., and F. Baneyx. 1999. Hyperosmotic shock induces the σ32 and σE stress regulons of Escherichia coli. Mol. Microbiol. 34:1029-1038. - PubMed
    1. Camilli, A., D. A. Portnoy, and P. Youngman. 1990. Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions. J. Bacteriol. 172:3738-3744. - PMC - PubMed

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