Temperature-dependent conjugative transfer of R27: role of chromosome- and plasmid-encoded Hha and H-NS proteins

Abstract

IncHI plasmids encode multiple-antibiotic resistance in Salmonella enterica serovar Typhi. These plasmids have been considered to play a relevant role in the persistence and reemergence of this microorganism. The IncHI1 plasmid R27, which can be considered the prototype of IncHI plasmids, is thermosensitive for transfer. Conjugation frequency is highest at low temperature (25 to 30 degrees C), decreasing when temperature increases. R27 codifies an H-NS-like protein (open reading frame 164 [ORF164]) and an Hha-like protein (ORF182). The H-NS and Hha proteins participate in the thermoregulation of gene expression in Escherichia coli. Here we investigated the hypothetical role of such proteins in thermoregulation of R27 conjugation. At a nonpermissive temperature (33 degrees C), transcription of several ORFs in both transfer region 1 (Tra1) and Tra2 from R27 is upregulated in cells depleted of Hha-like and H-NS-like proteins. Both chromosome- and plasmid-encoded Hha and H-NS proteins appear to potentially modulate R27 transfer. The function of R27-encoded Hha-like and H-NS proteins is not restricted to modulation of R27 transfer. Different mutant phenotypes associated with both chromosomal hha and hns mutations are compensated in cells harboring R27.

FIG. 1.

Comparison of the amino acid sequences of R27 ORF182 and E. coli protein Hha and of R27 ORF164 and E. coli protein H-NS by using the ClustalW 1.81 program of the European Bioinformatics Institute.

FIG. 2.

(A) RT-PCR analysis of transcription of the traH, traJ, and trhR (Tra1) genes and the trhA and trhE (Tra2) genes from plasmid R27 in WT and hha hns backgrounds. Cells were grown at 25°C (lanes 1 and 2) or 33°C (lanes 3 and 4). Lanes 1 and 3, strain 5K(R27); lanes 2 and 4, strain Hha3-hns(R27 hha hns). (B) RT-PCR analysis of transcription of trhR in cells lacking Hha-like proteins. Cells were grown at 25°C (lanes 1 and 2) and at 33°C (lanes 3 and 4). Lanes 1 and 3, strain 5K(R27); lanes 2 and 4, strain Hha3 ydgT(R27 hha). RT-PCR of 16S rRNA was used as a control to confirm equivalent quantities of template loading.

FIG. 3.

(A) Effect of increasing amounts of H-NS protein (0.1625, 0.325, 0.4875, 0.65, and 1.3 μg) on the electrophoretic mobility of DNA fragments corresponding to the promoter regions of the traH, traJ, and trhR (Tra1) genes, the trhA and trhE (Tra2) genes, or the oriT region. Arrows point to the R27 DNA fragment used in each test. (B) Effect of H-NS protein plus Hha protein on the electrophoretic mobility of a DNA fragment corresponding to the oriT region. Lane 1, no protein added; lane 2, Hha protein added (48.44 μg); lane 3, Hha protein (48.44 μg) and H-NS protein (0.325 μg) added; lane 4, H-NS protein (0.325 μg) added; lane 5, Hha (48.44 μg) and H-NS (0.65 μg) proteins added; lane 6, H-NS protein (0.65 μg) added. As nonspecific competitor DNA, an S/E (in thrR assay), S/P (in traH, trhA, trhE, and oriT assays), or S/H (in traJ assay) fragment corresponding to the upstream regulatory region of the hly operon of plasmid pHly152 was used (see text for details). The DNA standard used was a 100-bp ladder (Biotools) in all cases, except for the thrR assay, where λ/HindIII (Biotools) was used.

FIG. 4.

Plasmid R27 compensates for some hns-induced phenotypes. (A) Growth at low temperature of strains 5K (squares), 5K hns (circles), 5K hns(R27) (triangles), and 5K hns(R27 hns) (diamonds). (B) Hemolytic activity in culture supernatants of the mutant and WT strains transformed with hemolytic plasmid pANN202-312. The hemolytic activity of strain 5K hns(pANN202-312), referred to as 100%, was 1,327 U. OD_600 nm, optical density at 600 nm.

FIG. 5.

Plasmid R27 compensates for the deregulation of hemolysin production that hha mutants show. Hemolytic activity in culture supernatants of strains 5K, Hha3, Hha3(R27), and Hha3(R27 hha) transformed with hemolytic plasmid pANN202-312. The hemolytic activity of strain Hha3(pANN202-312), referred to as 100%, was 436 U.

FIG. 6.

Plasmid R27 compensates for hha-hns-induced deregulation of hemolysin expression (B) and for alterations of the growth rate at 37°C (A). The strains tested were 5K(pANN202-312) (squares), Hha3 hns(pANN202-312) (circles), Hha3 hns(pANN202-312 R27) (triangles), and Hha3 hns(pANN202-312 R27 hha hns) (diamonds). Hemolytic activity of strain Hha3 hns(pANN202-312), referred to as 100%, was 7,630 U. OD₆₀₀, optical density at 600 nm.