Expression and assembly of a functional type IV secretion system elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli

Abstract

Conditions perturbing protein homeostasis are known to induce cellular stress responses in prokaryotes and eukaryotes. Here we show for the first time that expression and assembly of a functional type IV secretion (T4S) machinery elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. After induction of T4S genes by a nutritional upshift and assembly of functional DNA transporters encoded by plasmid R1-16, host cells activated the CpxAR envelope stress signaling system, as revealed by induction or repression of downstream targets of the CpxR response regulator. Furthermore, we observed elevated transcript levels of cytoplasmic stress genes, such as groESL, with a concomitant increase of sigma(32) protein levels in cells expressing T4S genes. A traA null mutant of plasmid R1-16, which lacks the functional gene encoding the major pilus protein pilin, showed distinctly reduced stress responses. These results corroborated our conclusion that the activation of bacterial stress networks was dependent on the presence of functional T4S machinery. Additionally, we detected increased transcription from the rpoHp(1) promoter in the presence of an active T4S system. Stimulation of rpoHp(1) was dependent on the presence of CpxR, suggesting a hitherto undocumented link between CpxAR and sigma(32)-regulated stress networks.

FIG. 1.

A nutritional upshift leads to rapid induction of T4S genes. TraM protein and traA mRNA levels as well as DNA transfer frequencies were determined in different phases of bacterial growth. Analyses were performed in stationary phase as well as at the indicated time points after providing E. coli MG1655 cells harboring plasmid R1-16 with fresh media. (A) Analysis of the 14-kDa TraM protein by Western blotting. A part of the Coomassie-stained SDS gel with a 40-kDa protein is shown to demonstrate equal loading. (B) Northern blot analysis of traA mRNA levels. 23S rRNA is shown as a loading control. (C) Mating assays. The transfer frequencies (filled diamonds) and culture densities (OD₆₀₀; open circles) are shown. Values represent means of results from three independent experiments. No error bar indicates that the standard deviation was below the graphical resolution of the drawing program. Numbers on the x axis indicate minutes after dilution of the cultures into fresh medium. S, stationary phase.

FIG. 2.

Induction of a CpxAR-mediated extracytoplasmic stress response in E. coli cells expressing an active T4S system. E. coli cells without plasmid or harboring the repressed plasmid R1 or the derepressed plasmid R1-16 were analyzed by Northern blotting (A to C) or β-galactosidase assays (D). Analyses were performed in different phases of bacterial growth as indicated. Numbers indicate minutes after dilution of the cultures into fresh medium. S, stationary phase. (A to C) The visualized transcripts are indicated. The apparent size of the major mRNA species is given, when appropriate. 23S rRNA is shown as a loading control. Numbers at the bottom of the Northern blot shown in panel B indicate the relative signal intensities (n-fold) compared to the signal intensity in stationary phase. (D) The extracytoplasmic stress response mediated by the T4S system is abolished in a cpxR mutant genetic background. The activity of a transcriptional degP-lacZ fusion was measured 15 and 30 min after feeding stationary-phase cells with fresh medium. Shown are the results of two independent measurements.

FIG. 3.

Expression of the plasmid R1-encoded T4S system elicits a cytoplasmic stress response. E. coli MG1655 cells without plasmid or harboring the repressed plasmid R1 or the derepressed plasmid R1-16 were analyzed by Northern blotting. groESL (A), dnaJK (B), and hslVU (C) transcript levels are increased in cells expressing the R1-16-encoded T4S genes. 23S rRNA is shown as a loading control. Numbers above the blots indicate minutes after dilution. Numbers at the bottom of the Northern blot shown in panel B indicate the relative signal intensity compared to the signal intensity in stationary phase. S, stationary phase.

FIG. 4.

Both extracytoplasmic and cytoplasmic stress responses are dependent on the presence of a functional pilin gene. E. coli MG1655 cells without plasmid or harboring plasmid R1-16 or the traA null variant R1-16/A0 were analyzed by Northern blotting. (A) Analysis of degP mRNA levels. (B) Analysis of groESL mRNA levels. 23S rRNA is shown as a loading control. Numbers indicate minutes after dilution. S, stationary phase.

FIG. 5.

The T4S system-dependent cytoplasmic stress response is mediated by σ³². (A) Schematic representation of the experimental setup used for primer extension analyses of the groESL transcript which is represented by the waved line. For reverse transcription, a Cy5-labeled oligonucleotide (indicated by an arrow) complementary to the sequence downstream from the σ³² transcription start site was used. AUG indicates the translation start site for the GroES protein within the groESL transcript. (B) Primer extension analyses with RNA isolated 15 min after dilution into fresh medium. The signal indicated by an arrowhead corresponds to transcripts originating from the transcription start site (+1) of the groESL σ³² promoter. The four lanes on the left show the DNA sequence surrounding the σ³²-dependent transcription start site. Corresponding nucleotides in the DNA and RNA sequences are boxed. (C) Steady-state levels of σ³² protein are elevated when cells express a functional T4S system. Western blot analyses detecting σ³² (32 kDa) and maltose binding protein (45 kDa) are shown. S, stationary phase; 15, number of minutes after feeding cells with fresh medium.

FIG. 6.

Analyses of rpoH mRNA levels and promoter activities. (A) rpoH mRNA levels are increased in cells expressing the T4S system. E. coli MG1655 cells without plasmid or harboring plasmid R1-16 were analyzed by Northern blotting. 23S rRNA is shown as a loading control. Numbers at the top indicate minutes after dilution. Numbers at the bottom of the Northern blot indicate the relative signal intensity compared to the signal intensity in stationary phase. S, stationary phase. (B) Schematic representation of the experimental setup used for primer extension analyses of the rpoH transcripts (waved lines). For reverse transcription, a Cy5-labeled oligonucleotide (indicated by an arrow) complementary to the sequence downstream from the initiation codon (indicated by AUG) of the rpoH coding sequence was used. The promoters p₁, p₃, p₄, and p₅ as well as the sigma factors which are known to stimulate transcription from these promoters are indicated. The transcription start site is at +1. (C and D) Quantitative representation of primer extension analyses of rpoH transcripts isolated from cells at the indicated time points after feeding cells with fresh media. The relative abundance (reflected by the peak area) of transcripts starting at the indicated rpoH promoters p₁ and p₄ is shown. P1/P4, p₁ promoter activity normalized to the unregulated promoter p₄. (C) Comparison of specific rpoH transcripts from E. coli MG1655 cells without plasmid (n.p.) or harboring plasmid R1-16. (D) Comparison of specific rpoH transcripts from wild-type (wt) and cpxR mutant cells (cpxR).

FIG. 7.

Sex is stress. In the depicted model, expression and assembly of the conjugative T4S complex activate extracytoplasmic and cytoplasmic stress regulons. The assembly of a functional T4S complex leads to extracytoplasmic stress that is sensed by the CpxAR system. The phosphorylated CpxR response regulator (CpxR-P) stimulates the transcription of periplasmic folding factors and proteases. These proteins may play a protective role or may be needed for efficient assembly of the T4S machinery or for degradation of excessive Tra proteins. A cytoplasmic stress response which induces the transcription of classical heat shock genes is mediated via increased steady-state levels of the alternative sigma factor σ³² (encoded by the rpoH gene). We assume that chaperons such as DnaJK-GrpE and/or GroEL/S bind to Tra proteins in the cytoplasm, thereby increasing the half-life of σ³². The induced proteins might be involved in degradation of regulatory Tra proteins and in conferring export competence to precursors of T4S proteins. Our data suggest that the two stress networks are connected via CpxR-P-dependent transcriptional activation of rpoH. OM, outer membrane; IM, inner membrane; PG, peptidoglycan.