Temperature Dependent Control of the R27 Conjugative Plasmid Genes

Abstract

Conjugation of R27 plasmid is thermoregulated, being promoted at 25°C and repressed at 37°C. Previous studies identified plasmid-encoded regulators, HtdA, TrhR and TrhY, that control expression of conjugation-related genes (tra). Moreover, the nucleoid-associated protein H-NS represses conjugation at non-permissive temperature. A transcriptomic approach has been used to characterize the effect of temperature on the expression of the 205 R27 genes. Many of the 35 tra genes, directly involved in plasmid-conjugation, were upregulated at 25°C. However, the majority of the non-tra R27 genes-many of them with unknown function-were more actively expressed at 37°C. The role of HtdA, a regulator that causes repression of the R27 conjugation by counteracting TrhR/TrhY mediated activation of tra genes, has been investigated. Most of the R27 genes are severely derepressed at 25°C in an htdA mutant, suggesting that HtdA is involved also in the repression of R27 genes other than the tra genes. Interestingly, the effect of htdA mutation was abolished at non-permissive temperature, indicating that the HtdA-TrhR/TrhY regulatory circuit mediates the environmental regulation of R27 gene expression. The role of H-NS in the proposed model is discussed.

Keywords: R27; TrhR/TrhY-HtdA; plasmid conjugation; temperature-dependent control; transcriptional regulation.

Figure 1

Expression profile of the R27 genes at permissive and non-permissive temperatures. (A) Conjugation rates were calculated using cultures of both donor [AAG1(R27)] and recipient (MG1655) strains grown at either 25 or 37°C. Average values and standard deviation of three independent experiments is shown. (B) Summary of the genes with an altered transcriptional expression in cultures of the strain AAG1(R27) grown at either 25 or 37°C. R27 genes are also divided in tra and non-tra genes. Attending to the fold change (FC), genes are classified in derepressed at 25°C (FC>+2), repressed at 25°C (FC < -2), no thermoregulated (-2 < FC < +2) and genes with signal values lower than 100 fluorescence units in both culture conditions were arbitrarily considered as non-expressed. (C) Fold change expression of the 205 ORFs encoded in R27 plasmid between AAG1(R27) cells grown at either 25 or 37°C. Expressed genes were classified as tra genes (black bars) and non-tra genes (gray bars). Non-expressed genes are indicated as white bars.

Figure 2

HtdA represses expression of R27 genes primarily at 25°C. Summary of the genes showing an altered transcriptional expression in cultures of the strains AAG1(drR27) and AAG1(R27) grown at either 25°C (A) or 37°C (B). R27 genes are also divided in tra and non-tra genes. Attending to the fold change (FC), genes are classified in derepressed at (FC>+2), repressed (FC < -2), no thermoregulated (-2 < FC < +2) and genes with signal values lower than 100 fluorescence units in both culture conditions were arbitrarily considered as non-expressed. (C) Fold change expression of the 205 ORFs encoded in R27 plasmid between AAG1(drR27) cells and AAG1(R27) cells grown at either 25°C (upper panel) or 37°C (lower panel). Expressed genes were classified as tra genes (black bars) and non-tra genes (gray bars). Non-expressed genes are indicated as white bars.

Figure 3

TrhR and TrhY overexpression causes induction of the F operon even at non-permissive temperature. Transcriptional expression of the F operon of R27 was monitored in cultures of the strain AAG1-F, carrying a chromosomal lacZ fusion with the promoter sequence of the F operon. The effect of the pBADtrhRY plasmid in the presence of either R27 or its htdA derivative drR27 was assessed. Cultures were grown at either 25 or 37°C to mid-logarithmic phase in the absence (gray bars) or presence (black bars) of arabinose (0.02%). The β-galactosidase activity (Miller units) was determined in three independent cultures and mean values with standard deviations are plotted.

Figure 4

Transcriptional organization of the AN operon. (A) M-plots of the temperature dependent expression of the tra operons. The M value (log₂FC 25/37°C) for the expressed genes of the different tra operons is depicted. An M value between +1 and−1, equivalent to a FC of +2 and−2, is considered no alteration in the expression and is shadowed in gray. (B) Representation of the AN operon. Dashed lines indicate the different putative transcripts (#1 to #5) derived from the AN operon. A thick line indicates the fragments (a to d) cloned in pRS551 to construct the lacZ fusions used. (C) The positions of 5′- and 3′ ends of the transcripts #2, #3 and #4, labeled in bold, were determined by circRNA. The putative 5′ ends of transcript #5 identified by 5′RACE assays. (D) RT-PCR using primers within the ORFs flanking the intergenic regions indicated. In all cases RNA not-RT AMV-treated was used as negative control. (E) Transcriptional expression of the lacZ fusions with the indicated fragments of AN operon was determined in cultures grown at 25°C to mid-logarithmic phase. The β-galactosidase activity (Miller units) was determined in three independent cultures and mean values with standard deviations are plotted.

Figure 5

Schematic model of regulation of the tra operons at permissive and non-permissive temperatures. Green arrows indicate expression is promoted, whereas red crosses indicate expression is repressed.