H-NS is a part of a thermally controlled mechanism for bacterial gene regulation

Abstract

Temperature is a primary environmental stress to which micro-organisms must be able to adapt and respond rapidly. Whereas some bacteria are restricted to specific niches and have limited abilities to survive changes in their environment, others, such as members of the Enterobacteriaceae, can withstand wide fluctuations in temperature. In addition to regulating cellular physiology, pathogenic bacteria use temperature as a cue for activating virulence gene expression. This work confirms that the nucleoid-associated protein H-NS (histone-like nucleoid structuring protein) is an essential component in thermoregulation of Salmonella. On increasing the temperature from 25 to 37 degrees C, more than 200 genes from Salmonella enterica serovar Typhimurium showed H-NS-dependent up-regulation. The thermal activation of gene expression is extremely rapid and change in temperature affects the DNA-binding properties of H-NS. The reduction in gene repression brought about by the increase in temperature is concomitant with a conformational change in the protein, resulting in the decrease in size of high-order oligomers and the appearance of increasing concentrations of discrete dimers of H-NS. The present study addresses one of the key complex mechanisms by which H-NS regulates gene expression.

Figure 1. H-NS controls the expression of 77% of the thermoregulated genes of S. typhimurium

The cluster diagram shows the expression profile of S. typhimurium LT2 and the hns null mutant JH4000 grown at 25 and 37 °C, relative to the expression in LT2 at 25 °C. Out of 4451 genes, 531 showed an expression differential of ≥3-fold between incubation temperatures of 25 and 37 °C in LT2 and were therefore defined as temperature-responsive. The temperature response of 408 of these genes was found to be H-NS-regulated, as demonstrated by the similar expression levels observed at the two temperatures in JH4000. Each horizontal line represents one gene; red indicates an increase in expression, yellow indicates no change, and blue indicates a decrease in expression (relative expressions levels are indicated on the left-hand side).

Figure 2. Kinetics of temperature induction

A culture of LT2a was incubated at 25 °C in LB broth to a D₆₀₀ of 0.60 and transferred to 37 °C. Samples were harvested just prior to temperature up-shift and at intervals thereafter for RNA extraction. Expression of hilA (▲), hilC (◆) and hilD (■) was measured by quantitative RT-PCR and normalized to an LT2 culture grown at 37 °C throughout, and harvested at D₆₀₀ of 0.60.

Figure 3. Competitive gel-shift assays with H-NS at 25 and 37 °C

A 2128 bp DNA fragment encoding hilC and the up-stream gene STM2868 were digested with DraI. Digested DNA (0.5 μg) was incubated with a range of concentrations of H-NS at 25 °C (A) or 37 °C (B) and electrophoresis was carried out at the respective temperatures. A restriction map (C) shows the location of the genes and DraI restriction sites. Arrows indicate the fragments displaying a high affinity for H-NS.

Figure 4. Change in H-NS oligomerization with increasing temperature over the range 17.5–45 °C (A) and reversibility of H-NS oligomerization between 25 to 45 °C (B)

(A) SEC graphs for H-NS^1–89 at a range of temperatures (lines: 1, 17.5 °C; 2, 25 °C; 3, 30 °C; 4, 35 °C; 5, 40 °C; 6, 45 °C). The inset shows an expansion of the graph demonstrating the increase in the peak size at an elution volume of approx. 71.5 ml corresponding to dimeric H-NS^1–89. (B) SEC graphs for H-NS^1–89. H-NS^1–89 was initially run on the column at 25 °C (line 1). The sample was then kept at 45 °C for 15 h and re-run on the column at that temperature (line 3). It was then subsequently cooled to 4 °C and incubated at this temperature for 6 h before being heated to 25 °C and injected on to the chromatographic column (line 2). The sample shows almost complete reversibility over the temperature range studied. The inset shows that the formation of dimer (elution volume 71.5 ml) is also highly reversible.

Figure 5. Change in observed enthalpy with temperature

Plot of the change in observed enthalpy, ΔH, against temperature for the interaction of full-length H-NS with an approx. 300 bp oligonucleotide derived from sonicated calf thymus.

Figure 6. Ribbon representation of the structures of H-NS^1–57 (left-hand panel; PDB code, 1LR1 [22]) and H-NS^1–46 (right-hand panel; PDB code, 1NI8 [38])

Helix 3 (H3) is the longest helix in both structures.

Figure 7. Representation of the proposed mechanism for H-NS responding to a temperature rise from 25 to 37 °C

In the left-hand panel (25 °C) full-length H-NS is able to bind to strands of DNA in a co-operative manner based on the high-order oligomeric structure. The right-hand panel (37 °C) shows the effect of the conformational change on one of the H-NS dimers due to increased temperature. The dimer is no longer able to interact with the high-order protein oligomer and therefore no longer binds to DNA in a co-operative manner. The resulting loss in affinity affects the DNA topology and makes it more accessible to RNA polymerase, leading to gene transcription.