H-NS Controls pap and daa Fimbrial Transcription in Escherichia coli in Response to Multiple Environmental Cues

Strains and media.

The strains, plasmids, and bacteriophages used in this study are described in Table 1. Luria-Bertani (LB) broth, tryptone broth (TB), M9 minimal (M9) broth, and M9 agar were prepared as described previously (37, 50). Antibiotics, when used, were at final concentrations of 25 μg ml⁻¹ (kanamycin) and 25 μg ml⁻¹ (tetracycline). M9 agar-based media contained the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal) at a final concentration of 40 μg ml⁻¹.

Construction of mutant strains.

P1 transduction was used to introduce the hns651 mutation (19) into strains DL812 and DL1530 (Table 1). The preparation of P1 lysates and P1 transductions were carried out as described previously (50). hns651 transductants with a pink colony phenotype were selected on MacConkey medium containing tetracycline and salicin as described elsewhere (19). The hns651 mutation is an IS1 insertion in the 13th codon of the hns gene (N. P. Higgins, personal communication). Based on Western blot analysis, no H-NS protein was detected in strains containing the hns651 mutation (57).

Similarly, P1 transduction was used to introduce the Δcrp-45 mutation (48) into strains DL812 and DL1530 to create CWZ369 and CWZ370, respectively (Table 1). The Δcrp-45 mutation is a deletion mutation in the cyclic AMP (cAMP) receptor protein (CRP) (48). A P1 lysate was grown on strain DL3087, which contains a Tn10 insertion linked to the Δcrp-45 mutation (Table 1). Δcrp-45 transductants with a white colony phenotype were selected on MacConkey medium containing tetracycline and maltose.

Growth conditions.

For standard growth conditions, the bacteria were cultured in 10 ml of M9 glyc (M9 minimal liquid medium containing 2.45 μM ferric citrate, 30 μM thiamine, 100 μM calcium chloride, 1 mM magnesium sulfate, and 0.2% glycerol as a carbon source, pH 7) in a 37°C shaking water bath in a 50-ml Erlenmeyer flask. To test the effect of low temperature on fimbrial transcription, bacteria were grown at 18 to 20°C in a shaking water bath in M9 glyc. The effect of low oxygen levels was assessed by growing standing cultures in M9 glyc at 37°C. The effect of growth on a solid substrate was measured by plating cells on M9 glyc agar medium that did not contain X-Gal. The bacteria were collected by rinsing the plate with 10 ml of M9 salts at approximately the same time as the liquid M9 glyc culture was harvested. Bacteria were cultured in LB broth at 37°C in a shaking water bath to determine the effect of rich medium.

To measure the effects of the other stimuli, M9 glyc was modified as follows. For M9 gluc, glucose was substituted, at a final concentration of 0.2%, for glycerol in the standard medium to test a change in carbon source. To determine the effect of high osmolarity, the sodium chloride concentration was increased by 300 mM, compared to 8.5 mM in M9 glyc (making M9 NaCl). High pH was tested by using M9, pH 8.0, consisting of M9 glyc buffered by the addition of TAPS [tris(hydroxymethyl)methylaminopropanesulfonic acid] to a final concentration of 100 mM and adjusted to a pH of 8.0 using 4 M NaOH as described previously (51). Similarly, to make M9 pH 5.5, MES (2-(N-morpholino)ethanesulfonic acid) was added to M9 glyc at a final concentration of 100 mM and the pH was adjusted to 5.5 using 1 N HCl as described previously (51). M9 CAA was M9 glyc supplemented with Casamino Acids at a final concentration of 0.2% to assess the effect of amino acids on transcription. To test the effect of high iron concentrations (in M9 Fe), the concentration of ferric citrate was increased to 98 μM, compared to 2.45 μM in M9 glyc. All cultures except those used to measure the effects of low temperature and low oxygen levels were incubated at 37°C in a shaking water bath.

Measurement of β-galactosidase activity.

For assays determining the effects of environmental stimuli on fimbrial transcription, each bacterial strain was inoculated from a frozen −70°C stock onto M9 glyc agar, incubated at 37°C, and passaged once. After growth for approximately 36 h, a single Lac⁺ colony was isolated and resuspended in 1 ml of M9 salts. Since expression of β-galactosidase served as a reporter of fimbrial gene transcription in each strain used, a Lac⁺ colony was chosen to ensure that the cultures were started with bacteria that were actively transcribing the fimbrial genes. Flasks containing 10 ml of the appropriate prewarmed medium were inoculated with 140 μl of the colony suspension. Experiments testing different environmental conditions were frequently conducted in parallel, using the same colony suspension to inoculate different medium.

The bacterial cultures were grown to log phase (optical density at 600 nm of 0.25 to 0.9), and β-galactosidase activities were measured as described previously (37). This inoculation method ensured that all bacterial strains had grown for approximately 9 to 11 generations prior to the measurement of β-galactosidase activity. For cultures grown at pH 5.5 and 7.0, bacteria were centrifuged and then resuspended in M9 salts before proceeding with the assay. Each β-galactosidase activity value represents an average from two or more separate cultures grown under identical conditions.

For experiments assessing the effect of the Δcrp-45 mutation on fan and daa transcription, each bacterial strain was inoculated onto LB agar (DL812 and CWZ369) or TB agar (DL1530 and CWZ370) and incubated at 37°C. A single colony from each strain was isolated and resuspended in 1 ml of LB. Five milliliters of broth (LB or TB) was inoculated from the colony suspension and grown to exponential phase. β-Galactosidase activity was measured as described above, each value representing an average from two separate cultures grown under identical conditions.

Calculation of switch frequencies.

Phase transition rates were calculated as described previously (9). Each switch frequency is based on data from two or more separate colonies. To determine the switch frequency, each strain was streaked on the medium to be tested. An initial colony, phase ON (Lac⁺) or phase OFF (Lac⁻), was excised, resuspended and diluted in M9 salts, and plated on the same medium for the determination of switch frequencies.

Experimental design.

To study transcriptional regulation for the pap, daa, and fan fimbrial genes, we used transcriptional fusions that place lacZYA expression under the control of the promoter that drives transcription of the major fimbrial subunit gene (Fig. 1). In previous studies, three separate strains, each containing one of the fimbrial operon fusions as a lambda lysogen on the chromosome of MC4100, were created (11, 12, 55). DL1504 contains the papBA-lacZYA fusion (λ354), DL1530 contains the daa-lacZYA fusion (λ366), and DL812 contains the fanABC′-lacZYA fusion (λMW01) (Table 1). These fusions were constructed to measure transcription initiated from the pBA (pap), pA (daa), and pA (fan) promoters, respectively.

To assess changes in fimbrial gene expression under different conditions, transcription was quantitated by determining levels of β-galactosidase expression and comparing them to transcription levels of cultures grown in M9 glyc in a 37°C shaking water bath. These conditions were chosen as a reference point, as they yield high levels of fimbrial transcription for each of the operons tested.

To ensure that a given culture was initiated with cells that were transcriptionally active for fimbrial gene expression, the cultures were inoculated with a Lac⁺ colony grown on M9 glyc agar at 37°C. Because the pap and daa operons are subject to a phase variation mechanism, both phase ON (Lac⁺) and phase OFF (Lac⁻) colonies are observed under these conditions. Within a Lac⁺ colony, the percentage of cells in a phase ON state may vary between approximately 20 and 50 when grown on M9 glyc at 37°C (data not shown). To limit the variability due to phase variation, experiments in which the same colony suspension was used to start several cultures grown under different environmental conditions were conducted in parallel as often as possible. However, because of this inherent variability, changes in transcriptional levels for the pap and daa operons that were less than twofold were not considered significant in this study. fan gene expression is not known to be subject to phase variation, and all colonies displayed a Lac⁺ phenotype when grown on M9 glyc agar at 37°C.

H-NS controls pap and daa transcription.

Before investigating the effect of environmental stimuli on fimbrial transcription and the potential role of H-NS in these processes, it was important to determine whether H-NS controlled transcription of the daa and fan operons and, if so, if it altered fimbrial transcription in the absence of a change in environmental conditions. It was shown previously that introduction of the hns651 mutation significantly decreases transcription of the pap operon when environmental conditions remain unchanged (54, 57). Our results confirm this, showing an approximately 7.6-fold decrease in pap transcription in the hns651 mutant strain DL1947 compared to the wild-type strain DL1504 when grown in M9 glyc (Fig. 2). Studies have indicated that the repressive effect of the hns651 mutation on pap transcription is due, at least in part, to alterations in the rates at which cell transit between phase ON and phase OFF states, with the overall effect of decreasing the number of cells in a phase ON state at 37°C in M9 glyc (Table 2; references 54 and 57).

To determine whether H-NS is involved in controlling transcription of the daa and fan operons, the hns651 mutation was introduced into strains containing each of these transcriptional operons. The hns651 mutation is an insertion element in hns, and no H-NS protein is detected by Western blot analysis in strains containing this mutation (57). The hns651 mutation was introduced into DL1530 to create CWZ263 (Table 1). In contrast to the pap operon, transfer of the hns651 mutation into strain DL1530 containing the daa transcriptional fusion caused an increase in transcription of this operon in M9 glyc, indicating that H-NS plays only a negative role in controlling daa transcription (Fig. 2). Because of the effect of the hns651 mutation on pap phase variation (Table 2; references 54 and 57), phase transition rates were calculated in strain CWZ263 on M9 glyc plates at 37°C to determine if the stimulatory effect of the hns651 mutation on daa transcription was through influencing the phase variation mechanism (Table 2). While the phase ON→OFF transition rate was unchanged, the phase OFF→ON transition rate was 11.5-fold higher in the hns651 mutant strain CWZ263 than in the wild-type strain. Thus, cells transit from a phase OFF to a phase ON state more frequently than in the wild-type strain, accounting for the increased β-galactosidase activities measured in mutant strain CWZ263 compared to wild-type strain DL1530 grown on M9 glyc at 37°C.

In a similar manner, the hns651 mutation was introduced into strain DL812 containing the fan transcriptional fusion. Transductants were initially streaked on MacConkey-salicin to determine if they could utilize salicin, a trait indicative of an hns mutant strain due to derepression of the cryptic bgl operon (19). The hns651 transductants demonstrated an unstable phenotype in which both pink and white colonies were seen; in contrast pap and daa hns651 transductants retained a uniform pink colony phenotype. Because of this result, fan transcription was measured in five separate hns651 transductants after growth in various media to assess whether transcription was consistent between them. The transductants demonstrated a high level of variability for fan transcription (data not shown); thus, no quantitative data are shown. The results suggest that the transductants may have harbored secondary mutations and thus were genetically unstable (see Discussion).

H-NS controls fimbrial transcription in response to multiple environmental cues.

In the wild-type and hns651 mutant strains, various environmental stimuli were tested for their effects on pap, daa, and fan transcription and to determine the role of H-NS in responding to these environmental cues.

Temperature.

Temperature has been shown to be an important regulator of virulence gene expression in several genera of bacteria (34, 36). To determine the effect of temperature on fimbrial transcription, cultures were grown to exponential phase at 18 to 20°C in M9 glyc, and β-galactosidase activities were measured to determine pap, daa, and fan gene expression.

pap and fan transcription had previously been shown to be thermoregulated (9, 26, 53)); results of this study confirm those findings, demonstrating 380- and 121-fold reductions in pap and fan transcription, respectively, in response to low temperature (Fig. 2). In this study, daa transcription was also shown to be thermoregulated, demonstrating that low temperature is an important environmental cue for all three fimbrial operons. Transcription of daa was reduced 5.2-fold at low temperature compared to 37°C (Fig. 2).

Introduction of the hns651 mutation relieves the repression of low temperature on pap and daa fimbrial transcription, demonstrating that H-NS acts as a thermoregulator in each of these operons. For the daa operon, the hns651 mutation caused an increase in daa expression at 23°C to levels equivalent to those seen in the wild-type strain grown at 37°C (Fig. 2). H-NS was shown previously to control thermoregulation of the pap operon, and our results confirm this observation (Fig. 2) (25, 57).

Osmolarity.

Changes in osmolarity have been found to influence the transcription of virulence genes in several genera of bacteria, including Escherichia, Salmonella, Shigella, Pseudomonas, and Vibrio (34). To test the effect of high osmolarity on fimbrial transcription, β-galactosidase activities were measured after growth in M9 NaCl. Transcription of all three fimbrial operons was repressed by growth at high osmolarity. pap, daa, and fan transcription levels were repressed 3.5-, 2.1-, and 3.9-fold, respectively, compared to growth at low osmolarity (in M9 glyc) (Fig. 2).

For the daa operon, introduction of the hns651 mutation fully abrogates the transcriptional repression by high osmolarity. Transcription in the hns651 mutant strain grown under high-osmolarity conditions exceeds that seen for the hns651 mutant strain grown under conditions of low osmolarity (Fig. 2). For the pap operon, the level of transcription in the hns651 mutant strain at high osmolarity is decreased 1.6-fold compared to the hns651 mutant strain under low osmolarity, indicating that in the hns651 mutant strain high osmolarity may still have a repressive effect (Fig. 2). However, the magnitude of the effect of high osmolarity in the hns651 mutant strain is not as great as in the wild-type strain, where pap transcription is decreased 3.5-fold in response to increased osmolarity.

Rich medium.

Growth in LB, a rich medium, was also shown to repress transcription of all three operons tested. LB had the largest effect on pap transcription, which was repressed 22.4-fold compared to growth in M9 glyc (Fig. 2). Growth in LB reduced daa and fan transcription 4.5- and 3.7-fold, respectively, compared to growth in M9 glyc (Fig. 2).

Growth in LB has a repressive effect that is relieved for the pap and daa operons by the hns651 mutation. Transcription levels in the hns651 mutant strains grown in LB were equivalent to those seen when cells were grown in M9 glyc (Fig. 2).

Carbon source.

Glucose as a carbon source serves as another stimulus that controls transcription of all three fimbrial operons tested. The effect of glucose was tested by growing cultures in M9 gluc. Glucose was shown to repress fan transcription 3.5-fold (Fig. 2). Similarly, pap transcription was reduced 7.3-fold and daa transcription was reduced 6.6-fold compared to growth at 37°C in M9 glyc medium (Fig. 2), confirming results of previous studies (3, 5).

pap transcription has been shown to be dependent on the cAMP-catabolite gene activator protein (CAP) complex (3, 21, 22). To determine if daa and fan transcription was dependent on CAP, the Δcrp-45 deletion mutation (48) was transduced into strains DL812 and DL1530 to create CWZ369 and CWZ370, respectively. fan transcription in CWZ369 (295 ± 14 Miller units [MU]) was reduced 5.1-fold compared to transcription in the wild-type strain DL812 (1,502 ± 96 MU). Similarly, daa transcription was reduced in the Δcrp-45 strain CWZ370 (1 ± 0 MU) compared to the wild-type strain DL1530 (19 ± 2 MU). These results indicate that fan and daa transcription is dependent on CAP and are supported by other studies indicating cAMP-CAP is an important regulator of daa and fan gene expression (6, 32).

For the pap and daa operons, glucose is still somewhat repressive in the absence of H-NS. pap and daa transcriptional levels are 2.9- and 2.3-fold lower, respectively, in the hns651 mutant strains grown in M9 gluc compared to the mutant strains grown in M9 glyc (Fig. 2). However, while transcription is still decreased in the hns651 mutant strains grown in glucose, the repression is not as extensive as that in the wild-type strains, where transcriptional levels are reduced 7.3- and 6.6-fold, respectively, in response to glucose (Fig. 2).

Low oxygen.

Low oxygen levels were tested to determine their effects on fimbrial transcription by growing standing cultures in M9 glyc. The cultures were harvested in exponential phase, with the growth times being two to three times as long as in the cultures grown in M9 glyc with aeration. Low oxygen levels were shown to decrease fan transcription 3.7-fold (Fig. 2) compared to growth with aeration, whereas for the pap and daa operons, transcription levels varied less than 2-fold between conditions of low and high aeration (Fig. 2). Introduction of the hns651 mutation did not significantly alter pap or daa transcription in response to low-oxygen conditions.

pH.

Because pH has been shown to regulate the expression of several virulence genes (34), we tested the effects of low and high pH on fimbrial transcription. High pH (8.0) decreased fan transcription 2.3-fold, whereas pap and daa transcription was similar to that measured after growth at pH 7.0 (in M9 glyc) (Fig. 2). While high pH repressed fan transcription, low pH increased fan transcriptional levels 1.6-fold compared to growth at pH 7.0 (Fig. 2). pap and daa transcription at pH 5.5 was equivalent to that seen at pH 7.0 (Fig. 2).

While low pH did not greatly influence transcription of the fimbrial operons in the wild-type strains, it had a greater effect on fimbrial transcription in the hns651 mutant strains. Growth at a pH of 5.5 was found to stimulate pap and daa transcription 4.7- and 2.4-fold, respectively, above the level measured in the hns651 mutant strains grown at pH 7.0, indicating that low pH stimulates pap and daa transcription in the absence of H-NS (Fig. 2).

Casamino Acids.

The addition of Casamino Acids to M9 glyc was found to cause a 2.1-fold increase in daa transcription compared to growth in M9 glyc (Fig. 2). pap and fan transcription levels varied less than twofold after growth in M9 CAA compared to growth in M9 glyc (Fig. 2).

In contrast, growth of the hns651 mutant strains in Casamino Acids repressed fimbrial transcription for the pap and daa operons. Transcription in the respective hns651 mutant strains was decreased 3.9-fold for pap and 2.2-fold for daa in response to the addition of Casamino Acids compared to transcription of the hns651 mutant strains in M9 glyc (Fig. 2).

Solid medium.

For S pili, growth on solid medium was shown to stimulate sfa transcription fourfold above growth in liquid medium (49). For the pap operon, transcriptional levels were similar to those measured after growth in M9 glyc liquid medium (Fig. 2). In contrast, growth on solid agar decreased transcription twofold for the fan and daa operons (Fig. 2).

For the daa operon, introduction of the hns651 mutation caused a loss of repression such that transcriptional levels in the hns651 mutant strain grown on solid medium were equivalent to those when cells were grown in liquid M9 glyc (Fig. 2).

Iron.

In the case of CFA/I fimbriae, high iron levels have been shown to repress production at the bacterial surface (28). In contrast, high iron levels did not dramatically alter transcription of the operons tested in this study (data not shown). In the hns651 mutant strains, pap transcription levels were increased 2.2-fold in response to increased iron concentration, whereas daa transcription levels in the hns651 mutant strain remained consistent with those measured for cells grown in M9 glyc (data not shown).

Effect of environment and H-NS on pap and daa phase transition rates.

Because transcription of both pap and daa is subject to a methylation-dependent phase variation mechanism (33, 55), we analyzed pap and daa phase transition rates to determine if the repressive effects of glucose and high osmolarity could be attributed to alterations in switch frequencies. In addition, the phase transition rates were determined for strains DL1947 and CWZ263 on M9 glyc, M9 gluc, and M9 NaCl to assess the influence of H-NS on switch frequencies under these conditions. While growth on LB is another condition that is repressive for pap and daa transcription, all of the colonies had a uniform colony phenotype on LB such that phase transition rates could not be calculated on this medium.

For both pap and daa, the phase ON→OFF transition rates are not greatly influenced by the hns651 mutation, glucose as a carbon source, or high osmolarity. The phase ON→OFF rates are similar for the hns651 mutant strains DL1947 and CWZ263 compared to their respective wild-type strains DL1504 and DL1530 grown under all conditions tested (Table 2).

In contrast, the phase OFF→ON transition rate for the pap operon is altered by glucose as a carbon source, high osmolarity, and the hns651 mutation (Table 2). On glucose, a phase transition rate could not be calculated for the wild-type strain DL1504, as no phase ON (Lac⁺) colonies were observed in a screening of over 37,000 colonies plated from four individual phase OFF (Lac⁻) colonies grown on glucose. This result is in agreement with earlier results for a similar, but not identical, papBA-lacZYA transcriptional fusion in which only three Lac⁺ colonies were seen in the screening of 119,000 colonies, yielding a phase transition frequency of 4.51 × 10⁻⁶/cell/generation (9). Growth at high osmolarity also decreases the phase OFF→ON transition rate 2.2-fold compared to growth on M9 glyc in DL1504, indicating that high osmolarity, like glucose, inhibits transcription by decreasing the rate at which cells transition to a phase ON state (Table 2). As described previously (54, 57) and shown in Table 2, the phase OFF→ON transition rate is lower in the hns651 mutant strain DL1947 than in the wild-type strain grown on M9 glyc.

In the hns651 mutant strain DL1947, the phase OFF→ON transition rates on M9 glucose and M9 NaCl are increased to levels similar to those for wild-type strain DL1504 grown on M9 glyc, demonstrating that the hns651 mutant transitions more frequently to a phase ON state on these media compared to the wild-type strain. However, while the phase OFF→ON transition rates are increased, the overall level of transcription does not increase to that seen for wild-type strain DL1504 grown in M9 glyc. Instead, it approximates transcription seen for the hns651 strain grown on M9 glyc. These results suggest that while phase ON colonies are seen, the level of transcription within the phase ON cells of DL1947 cannot be at the same level as in the wild-type strain (see Discussion).

For the daa operon, the phase OFF→ON transition rates are increased by the hns651 mutation under each of the conditions tested. In hns651 mutant strain CWZ263 grown on M9 glyc, the phase OFF→ON rate is increased 11.5-fold compared to the wild-type strain DL1504. Similarly, under conditions of high osmolarity and glucose as a carbon source, the phase OFF→ON rates are increased 8.0- and 3.1-fold, respectively, in the hns651 mutant strain compared to wild-type strain DL1530 grown under the same conditions. The phase OFF→ON rate for CWZ263 grown on glucose is not as high as that seen in the hns651 strain grown on M9 glyc, supporting the transcriptional evidence that glucose is still partially repressive in an hns651 mutant strain (Fig. 2). Taken together, these results indicate that the hns651 mutation relieves the repression of glucose and high osmolarity, at least in part, by increasing the rate at which cells transit into the phase ON state. While it is clear that the hns651 mutation influences the phase OFF→ON transition rates, it is not evident that the environmental stimuli affect the phase transition rates in the wild-type strain. The phase OFF→ON rates do not vary significantly between growth on M9 glyc, M9 gluc, or M9 NaCl.