Abstract
MR/P fimbriae of uropathogenic Proteus mirabilis undergo invertible element-mediated phase variation whereby an individual bacterium switches between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Under different conditions, the percentage of fimbriate bacteria within a population varies and could be dictated by either selection (growth advantage of one phase) or signaling (preferentially converting one phase to the other in response to external signals). Expression of MR/P fimbriae increases in a cell-density dependent manner in vitro and in vivo. However, rather than the increased cell density itself, this increase in fimbrial expression is due to an enrichment of fimbriate bacteria under oxygen limitation resulting from increased cell density. Our data also indicate that the persistence of MR/P fimbriate bacteria under oxygen-limiting conditions is a result of both selection (of MR/P fimbrial phase variants) and signaling (via modulation of expression of the MrpI recombinase). Furthermore, the mrpJ transcriptional regulator encoded within the mrp operon contributes to phase switching. Type 1 fimbriae of Escherichia coli, which are likewise subject to phase variation via an invertible element, also increase in expression during reduced oxygenation. These findings provide evidence to support a mechanism for persistence of fimbriate bacteria under oxygen limitation, which is relevant to disease progression within the oxygen-restricted urinary tract.
Fimbriae (also termed pili) are adhesive surface filaments produced by many bacterial species (55). Fimbria-mediated adherence plays an important role in bacterial pathogenesis (16). Many fimbrial operons undergo phase variation whereby bacteria switch between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Expression of some fimbriae is tied to environmental cues via bacterial two-component regulatory systems such as the BvgAS-activated fimbrial expression in Bordetella pertussis (59) and the ToxR/ToxS/ToxT-regulated fimbrial expression in Vibrio cholerae (38). Some fimbrial phase variations are results of molecular events such as site-specific DNA recombination, slipped-strand mispairing, gene conversion, and DNA methylation (1, 57-59). It is believed that the advantage of these random molecular events is to allow phase variation in a bacterial population, which in turn allows bacteria to readily adapt to environmental changes or seek a new niche.
MR/P fimbriae of uropathogenic Proteus mirabilis and type 1 fimbriae of uropathogenic Escherichia coli share many features. Both belong to the family of fimbriae that are assembled through the chaperone-usher pathway (55). Both types of fimbriae are important bladder colonization factors (18, 36, 39) despite their differences in receptor; type 1 fimbriae bind to the mannose moieties of various glycoproteins, whereas MR/P fimbria-mediated hemagglutination is mannose resistant (3, 32). These fimbriae undergo phase variation dependent on a site-specific DNA recombination event (1, 40, 62) and are highly expressed during experimental urinary tract infections (28, 54). In both cases, the promoter for the fimbrial operon resides on a DNA element that is flanked by inverted repeats. DNA recombination between the inverted repeats results in inversion of the DNA element carrying the promoter. Hence, this DNA element is termed the invertible element (IE). The orientation of the IE that allows transcription of the fimbrial operon is defined as the ON orientation and the opposite orientation as the OFF orientation.
Switching of the IE is catalyzed by site-specific DNA recombinases. FimB and FimE are the first and most extensively studied recombinases for type 1 fimbriae of E. coli (31). Studies show that FimB is able to switch the IE from ON to OFF and from OFF to ON, whereas FimE mediates only ON-to-OFF switching (17, 40). Recently, there have been additional FimB- and FimE-like recombinases characterized in clinical E. coli isolates that also mediate fim IE switching, including IpuA and IpbA of uropathogenic E. coli strain CFT073 (7), FimX of uropathogenic E. coli strain UTI89 (21), and HbiF of meningitis-causing E. coli K1 strain RS218 (60). It is postulated that the relative abundance of each recombinase under different environmental conditions is a key factor in determining the expression level of type 1 fimbriae in an individual bacterium. However, in the case of MR/P fimbriae of uropathogenic P. mirabilis, MrpI is the sole recombinase involved in IE switching (36), suggesting that other mechanisms for MR/P fimbrial regulation likely exist.
Differential fimbrial expression can be achieved, in theory, by two means: signaling and selection. In a signaling process, environmental cues modulate the expression or activity of the recombinases to change the frequencies of the ON-to-OFF and the OFF-to-ON switches. As a result, fimbrial expression decreases in an environment where the recombinases favor the ON-to-OFF switch and increases in an environment where the recombinases favor the OFF-to-ON switch. In a selection process, the growth rates of the fimbriate bacteria and the nonfimbriate bacteria vary when the environment changes. Therefore, fimbrial expression decreases under conditions where the nonfimbriate bacteria outgrow the fimbriate bacteria and increases under conditions where the fimbriate bacteria are more fit than the nonfimbriate bacteria. There is evidence for both processes. Early studies of type 1 fimbriae of Salmonella enterica serovar Typhimurium reported selective outgrowth of fimbriate strains over nonfimbriate strains in static liquid medium, providing evidence for the selection process (47). LeuX, a tRNA for the rare leucine codon UUG, upregulates E. coli type 1 fimbrial expression, presumably by increasing translation of FimB because the fimB gene contains five UUG codons whereas the fimE gene has only two (44, 52). Additionally, other studies of type 1 fimbriae of E. coli demonstrate that global regulators, including integration host factor, leucine-responsive regulatory protein (Lrp), and histone-like nucleoid-structuring protein (H-NS), could affect the switching of the IE (5, 13, 15, 22, 30, 56). Together, these studies provide evidence for a signaling process. Here, we address how signaling and selection, separately and together, affect the expression of MR/P fimbriae in P. mirabilis and type 1 fimbriae in E. coli.
MATERIALS AND METHODS
Bacterial strains and media.
Uropathogenic P. mirabilis strain HI4320 was isolated from the urine of an elderly, long-term-catheterized woman with significant bacteriuria (>105 CFU/ml) (43) and has been sequenced (50). The phase-locked mutants of P. mirabilis HI4320 were constructed by inserting a kanamycin resistance cassette within the mrpI gene (37). The isogenic mrpJ mutant was constructed from P. mirabilis HI4320 by replacing a 180-bp BspHI-BglII fragment containing the 5′ end of the mrpJ gene with a kanamycin resistance cassette (37). The mrpABCDEFGHJ mutant and the mrpAΩlacZ and mrpIΩlacZ merodiploid strains were constructed for this study (see below). The dsbA mutant of P. mirabilis HI4320 was constructed using the Targetron gene knockout system (Sigma) with modifications (49); the mrpJ dsbA double mutant was made by using the dsbA knockout construct on the mrpJ mutant. Uropathogenic E. coli strain CFT073 was isolated from the blood and urine of a patient diagnosed with pyelonephritis (42). The phase-locked mutants of E. coli CFT073 were constructed by mutating the left inverted repeat of the IE (19). E. coli DH5α was used as the host strain for transformation of plasmids other than the pR6K-derived suicide vector pGP704 (41) and its derivatives, for which E. coli DH5αλpir was used as the host strain. Unless otherwise stated, Luria broth and nonswarming agar (4) were routinely used to culture bacteria, and ampicillin (100 μg/ml) or kanamycin (50 μg/ml) was added when necessary.
Atmospheric and oxygen-limited cultures.
To select for a population of mixed ON and OFF fimbrial expression, strains were cultured for 24 h in 60 ml Luria broth in a 125-ml flask with agitation (200 rpm) under atmospheric oxygen. This culture was diluted 1:100 into identical 125-ml flasks containing 25 ml Luria broth. One flask was placed in the same shaker (atmospheric oxygen), and the other flask was placed in a shaker inside a hypoxic glove box (Coy Laboratory Products Inc, Grass Lake, MI) containing 5.0% oxygen. Oxygenation was controlled by replacement of the atmosphere with nitrogen gas. Both cultures were grown for 24 h.
Construction of an isogenic mrpABCDEFGHJ mutant from P. mirabilis strain HI4320.
A 3.1-kb BamHI-PvuII DNA fragment containing sequences upstream of the mrpA gene and the 5′ end of the mrpA gene and a 2.2-kb PflMI-SphI DNA fragment containing the 3′ end of the mrpJ gene and sequences downstream of the mrpJ gene were used for homologous recombination to replace the mrp operon (mrpABCDEFGHJ) with a kanamycin resistance (encoded by aphA) cassette. Homologous recombination using the suicide vector pGP704 was carried out as described previously (37). The mrpABCDEFGHJ mutation was verified by Southern blot analysis (data not shown).
Construction of merodiploid transcriptional lacZ fusions in the wild-type P. mirabilis strain HI4320.
The merodiploid transcriptional fusions, mrpAΩlacZ and mrpIΩlacZ, were constructed using the same strategy described previously (37). Briefly, a promoterless lacZ gene was cloned into the suicide vector pGP704. For the mrpAΩlacZ fusion, a 1.9-kb DNA fragment containing the mrpI gene, the IE, and the 5′ end of the mrpA gene was cloned in front of the lacZ gene; the resulting suicide construct was designated pLX6111. Integration of pLX6111 into the chromosome resulted in the merodiploid fusion mrpAΩlacZ, which contains two copies of the mrpI gene and the IE, one controlling transcription of the lacZ gene and the other controlling transcription of the mrp operon. For the mrpIΩlacZ fusion, a 522-bp DNA fragment containing the mrpI gene excluding the first 14 codons was PCR amplified and cloned in front of the lacZ gene; the resulting suicide construct was designated pLXA2404. Integration of pLXA2404 into the chromosome resulted in the merodiploid fusion mrpIΩlacZ. Both merodiploid fusions, mrpAΩlacZ and mrpIΩlacZ, were verified by Southern blot analysis (data not shown). Merodiploid strains were maintained in Luria broth supplemented with 50 μg/ml ampicillin.
Flow cytometry and immunofluorescence microscopy.
P. mirabilis was cultured in atmospheric or 5.0% oxygen as described above, and after 4, 8, 12, and 24 h, samples were collected and processed for flow cytometry or immunofluorescence microscopy.
For flow cytometry, cells were prepared as described previously (24) with the following modifications. To detect surface expression of MR/P fimbriae, polyclonal rabbit antiserum raised against purified MrpA of P. mirabilis HI4320 (35) was added to ∼5 × 108 bacterial cells to a final dilution of 1:1000 in phosphate-buffered saline (PBS) with 2% normal goat serum (PBS-GS) (Gibco). Cells were washed once in PBS and subsequently incubated with 5 μM Syto 9 (nucleic acid stain; Molecular Probes) and Alexa Fluor 633-conjugated goat anti-rabbit immunoglobulin G (IgG) (Molecular Probes) at a final dilution of 1:250 in 0.85% NaCl with 2% normal goat serum (NaCl-GS). Each sample was then pelleted, resuspended in 0.85% NaCl to approximately 107 cells ml−1, and analyzed by flow cytometry (FACSCanto; Becton Dickinson). Syto 9 fluorescence emission was collected though a 30-nm band pass filter centered at 530 nm and Alexa Fluor 633 fluorescence was collected through a 20-nm band-pass filter centered at 660 nm. Ten thousand Syto 9-positive events (bacterial cells) were collected and analyzed for MrpA expression as indicated by Alexa Fluor 633 fluorescence. All flow cytometry experiments were done in triplicate and were analyzed using FlowJo software (Tree Star, Inc.). Significant differences in MrpA expression of bacteria cultured in atmospheric and 5.0% oxygen were determined using a two-tailed paired Student t test (GraphPad; InStat).
For immunofluorescence microscopy, bacteria were spotted onto glass coverslips coated with poly-l-lysine (Becton Dickinson) situated in a 24-well plate. Cells were dried and fixed in PBS with 4% paraformaldehyde for 20 min at room temperature. The coverslips were washed once with PBS and blocked for 16 h at 4°C with PBS-GS. The blocking solution was aspirated, and the coverslips were incubated with polyclonal rabbit serum raised against purified MrpA (diluted 1:5,000 in PBS-GS) for 2 h at 30°C. The coverslips were washed three times with PBS-GS and then incubated in the dark for 45 min at 30°C with Syto 9 (to stain bacterial nucleic acid) and secondary antibody (Alexa Fluor 594-conjugated goat anti-rabbit IgG; Molecular Probes) at final dilutions of 1:5,000 and 1:1,000, respectively, in NaCl-GS. Coverslips were washed three times with NaCl-GS and once with 0.85% NaCl to remove serum and then mounted with ProLong Gold Antifade mounting medium according to the manufacturer's specifications (Molecular Probes). The slides were examined at a magnification of ×1,000 with an Olympus BX60 system microscope equipped for fluorescence with filters for fluorescein isothiocyanate and Texas Red detection. All images were obtained and analyzed with an Olympus DP70 color digital video camera and DP Controller/Manager software.
CBA/J mouse model of ascending urinary tract infection.
The CBA/J mouse model of ascending urinary tract infection was a modification of the method originally developed by Hagberg's group (20, 29). Twenty female CBA/J mice were each transurethrally challenged with 5 × 107 CFU (in a volume of 50 μl) of the wild-type P. mirabilis strain HI4320. Seven days after challenge, the mice were sacrificed and bacteria in bladder and kidneys were quantitatively cultured on nonswarming agar. Values below 102 CFU/g tissue were set to 102 (limits of detection) for statistical analysis. Homogenized bladder and kidney samples were also subjected to the IE assay as described previously (36).
RESULTS
Proteus mirabilis MR/P fimbrial expression in vitro and in vivo.
A PCR-based IE assay (62) was designed to quantitatively determine the orientation of the IE. When P. mirabilis was cultured in Luria broth, the percentage of IE in the ON orientation increased over time; higher levels of MR/P fimbrial expression correlated with higher percentages of IE in the ON orientation (Fig. 1A). Immunogold labeling of MrpH, the tip adhesin of MR/P fimbriae, revealed phase variation of MR/P fimbriae in a broth culture of P. mirabilis: some bacteria expressed MR/P fimbriae (phase ON), and some did not (phase OFF) (Fig. 1B).
FIG. 1.
Phase variation of P. mirabilis MR/P fimbriae. (A) Differential expression of MR/P fimbriae in 5-ml broth cultures of P. mirabilis. All cultures were standardized to the same OD600, and whole-cell lysates were subjected to the IE assay and Western blot analysis with affinity-purified antibodies against MrpA. (B) Electron micrograph showing the phase variation of MR/P fimbrial expression in a broth culture of P. mirabilis. Immunogold labeling of MrpH, the tip adhesin of MR/P fimbriae, was performed as described previously (35). Note that the top left bacterium is gold labeled, while the top right bacterium is unlabeled. Scale bar, 500 nm. (C) Correlation between MR/P fimbrial expression and bacterial colonization in the bladder. Female CBA/J mice were transurethrally challenged with 5 × 107 CFU of the wild-type P. mirabilis strain HI4320. Seven days after challenge, mice were sacrificed. Bacteria in the bladder were both quantitatively cultured and subjected to the IE assay using the technique described previously (36). A positive correlation was found between MR/P fimbrial expression (percentage of IE in the ON orientation; y axis) and bacterial colonization in the bladder (log10 CFU/g tissue; x-axis): y = 17x − 30, r2 = 0.9, n = 18, P < 0.0001.
The MR/P fimbrial phase variation in broth appeared to be growth phase related, with the highest percentage of bacteria with the IE in the ON orientation during stationary phase (Fig. 1A). This seemingly cell density-dependent increase of MR/P fimbrial expression was also evident during experimental urinary tract infections (Fig. 1C). Analyzing P. mirabilis-infected mouse bladders, we found a positive correlation between bacterial colonization and MR/P fimbrial expression: a higher number of bacteria in the bladder correlated with a higher level of MR/P fimbrial expression (Fig. 1C) (r2 = 0.9, n = 18, P < 0.0001).
MR/P fimbrial expression under various culturing conditions.
To determine whether increased MR/P fimbrial expression directly resulted from increased cell density, we cultured P. mirabilis in broth to stationary phase under various conditions (Fig. 2A). In highly aerated broth cultures (1-ml culture in a tilted 14-ml tube), despite the cell density reaching the highest value (optical density at 600 nm [OD600] = 5), the IE remained primarily in the OFF orientation (<2% ON). Increases in broth volume resulted in higher percentages of the IE being in the ON orientation (∼20% ON in 3-ml cultures and ∼40% ON in 5-ml cultures). It is known that good aeration results from constant agitation and a large surface-to-volume ratio of the culture medium to the air and that poor aeration results from culturing a large volume in a small tube. Therefore, we were interested in determining if the decrease in oxygen rather than the increase in broth volume could be responsible for the increase in MR/P fimbrial expression. To address this issue, we cultured bacteria in upright (rather than tilted) culture tubes or added a mineral oil overlay, which prevents direct oxygen exchange between broth and air. For 1-ml cultures, the IE was 15 to 20% ON in upright tubes, compared to <2% ON in tilted tubes. Adding a mineral oil overlay led to a further increase in the ON population to 70% (Fig. 2A). For 10-ml cultures in 110-ml flasks, despite the increase in broth volume, good aeration in the flask kept MR/P fimbrial expression low (<2% ON). These data indicated that reduced aeration (and not an increase in broth volume) led to an increase in MR/P fimbrial expression.
FIG. 2.
Correlation between MR/P fimbrial expression and oxygenation. (A) As depicted in the diagram, P. mirabilis was incubated in triplicate as 1-ml, 3-ml, or 5-ml cultures in 14-ml tubes or as 10-ml cultures in 110-ml flasks. All cultures were incubated for 24 h at 37°C with constant agitation (200 rpm). Tubes were placed either tilted (45°) or upright in the incubator. Some 1-ml cultures were placed in microaerobic jars or were overlaid with 3-ml mineral oil to further reduce oxygen in the broth. (B) P. mirabilis was cultured in identical 125-ml flasks under atmospheric oxygen (top panel) or 5.0% oxygen (bottom panel). Samples were collected over time and subjected to the IE assay. Nucleotide size markers are shown on the right side of each panel.
Reduced aeration has two direct consequences: a reduction in oxygen and an increase in CO2. To discriminate between these two conditions, we utilized two different incubation conditions. Bacteria were cultured in microaerobic jars using CampyPaks (Becton Dickinson), which produce a microaerobic environment with reduced O2 (5 to 15%) and increased CO2 (5 to 12%). In addition, bacteria were cultured in a 5% CO2 incubator, which has increased CO2 levels but atmospheric levels of oxygen. Cultures grown in microaerobic jars showed higher levels of MR/P fimbrial expression than those grown under aerobic conditions (Fig. 2A). However, there was no effect on MR/P fimbrial expression when bacteria were cultured in the CO2 incubator (data not shown). Together, these data suggest that the increased MR/P fimbrial expression under microaerobic conditions is due to reduced oxygen but not increased carbon dioxide.
Although the culture conditions used up to this point strongly indicated that reduced oxygen levels affected the orientation of the MR/P IE, the precise oxygenation of the cultures shown in Fig. 2A was not known. Thus, to confirm the contribution of restricted oxygenation to MR/P fimbrial IE switching in a more controlled setting, P. mirabilis was cultured in identical flasks with agitation in either atmospheric oxygen or a hypoxic glove box with 5.0% oxygen (Fig. 2B). Samples were collected at 2, 4, 6, 8, 10, 12, and 24 h postinoculation (hpi) and subjected to the IE assay. Although the initial inoculum had a mixed population of ON and OFF P. mirabilis, bacteria in the flask cultured under atmospheric oxygen switched to mostly OFF within 2 h and remained mostly OFF for the duration of the experiment. In contrast, P. mirabilis cultured in 5.0% oxygen displayed an increasing ON population by 8 h, although the OFF population rose slightly during the first 2 h. The ON population peaked by 12 h and maintained this highest level through 24 h (Fig. 2B).
In summary, the MR/P fimbrial expression in the wild-type P. mirabilis was optimal (up to 70% ON) under oxygen-limiting conditions. MR/P fimbrial expression increased when oxygen in broth decreased. Hereafter, we cultured bacteria in identical flasks with agitation under either atmospheric oxygen (approximately 21% O2; aerobic) or 5.0% oxygen (microaerobic) to further assess the role of oxygenation in fimbrial expression.
Surface expression of MR/P fimbriae correlates with IE orientation and is optimal under oxygen-limiting conditions.
Previously, we demonstrated that the MrpI recombinase was required for switching the orientation of the IE (36). A mutation in the mrpI gene abolished phase variation of MR/P fimbriae and gave rise to two different mutants, one with the IE locked in the ON orientation and the other with the IE in the OFF orientation. As expected, the OFF mutant (L-OFF) did not express MR/P fimbriae under either aerobic or oxygen-limiting conditions, and the ON mutant (L-ON) expressed MR/P fimbriae under both conditions (Fig. 2B).
To confirm that the orientation of the IE observed in the IE assay relates to surface expression of MR/P fimbriae, we employed flow cytometry and immunofluorescence microscopy. For these studies, wild-type strain HI4320 was cultured under atmospheric oxygen or 5.0% oxygen as for the IE assay, and samples were collected at 4, 8, 12, and 24 hpi. In the following experiments, the MR/P L-ON and L-OFF mutants were used as positive and negative controls, respectively, of MR/P IE orientation.
For the flow cytometry experiments, bacteria were labeled with Syto 9 for the detection of bacterial nucleic acid and with rabbit anti-MrpA antiserum plus goat anti-rabbit IgG Alexa Fluor 633 conjugate for the detection of MR/P fimbria expression. Bacteria were distinguished from dust and debris based on their forward scatter (size) and Syto 9 fluorescence properties. Then, a gate was set on this bacterial cell population using the MR/P OFF mutant such that cells of wild-type HI4320 were considered positive for expressing MrpA when their Alexa Fluor 633 fluorescence intensity exceeded nearly all (>98%) of the OFF mutant control population. Using this gate, the percentage of bacteria that were positive for MrpA expression was determined for wild-type HI4320 cultured under atmospheric oxygen versus 5.0% oxygen. Histograms from a representative experiment are shown in Fig. 3A. As the percentage of MrpA-positive bacteria increases, the histogram of the population shifts to the right of the indicated gated regions (which is measured by an increase in Alexa Fluor 633 fluorescence intensity). As observed with the IE assay, the proportion of MR/P fimbriate bacteria decreased after 4 h for bacteria cultured under atmospheric oxygen (Fig. 3A, blue histograms) but increased after 4 h for bacteria cultured under 5.0% oxygen (Fig. 3A, red histograms). This experiment was repeated three times, and the average percentages of MrpA-positive bacteria from each time point are shown in Fig. 3B. As before, we found that during culture under atmospheric oxygen, the percentage of wild-type P. mirabilis expressing MrpA decreased over time (Fig. 3B) (15.18% at 4 hpi, 3.68% at 8 hpi, 2.14% at 12 hpi, and 2.48% at 24 hpi), in contrast to bacteria cultured under 5.0% oxygen (Fig. 3B) (15.10% at 4 hpi, 13.31% at 8 hpi, 24.69% at 12 hpi, and 29.72% at 24 hpi). Moreover, the difference in the proportions of MR/P fimbriate bacteria cultured under atmospheric and 5.0% oxygen at 12 and 24 hpi was determined to be statistically significant as determined by Student's t test (P = 0.0037 and 0.0040, respectively).
FIG. 3.
Confirmation of surface expression of MR/P fimbriae by flow cytometry (A and B) and immunofluorescence (C). P. mirabilis was cultured as described in Fig. 2B. Samples were collected over time and processed for flow cytometry or immunofluorescence. (A) Flow cytometry histograms depicting the number of bacterial cells (y axes) and increasing MrpA expression (or log of Alexa Fluor 633 relative fluorescence intensity; x axes). Blue histograms represent bacteria cultured in atmospheric oxygen, while red histograms represent bacteria cultured in 5.0% oxygen. A representative of three experiments is shown. The percentage of MrpA-positive cells that lie within the indicated gated regions for each bacterial population is depicted in the top right corner of each graph. (B) Bars represent the average (n = 3) surface expression of MrpA for each population. Error bars represent the standard errors of the means. *, P < 0.05. WT, wild type. (C) Immunofluorescence images of HI4320 cultured in atmospheric (atm) or 5.0% oxygen. Bacterial DNA is labeled with Syto9 (green), and MR/P fimbriae are labeled with rabbit anti-MrpA antiserum and anti-rabbit IgG conjugated to Alexa Fluor 594 (red).
To further examine the surface expression of MR/P fimbriae, we also employed immunofluorescence microscopy. For these studies, bacteria were labeled with Syto 9 for the detection of bacterial nucleic acid and with rabbit anti-MrpA antiserum plus goat anti-rabbit IgG Alexa Fluor 594 conjugate for the detection of MR/P fimbria expression. Representative immunofluorescence images are shown in Fig. 3C. As observed in the flow cytometry and IE assay experiments, wild-type P. mirabilis cultured under 5.0% oxygen expressed more MR/P fimbriae over time than bacteria cultured under atmospheric oxygen (Fig. 3C). Together, the flow cytometry and immunofluorescence data are in agreement with the IE assay, further substantiating the finding that MR/P fimbrial expression of P. mirabilis is increased under oxygen-limiting conditions.
Bacteria expressing MR/P fimbriae outgrow bacteria not expressing MR/P fimbriae under oxygen-limiting conditions.
Independent growth curves of mrpI mutants with the IE locked in either the OFF or ON position indicated that the L-OFF and the L-ON mutants grew equally well when cultured in either atmospheric or 5.0% oxygen (Fig. 4A). Since inoculating two strains in coculture can reveal subtle differences in fitness that may not be apparent in independent cultures (27, 33), we mixed L-ON and L-OFF strains in defined ratios. Because the IE assay provides a useful indicator of the levels of OFF and ON P. mirabilis in a population, we used this assay to assess the levels of L-ON and L-OFF bacteria at the end of each coculture experiment. If there is no advantage associated with presence or absence of MR/P fimbriae, we would expect the population to maintain the input ratios throughout the course of the experiment. However, when the two strains were cultured together, the L-ON mutant outcompeted the L-OFF mutant under oxygen-limiting conditions (Fig. 4B). For example, when the L-ON and L-OFF mutants were combined in a ratio of 1:100 (1% ON) and then cultured under 5.0% oxygen for 24 h, the percentage of the L-ON mutant in the culture visibly increased. A growth advantage of bacteria expressing MR/P fimbriae over bacteria not expressing MR/P fimbriae could account for the increase in MR/P phase-ON bacteria in the wild-type strain during oxygen limitation (Fig. 4B). Notably, the input ratios of L-ON and L-OFF bacteria remained constant when cocultured under atmospheric oxygen (Fig. 4B).
FIG. 4.
(A) Growth curves of the L-OFF and L-ON mutants of P. mirabilis. Bacteria were cultured at 37°C with constant agitation (200 rpm) in atmospheric oxygen (atm) or 5.0% oxygen. The OD600 was measured over time. Data points are the averages and standard errors of the means from three experiments. (B) In vitro growth competition between the L-ON and the L-OFF mutants. The L-ON and the L-OFF mutants of P. mirabilis were combined in specific ratios to obtain mixtures that contained 0, 0.01, 1, 10, 50, or 100% of the L-ON mutant. The wild-type (wt) strain and the mixtures of the mutants were adjusted to an OD600 of 1, diluted 1:100 into LB, and cultured in broth under atmospheric oxygen (middle panel), or in 5.0% oxygen (bottom panel). The inocula (top panel) and cultures were subjected to the IE assay. Nucleotide size markers are shown on the left.
MR/P fimbrial expression in an isogenic mrpABCDEFGHJ mutant of P. mirabilis.
Having established that cultures incubated under oxygen-limiting conditions are enriched for fimbriate bacteria, we sought to determine the role that the MR/P fimbriae themselves played in IE switching. To address this question, we asked whether a deficiency in MR/P fimbrial production would affect the IE being switched into the ON orientation under oxygen-limiting conditions. That is, is synthesis of fimbriae, per se, required for this enrichment of the ON population? An isogenic mrpABCDEFGHJ mutant was constructed by replacing the mrp operon with a kanamycin resistance cassette (see Materials and Methods). Under oxygen-limiting conditions, the IE of the mrpABCDEFGHJ mutant remained primarily in the OFF orientation (Fig. 5). This result suggested that the presence of intact MR/P fimbriae affects the switching of the IE.
FIG. 5.
MR/P fimbrial expression in isogenic mutants of P. mirabilis. Wild-type (WT) and mutant strains of P. mirabilis were cultured in atmospheric oxygen (atm) or 5.0% oxygen. The switch of the IE was measured. Nucleotide size markers are shown on the left.
Why do ON bacteria outgrow OFF bacteria under oxygen-limiting conditions?
The ON bacteria differ from the OFF bacteria in the transcription of the mrpABCDEFGHJ operon, which leads to expression of all the proteins required for the MR/P fimbrial production (MrpA, -B, -C, -D, -E, -F, -G, and -H) and a regulatory protein, MrpJ, that represses flagellum synthesis while MR/P fimbriae are assembled (37). To test whether MrpJ is responsible for the increase in ON bacteria during oxygen-limited growth, we examined the isogenic mrpJ null mutant constructed previously (37). As shown in Fig. 6, the percentage of IE in the ON orientation in the mrpJ mutant increased from <2% ON under aerobic conditions to ∼30% ON under oxygen-limiting conditions; this increase was not as great as that of the wild-type strain. Deletion of MrpJ, presumably leading to loss of coordination between MR/P fimbrial production and flagellum synthesis in the ON bacteria, diminished the increase in the ON population during oxygen-limited culture but did not completely abolish it. Nevertheless, in the absence of MrpJ, ON bacteria still increased in the percentage of the total population compared to OFF bacteria under oxygen-limiting conditions, suggesting that the growth advantage of L-ON versus L-OFF P. mirabilis was not completely attributable to MrpJ. We also examined isogenic mrpA, mrpB, mrpE, mrpF, mrpG, and mrpH mutants of P. mirabilis, and all of these mutants shared the same phenotype as the mrpABCDEFGHJ mutant: <2% of the IE was in the ON orientation regardless of the culturing conditions (data not shown). Therefore, it appeared that it was their ability to produce fimbriae that allowed the ON bacteria to outcompete OFF bacteria during oxygen limitation.
FIG. 6.
The mrpJ and the dsbA mutants of P. mirabilis strain HI4320. Wild-type (WT) and mutant strains of P. mirabilis were cultured in atmospheric or 5.0% oxygen. The orientation of the IE was measured. Nucleotide size markers are shown on the left.
One of many common features shared by MR/P fimbriae and type 1 fimbriae is their fimbrial assembly process, which requires disulfide bond formation in all pilins prior to assembly on the bacterial surface (55). Mutations in dsbA, the gene encoding the periplasmic thio-disulfide oxidoreductase DsbA, abolished P fimbrial production in uropathogenic E. coli and bundle-forming pilus production in enteropathogenic E. coli (26, 61). Studies by Klemm's group suggested that production of disulfide-containing fimbriae altered the thio-disulfide status of the global regulator OxyR and therefore affected expression of other genes (53). To investigate whether fimbrial production per se is responsible for the growth advantage of ON bacteria, we examined a dsbA null mutant of P. mirabilis strain HI4320. The dsbA mutant was originally identified using signature-tagged mutagenesis (8) and was notable because it showed significant attenuation in its ability to colonize bladder and kidneys in the CBA/J mouse model of ascending urinary tract infection. For the current study, an isogenic dsbA mutant was constructed. Western blot analysis indicated that the dsbA mutant was unable to produce MR/P fimbriae (data not shown). To our surprise, when the dsbA mutant was cultured under oxygen-limiting conditions, the percentage of IE in the ON orientation still increased (Fig. 6). Despite the inability of the dsbA mutant to produce MR/P fimbriae, bacteria with the IE in the ON orientation still increased in number relative to bacteria with the IE in the OFF orientation in an oxygen-limiting environment. Therefore, the growth advantage of the ON bacteria cannot be attributed solely to MR/P fimbrial production.
To further evaluate the role of mrpJ and dsbA in MR/P IE switching, a double mrpJ dsbA mutant was constructed. Whether cultured under atmospheric oxygen or 5.0% oxygen, the IE of this mutant was almost completely in the OFF orientation (Fig. 6).
The MrpI recombinase-dependent signaling process under aerobic conditions.
We observed that under aerobic conditions, the percentage of the L-ON mutant in the mixtures of the L-ON and L-OFF mutants remained the same, suggesting a lack of growth competition between the L-ON and the L-OFF mutants (Fig. 4B). When the wild-type strain was cultured in oxygen-limited broth and then subsequently cultured in aerated broth, the percentage of bacteria with the IE in the ON orientation decreased (Fig. 4B). Since there is no growth competition under aerobic conditions, we would have expected this ratio to remain constant. However, since the IE ratio changed, these results suggested that under aerobic conditions, the percentage of the population with the IE oriented to phase ON in the wild type remains low due to a signaling process that involves the MrpI recombinase.
Our data indicated that ON-to-OFF switching was favored only during the first 2 h (Fig. 2B). This could be related to the relatively aerobic conditions in fresh medium. It is well known that an increase in bacterial load will decrease the oxygen concentration in the broth. The more bacteria in the medium, the more quickly oxygen is consumed. To test this, we inoculated fresh LB broth with an increasing amount of the wild-type bacteria that were cultured in oxygen-limited broth. All cultures were incubated again under oxygen-limiting conditions and were monitored at 2 h and 24 h (Fig. 7). In cultures inoculated with 5 × 108 CFU of bacteria or more, the percentage of the ON bacteria remained unchanged (∼70%) at 2 h and increased to 90% by 24 h. Therefore, by increasing the amount of bacteria in an inoculum, we were able to bypass the relatively aerobic phase, during which the MrpI recombinase was normally able to switch the IE into the OFF orientation.
FIG. 7.
Wild-type P. mirabilis strain HI4230, cultured under oxygen-limiting conditions, was used to inoculate Luria broth at concentrations of 5 × 106, 5 × 107, 5 × 108, 1 × 109, 1.5 × 109, 2 × 109, and 2.5 × 109 CFU/ml. Cultures were then incubated under oxygen-limiting conditions. Individual cultures were sampled at indicated time points and subjected to the IE assay.
Expression of the MrpI recombinase is regulated by oxygen.
In this study, we observed that the IE is preferentially in the ON orientation in P. mirabilis cultured under oxygen limitation. This preference could be due to selection, that is, the fact that ON bacteria outgrew OFF bacteria under oxygen limitation (as demonstrated by using the L-ON and L-OFF mutants [Fig. 4]). However, another explanation or contributing factor could be that mrpI expression was itself regulated by oxygen (i.e., signaling). To monitor transcription of the mrpI gene and the mrpA gene, we constructed merodiploid transcriptional lacZ fusions in P. mirabilis strain HI4320 (see Materials and Methods). The β-galactosidase activity in the mrpAΩlacZ fusion strain was higher when bacteria were cultured in oxygen-limited broth (Fig. 8); this was consistent with an increase in MR/P fimbrial expression. On the other hand, the β-galactosidase activity in the mrpIΩlacZ fusion strain increased when bacteria were cultured under atmospheric oxygen, suggesting that the mrpI transcription itself may be oxygen regulated (Fig. 8). Further investigations are necessary to better detail the oxygen regulation of mrpI, as well as whether and how the MrpI recombinase activity, or IE binding preference, might be modulated by oxygen.
FIG. 8.
Transcriptional activities of mrpA and mrpI as measured by the β-galactosidase activities in mrpAΩlacZ and mrpIΩlacZ merodiploid strains, respectively. The mrpAΩlacZ and mrpIΩlacZ merodipoid strains were cultured in atmospheric oxygen (atm) or 5.0% oxygen for 24 h at 37°C. β-Galactosidase activities were assayed in triplicate; the paired one-tailed t test was used to assess significance. Error bars indicate standard errors of the means. Note the different y axes for mrpA and mrpI.
E. coli also exhibits increased type 1 fimbrial expression under oxygen-limiting conditions.
Static broth is more oxygen limited than aerated broth (47). Passaging bacteria in static cultures has been a common laboratory practice to enrich fimbrial production (2, 45, 46). This led us to ask whether increased fimbrial expression under oxygen-limiting conditions is a general phenomenon. We analyzed type 1 fimbrial expression in uropathogenic E. coli CFT073 (Fig. 9). When E. coli CFT073 was cultured under atmospheric oxygen, the IE was almost completely OFF, whereas when CFT073 was cultured under 5.0% oxygen, the IE-ON population increased over time. We conclude that, as with MR/P fimbriae, expression of type 1 fimbriae is increased during oxygen limitation.
FIG. 9.
Effect of oxygen limitation on expression of type 1 fimbriae in uropathogenic E. coli CFT073. Wild-type CFT073 was cultured in atmospheric or 5.0% oxygen. The inocula and cultures were subjected to the IE assay. Nucleotide size markers are shown on the left side of the panel.
DISCUSSION
In this study, we dissected the molecular basis for differential fimbrial expression. As described above, differential fimbrial expression can result from signaling or selection. There are two crucial findings in this report that argue for both selection and signaling. First, increased MR/P fimbrial expression under oxygen-limiting conditions results from a selection process where fimbriate bacteria outgrow nonfimbriate bacteria. Second, decreased MR/P fimbrial expression under aerobic conditions results from a signaling process where the MrpI recombinase actively converts fimbriate bacteria to nonfimbriate bacteria, as evidenced by an increase in mrpI transcript levels in atmospheric oxygen compared to a restricted-oxygen environment.
What makes ON bacteria different from OFF bacteria? A study by Old and Duguid showed selective outgrowth of S. enterica serovar Typhimurium strains expressing type 1 fimbriae over those not expressing type 1 fimbriae in static liquid medium (47). They attributed such a growth advantage of the fimbriate strains to their ability to form a pellicle on the broth surface, where better access to oxygen promoted their growth. In our study, all cultures were grown in a shaking incubator with constant agitation (200 rpm), yet pellicle formation was observed for all strains. Cultures formed pellicles adherent to flask walls regardless of oxygenation, and samples were taken from the broth phase. Therefore, the growth advantage of fimbriate bacteria appeared to be unrelated to their ability to form pellicles. Nevertheless, culturing bacteria under static conditions has long been used to select for fimbriation, and although reduced oxygenation in a static culture has been hypothesized to be the selective force for fimbrial expression (6), this is the first report where culture conditions that varied only in oxygen tension (identical flasks cultured for the same duration with identical agitation) proved that fimbrial expression was oxygen regulated.
Deletion of the mrp operon (mrpABCDEFGHJ) abolished the generation of ON bacteria; when the mrpABCDEFGHJ mutant was cultured under the oxygen-limiting conditions, the IE remained primarily in the OFF orientation. Therefore, the growth advantage of the ON bacteria in wild-type P. mirabilis can be attributed to transcription of the mrp operon, which subsequently leads to two main events: expression of all the proteins required for MR/P fimbrial biogenesis (MrpA, -B, -C, -D, -E, -F, -G, and -H) and expression of MrpJ, a regulatory protein that represses flagellum synthesis.
To investigate whether the expression of MrpJ itself, an event associated with the IE being in the ON orientation, was responsible for the growth advantage of the ON bacteria, we analyzed the mrpJ mutant. We previously demonstrated that MrpJ represses flagellum synthesis and therefore coordinates bacterial motility and adherence (37). When the mrpJ mutant was cultured under oxygen-limiting conditions, ON bacteria still outnumbered OFF bacteria, but to a lesser extent compared to the wild-type strain. This suggests that the increase in ON bacteria during oxygen-limited culture is partly but not completely attributable to MrpJ.
The complete loss of ON bacteria during limited oxygenation in separate mrpA, mrpB, mrpE, mrpF, mrpG, and mrpH mutants (data not shown) suggested that fimbrial assembly is largely responsible for the increase in ON bacteria in reduced-oxygen environments. This is why the phenotype of the dsbA mutant was initially unexpected. Fimbrial production requires DsbA, which catalyzes disulfide bond formation in pilin subunits (55). The dsbA mutant was unable to produce assembled MR/P fimbriae, but the percentage of IE in the ON orientation still increased from <2% to ∼30% when bacteria were cultured under oxygen-limiting conditions. This suggests that the growth advantage of the ON bacteria is partly but not completely attributable to their ability to produce fimbriae. What is the difference between the dsbA mutant and the mrpA, mrpB, mrpE, mrpF, mrpG, and mrpH mutants? None of these mutants is capable of producing MR/P fimbriae, yet the dsbA mutant strain retains some preference for switching the IE to ON during oxygen restriction, while the latter mutants completely lose this switching behavior. We believe that the difference resides in the expression of MrpJ. The insertion of a kanamycin resistance cassette in mrpA, mrpB, mrpE, mrpF, mrpG, or mrpH is more likely to pose a polar effect on the MrpJ expression than the mutation in dsbA. Our data indicated that MrpJ does contribute to the increase in ON bacteria during oxygen limitation. Therefore, the growth advantage of the ON bacteria may be a combinatory effect of all the events associated with the IE being in the ON orientation: fimbrial production involving disulfide bond formation and the repression of flagellum synthesis by MrpJ. This hypothesis was tested with a double mutant of P. mirabilis that lacked both MrpJ and DsbA, where the double mutant had almost no ON bacteria in the population, even when grown under oxygen-limiting conditions. It should be noted that while neither an mrpABCDEFGH mutant nor an L-OFF mutant produces MR/P fimbriae, a dsbA mutant should not assemble any of the 17 chaperone-usher fimbriae encoded by P. mirabilis (though a dsbA mutant is predicted to produce MrpJ). Likewise, it is possible that the small amount of ON bacteria in the mrpJ dsbA double mutant is caused by one or more of the 14 mrpJ paralogs in the P. mirabilis genome (48).
What is the connection between oxygen-limiting conditions and fimbrial production? We speculate that the electron transport chain may be the missing link. The electron transport chain passes electrons to oxygen and generates a proton gradient across the inner membrane, while protons returning to the cytosol via the proton-translocating ATPase drive ATP synthesis. It is known that electrons released from disulfide bond formation, a crucial step in the fimbrial assembly process, are transferred to oxygen via DsbA, DsbB, and components of the electron transport chain, ubiquinone and cytochrome oxidase (11). The paradox is that fimbriate bacteria have a growth advantage over nonfimbriate bacteria in a low-oxygen environment, where the oxygen limitation, in theory, should hinder fimbrial production. On the other hand, the momentum of the fimbrial assembly process could be a driving force for the electron transport and could help maintain the proton gradient under oxygen-limiting conditions.
In P. mirabilis, the repression of flagellar synthesis by MrpJ could also help maintain the proton gradient because flagellar rotation consumes proton motive force. Indeed, flagellar assembly and function have been tied to the respiratory protein fumarate reductase (10). Fumarate reductase is known to be involved in anaerobic respiration (9). Furthermore, we recently found that an sdhC mutant (succinate dehydrogenase, involved in aerobic respiration) was defective in MR/P fimbrial production (23). We propose that the ability of the ON bacteria to maintain the proton gradient under oxygen-limiting conditions gives them the advantage over the OFF bacteria. It is possible that other fimbriae produced by P. mirabilis also contribute to maintenance of the proton gradient, as 10 chaperone-usher fimbriae encoded in the P. mirabilis genome have an mrpJ paralog within the fimbrial operon (48).
The Cpx signaling pathway may play a role in DsbA control of IE switching. The Cpx pathway is upregulated in response to envelope stress (reviewed in reference 51), which in this study could be caused by accumulation of unassembled fimbrial subunits in the periplasm of a dsbA mutant. Additionally, DsbA itself is upregulated when the Cpx pathway is activated. A constitutively activated Cpx system downregulates chemotaxis and motility (motAB-cheAW and tsr) but not the production of flagella themselves (12); such a paralysis of the flagella could also preserve proton motive force. Conversely, P. mirabilis fimbrial and flagellar production are inversely linked via MrpJ and its paralogs. On the other hand, the Cpx-DsbA-motility link does not explain all the results of the current study. Constitutive activation of CpxR leads to increased piliation in E. coli (25). However, a dsbA mutant would be expected to have increased envelope stress and would therefore have upregulated Cpx signaling and presumably an increased signal for fimbriation. Instead, the P. mirabilis dsbA mutant has a lower proportion of the population with the mrp IE in phase ON.
MrpI recombinase synthesis is a target of a signaling process. That is, mrpI appears to be transcriptionally regulated by oxygen levels, with higher expression during aerobic growth. This finding suggests that the MrpI recombinase preferentially switches the IE from ON to OFF. It is also possible that MrpI recombinase activity or switching preference is modulated by oxygen, or an oxygen-responsive cofactor could interact with MrpI to dictate IE switching. A better understanding of how MrpI recombinase synthesis or activity is modulated in response to oxygen awaits further investigation.
We have demonstrated in this study that bacteria expressing MR/P fimbriae outcompete nonfimbriate bacteria under oxygen-limiting conditions. The fact that culturing bacteria in static broth, a relatively more oxygen-limiting condition, is a common laboratory practice used to enrich expression of many other types of fimbriae suggests that selection for fimbriate bacteria in reduced oxygen may be a general phenomenon. It has previously been reported that expression of E. coli type 1 fimbriae is reduced in stationary phase via the sigma factor RpoS (14). It should be noted that we found a significant amount of phase-ON E. coli only in oxygen-limited cultures (Fig. 9); thus, it is possible that RpoS and oxygen limitation play opposing roles in IE inversion. Alternatively, the previous report focused on a commensal strain of E. coli (a K-12 derivative), while our oxygen limitation studies were conducted using a uropathogenic isolate, E. coli CFT073, so it is possible that these results stem from different responses by commensal and pathogenic bacteria.
Specific niches within the host can be oxygen limited, especially in the urinary tract, where bacteria have no direct access to atmospheric oxygen and indeed consume oxygen. During experimental urinary tract infections, type 1 fimbrial expression and MR/P fimbrial expression increase significantly to >95% ON in some infected bladder tissue samples (Fig. 1C) (18, 36). The IE of the mrpABCDEFGHJ mutant, however, remained primarily in the OFF orientation (<5% ON) during experimental urinary tract infections (data not shown). This suggests that the increased fimbrial expression in vivo is also a result of the selection of fimbriate bacteria. What is the selective force for fimbrial expression in vivo? One answer comes from the adherence function of fimbriae. Fimbriate bacteria adhere more avidly to host epithelial cells than nonfimbriate bacteria, which gives them an advantage in avoiding host clearance by urination. In addition, fimbriate bacteria may outgrow nonfimbriate bacteria due to potentially oxygen-limiting conditions in vivo.
Adaptation to a low-oxygen environment in the host may represent a common theme of bacterial pathogenesis. Expression of invasion genes in S. enterica serovar Typhimurium increases under oxygen-limiting conditions as well (34). In general, quick adaptation of bacteria to an environment is often attributed to their abilities to recognize (signal transduction) and respond to (gene regulation) a variety of environmental cues. This report emphasizes that equally important is the ability of bacteria to maintain heterogeneity in a population.
Acknowledgments
We gratefully acknowledge C. V. Lockatell and D. E. Johnson for assistance with the mouse model of ascending urinary tract infection and J. Norman and M. O'Riordan for advice on fluorescence-activated cell sorter analysis. We thank B. Cormack, M. Donnenberg, J. Kaper, and members of the Mobley lab for helpful discussions.
This work was supported by Public Health Service grants DK49720 and AI43363 from the National Institutes of Health (to H.L.T.M.). M.M.P. was supported in part by National Research Service Award F32 AI068324.
Footnotes
Published ahead of print on 29 December 2008.
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