Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 710 East Pratt Street, Baltimore, MD 21202, USA
Correspondence
Robert Belas
belas{at}umbi.umd.edu
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ABSTRACT |
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INTRODUCTION |
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Proteus mirabilis swimmer cells generally have six to ten peritrichous flagella. Studies by Belas and others have shown that the flagellin locus contains two flagellin-encoding genes, flaA and flaB, which have domains of high homology at both the nucleotide and amino acid level (Belas & Flaherty, 1994; Murphy & Belas, 1999
). flaA possesses an upstream regulatory sequence with consensus homology to the flagellar class III
28 promoters (Helmann, 1991
). Biochemical analysis of isolated flagella indicates that they are composed of a sole flagellin species (Belas, 1994
). Indeed, transcriptional lacZ fusions of flaA and flaB suggest that, while flaA expression is co-ordinately upregulated with swarmer cell differentiation, flaB remains silent and is not expressed in either swimmer or swarmer cells (Belas, 1994
). This finding is corroborated by Northern blot analysis, with flaA yielding a single 1·2 kb mRNA, while several flaB-specific primers failed to yield a product. Furthermore, mutations constructed in flaB by allelic exchange were wild-type, while flaA mutants were defective in motility (Belas, 1994
).
In earlier studies, flaA mutants were found to be unstable and occasionally produced flares of motile cells on semisolid motility (Mot) agar (Belas, 1994). Twelve flaA Mot+ revertants (DF1002 to DF1013) were analysed and shown to behave like wild-type cells with respect to swarming and swimming, and other phenotypes (Belas & Flaherty, 1994
). Southern blot analysis of DF1002 and DF1003 revealed that each revertant was missing a section of the flaA-flaB region, though the missing region was different in each case. PCR amplification using primers specific to either flaA or flaB revealed a 740 bp amplicon, as opposed to a 2142 bp amplicon for the full flaA-flaB locus. Sequencing of the cloned 740 bp amplicon showed a hybrid flagellin gene composed of the 5' end of flaA and the 3' end of flaB. While the junction creating the hybrid flaAB gene was different for both strains, the size of the deleted region was constant at 1410 bp and the resulting flaAB gene, and its deduced amino acid product, were nearly identical in size to flaA and FlaA, respectively (Murphy & Belas, 1999
).
While originally hypothesized to involve evasion of host defences, the production of hybrid FlaAB filaments may also result in a change in the helical structure of the flagellar filament. Recent analysis of a Salmonella enterica serovar Typhimurium flagellin fragment known as F41 has demonstrated that the N and C termini form the densely packed core of the filament and are necessary for the axial intersubunit interactions between flagellin monomers in the protofilament (Samatey et al., 2001). The shape of the filament depends upon the arrangement of the flagellin monomers, which depends in turn upon the amino acid sequence of the protein, temperature, pH, ionic strength and torsional load, i.e. an increased load on the filament causing changes in shape (Kamiya & Asakura, 1974
, 1976a
, b
; Macnab, 1987
). In S. typhimurium, the flagellar filament undergoes changes in helical pitch, amplitude and handedness in response to pH changes, switching from a long-pitch, left-handed corkscrew to a shorter-pitch, right-handed one (Macnab & Ornston, 1977
). Changes in shape in turn affect motility, and hence a change in environment may in fact benefit a strain possessing helically different flagella.
In this study we examined the motility of DF1003 (FlaAB+) and the wild-type (FlaA+), and compared the swimming speed of each with the morphology of their respective flagella.
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METHODS |
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Growth curves of wild-type and DF1003 strains were carried out as follows. T-broth (2 ml) was inoculated in triplicate from isolated colonies on L-agar and incubated overnight with shaking at 37 °C. Cultures were diluted 1 : 100 in 10 ml T-broth in 125 ml conical flasks and initial OD600 readings were taken to equalize inoculum densities. Cultures were incubated as above and OD600 readings, beginning at 2 h, were recorded until stationary phase was detected.
Measurement of swimming velocity preparation of modified media
NaCl.
T-broth (170 mM NaCl) was adjusted to a final NaCl concentration of 255, 340, 425 or 510 mM NaCl by addition of 5 M NaCl. Cultures were adjusted to 85 mM NaCl by addition of an equal volume of T-broth containing no NaCl.
pH.
Aliquots of T-broth were adjusted to the required pH using 0·2 M stock sodium acetate solutions for pH 4·6 and 5·2, and 0·2 M stock potassium phosphate for pH 5·87·6 (Sigma-Aldrich). Tris buffer (1 M, pH 8·5) was used to buffer T-broth to pH 8·2 (final concentration 25 mM).
Viscosity.
PVP MW 360 000 (Sigma-Aldrich) was dissolved to 50 % (w/v) in dH2O and dialysed overnight in 25 mm diameter dialysis tubes (The Spectrum Companies) against several changes of dH2O. The total volume of dialysed PVP was measured and adjusted to a final concentration of 20 % (w/v) with dH2O. The PVP was autoclave-sterilized (20 min, 121 °C) and aliquots were mixed with appropriate volumes of either 2x or 4x T-broth to give the final concentrations of PVP (15, 10 and 5 %, w/v).
Microscopy and motility measurement.
For each motility measurement, 2 ml overnight cultures of FlaA- and FlaAB-expressing cells were diluted 1 : 100 in fresh T-broth in 125 ml conical flasks and incubated for about 3 h until the OD600 was 0·5 (mid-exponential phase) for each strain. The cell suspensions were then diluted in the respective medium, and 10 µl of the diluted cell suspension was removed, placed onto a microscope slide and covered with a grease-gasketed coverslip. The cells were immediately viewed with phase-contrast at x400 using an Olympus BX60 upright microscope. After choosing a field of view containing about 2550 actively motile cells, video images were acquired using a Canon Elura (40MC) digital camera attached to the microscope. Five minutes of cell motion from two separate fields of view was acquired per strain for each condition measured. Following review of the entire video period, a 1 min segment starting within the first 20 s was selected for detailed motion analysis using Adobe Premiere, VirtualDub (version 4.1; Avery Lee 19982001) and Adobe Photoshop. The result was a series of still images taken every 0·33 s depicting the location of each cell in the field of view. This series of digital images was subsequently used to locate and measure the position of randomly chosen cells by recording their pixel coordinates then converting to a micrometre scale. Individual cells (n=20 for NaCl, n=10 for others) were tracked across nine consecutive series for a total of 2·66 s and the coordinates recorded. Statistical reproducibility of the measurements was ensured by repeating each experiment a minimum of five times over subsequent days.
Data analysis.
The coordinates of each tracked cell were recorded onto Microsoft Excel spreadsheets and equations for total distance, mean distance, mean velocity, mean angle and tumble frequency were used to provide final measurements of these parameters for each cell. Representative cell tracks from each series were checked for accuracy by plotting the calculated coordinates and comparing them to the video track of the individual cell. Students' t-test (pairwise, independent, at P<0·05) was used to compare values obtained from FlaA- and FlaAB-expressing cells.
Measurement of filament length and the number of filaments per cell.
Samples of the standardized dilutions of cells for cell motion analysis were taken and stained with AlexaFluor 594 (Molecular Probes) following the method described by Turner et al. (2000). Fluorescently stained filaments were visualized with an Olympus BX60 upright microscope at an excitation of 480 nm and emission of 535 nm. Representative images were acquired using a Quantix model 1400 CCD camera (Photometrics) and analysed using the image-processing program IPlab 4.1a (Scanalytics) and Adobe Photoshop 7.0.
Measurement of flagellar helical parameters and preparation of isolated flagella.
Flagella were prepared using the method described by Belas (1994). Briefly, P. mirabilis strains were incubated on L-agar to produce swarmer cells. Flagella were sheared from cells and harvested by centrifugation, then purified through a sucrose step gradient. The protein concentration of each fraction was determined by the bicinchoninic acid (BCA) protein assay (Pierce Chemicals) and an aliquot from each was run on a 410 % gradient SDS-PAGE gel and stained with Coomassie blue (Sigma) to determine purity and the relative amounts of flagellin in each fraction. Fractions were stored at 4 °C and those showing the highest and cleanest yield of flagella for FlaA- and FlaAB-expressing cells were used for analysis.
Alexafluor staining of isolated flagella.
Isolated flagella were stained with AlexaFluor 594 (Molecular Probes) following the method described by Turner et al. (2000) with the following modifications. One vial of AlexaFluor free dye was dissolved in 1 ml of a 1 : 1 solution of dichloromethane/2-propanol and divided into 100 µl aliquots in screw-cap microcentrifuge tubes. A flagellar suspension containing 1 µg protein was added to 100 µl of the dye, together with 25 µl Na2HCO3 to shift the pH to 7·8. Dye binding was achieved by shaking the suspension on a flat bed rotary shaker (100 r.p.m.; RT) in the dark for 1 h. Samples were then dialysed overnight in several changes of 1x PBS using a microdialyser as per the manufacturer's instructions (Spectrum). The concentrations of FlaA- and FlaAB-expressed flagella were equalized by diluting aliquots of dialysed samples 1 : 10, 1 : 20 and 1 : 50, and checking 10 µl of each dilution to give about 2025 flagella per field of view. A volume of 610 µl of the appropriate dilution was then placed on glass coverslips and appropriate amounts of solutions or buffers designed to alter NaCl or pH levels to that required (85 mM NaCl, 425 mM NaCl, pH 5·8; prepared beforehand as described above), were added to the sample on the coverslip before inversion onto the slide.
Measurement of helical parameters.
Measurements of amplitude () and wavelength (
) (n=100), were made using the methods described by Kamiya & Asakura (1976b)
. Data analysis was carried out as described above, with significant difference between wild-type and DF1003 (FlaAB+) flagella calculated for a 95 % confidence interval (P<0·05).
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RESULTS |
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FlaAB+ cells are motile in viscous medium
The addition of the viscosifying agent PVP to liquid medium has the effect of slowing down the rotation of the flagella and thus reducing the velocity of peritrichously flagellated bacteria at levels above 2 % (w/v) (Greenberg & Canale-Parola, 1977; Schneider & Doetsch, 1974
). This effect was clearly exhibited by both strains as the concentration of PVP was increased from 5 to 10 % (w/v) (Table 1
). However, the FlaAB-expressing strain was less affected overall, with its mean velocity at both 5 and 10 % PVP being significantly higher than that of the FlaA-expressing strain (Table 1
). At higher PVP concentrations, i.e. 15 % PVP, both strains had almost equal swimming speeds, suggesting that there is an upper end to any advantage in viscous medium conferred by expression of FlaAB+ filaments.
FlaAB+ flagellar helix retains structure in high salt and acidic pH
We chose the three conditions that showed the greatest differences in swimming velocity between DF1003 and wild-type cells: 85 mM NaCl (pH 7·0; low NaCl concentration), 425 mM NaCl (pH 7·0; high NaCl concentration) and pH 5·8 (170 mM NaCl; low pH), and measured the amplitude and wavelength of the flagellar filaments of each strain. Filaments in 170 mM NaCl pH 7·0 (physiological norm) were used as a control for comparison. The results are presented in Table 2 and Figs 3 and 4
. There is a significant difference between wild-type and FlaAB+ filaments, in terms of both amplitude (Fig. 3
a) and wavelength (Fig. 3b
) under all conditions tested, including the physiological norm (control). Examples of individual flagella in pH 5·8 and 425 mM NaCl from both strains are compared side by side in Fig. 4
(a) and (b), respectively, with the changes in amplitude and wavelength illustrated in Fig. 4(c)
. Either an increase in the NaCl concentration or a change in the acidity/alkalinity of the medium led to a greater structural change in FlaA+ filaments as compared to FlaAB+ filaments. Conversely, a reduction in NaCl concentration to 85 mM (50 % of physiological norm) resulted in a greater change in the structure of FlaAB+ filaments compared to wild-type FlaA+ filaments. Thus FlaAB+ filaments appear to retain their helical structure at both high salt (425 mM NaCl) and acidic pH (5·8) when compared to wild-type filaments, but lose that structure at lower NaCl concentrations (85 mM NaCl). This phenomenon is illustrated in Fig. 5
, where the difference between the filaments of each strain is shown as a percentage change in wavelength from the physiological norm (set to 100 %). While percentage change in FlaAB+ remains near zero at 425 mM NaCl and pH 5·8, that for FlaA+ rises sharply to +12 % at pH 5·8. On the other hand, both strains undergo a similar level of change (about 6 %) when the salt concentration is halved to 85 mM.
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Flagellin structure may be affected by amino acid substitutions
Kanto et al. (1991) identified key mutations in strains of S. typhimurium compared to the wild-type SJW1103 that were responsible for changes in flagellar shape in that species. We aligned the deduced amino acid sequences of the wild-type FlaA protein and FlaAB from DF1003 with S. typhimurium SJW1103 to look for substitutions at these sites. The CLUSTAL W alignment (Thompson et al., 1994
) revealed a substitution between FlaA and FlaAB at one of these sites (K414N) located in the conserved C-terminal end and shown underlined in Fig. 6
. This substitution renders the site more compact and acidic in FlaAB due to the size and composition of the side chain. Analysis of the central domain (not shown) also revealed numerous substitutions between G-190 and D-266, including a 21 aa region containing 14 non-conserved substitutions between G-190 and V-212. At least three of these non-conserved substitutions (K198L, N200I and S210I) lead to replacement of hydrophilic with hydrophobic side chains, while a further three (A191D, T194D and A196N) introduce acidic side chains. Only one substitution (L205K) replaces a hydrophobic side chain with a hydrophilic one. Thus it is possible that while this region is not as critical to flagellin structure as the conserved termini, these resulting differences in secondary structure of the FlaAB protein may help it retain the helix morphology when exposed to high salt and acidic pH.
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DISCUSSION |
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How might the expression of FlaAB+ flagellin lead to enhanced motility in some environments? Amplitude and wavelength are inversely related; therefore a decrease in amplitude leads to a concomitant increase in wavelength and vice versa. In physical terms the former leads to a flattening out or relaxation in the helical flagellar structure, as portrayed in Fig. 4(a) and (b) and illustrated in the accompanying drawing (Fig. 4c
). This change in flagellar filament morphology occurs concomitant with the changes in motility observed for FlaA+ and FlaAB+ cells. To emphasize this relationship, the increase in helical wavelength of FlaA+ filaments from 1·44 to 1·62 µm at pH 5·8 is accompanied by a decrease in amplitude from 0·194 to 0·170 µm, producing a relaxed filament. However, at the same pH, the wavelength of FlaAB+ flagella remains almost unchanged (from 1·47 to 1·46 µm), while the amplitude increases slightly (from 210 to 226 µm), indicating a tightening of the FlaAB+ flagellar helix. In step with these morphological changes to the filament, the mean velocity of FlaA+ cells decreased by 50 % (from 23·14 to 11·77 µm s1), while that of FlaAB+ cells decreased by only 37 % (from 25·93 to 16·33 µm s1). Similarly, at 425 mM NaCl, the amplitude of the FlaAB+ filament increases to a greater extent than FlaA+ (210 to 251 µm against 194 to 215 µm). This structural change in the flagellum is reflected in greater retention of motility (50 % of normal velocity compared to only 12 % for FlaA+). Thus, a tightening of the filament is correlated with a smaller decrease in swimming velocity and vice versa. Interestingly, when the FlaAB-expressing strain is subjected to 85 mM NaCl, it is significantly less motile (9·05 against 19·13 µm s1) and measurements of the amplitude and wavelength of the FlaA+ filament show that it has tightened significantly, compared to the FlaAB+ filament. Thus, in this environment the FlaA+ filament is able to maintain a tighter helix than the FlaAB+ filament and it is this helix morphology that apparently helps FlaA-expressing cells gain a motility advantage at this salt level, rather than the relaxation of helical structure in FlaAB+. Therefore, environmental conditions, such as NaCl concentration and pH, alter flagellar filament morphology differently depending on whether a FlaA+ or FlaAB+ filament is produced and the tighter filament helix of FlaAB flagellin appears to be a better propeller under these conditions.
Measurements of the mean length of the filaments and the number of flagella per cell indicate that FlaA+ cells produce longer filaments and may possess slightly more flagella per cell than the FlaAB-expressing cells. It is therefore feasible that possession of more, longer flagella might be a factor in determining the swimming speed of FlaA+ cells. While a tantalizing hypothesis, it is unlikely for at least two reasons. First, for this hypothesis to be true, the length and number of flagella would have to change with the different environmental conditions tested. This is not the case, as the data indicate that both the number of flagella and length of the filaments were constant for each strain under the conditions tested. Second, the hypothesis predicts that FlaA-expressing cells would be better swimmers than cells expressing FlaAB+. This is not the case, as having fewer and shorter flagella would be expected to inhibit FlaAB+ motility rather than enhance it; yet the data show that under normal conditions the two strains have similar mean velocities, while under most amended conditions FlaAB-expressing cells are significantly faster than FlaA+ cells, which have longer filaments and slightly more flagella. It appears far more feasible based on these data that the differences in swimming speed are due to alterations in the flagellar filament helix shape rather than the length or abundance of flagella on the cell.
Altered helicity can result from single amino acid substitutions in the flagellin sequence. For example, about a dozen point mutations have been identified in S. typhimurium SJW1103 that affect filament morphology and all except one are found within the conserved terminal regions of flagellin (Hyman & Trachtenberg, 1991; Kanto et al., 1991
; Samatey et al., 2001
). These single residue changes resulted in straight, coiled and two types of curly flagella. Hydrophobic interactions between residues in different subunits of the filament have also been found to play a role in filament supercoiling. Yokekura et al. (2003)
examined intersubunit interactions in two mutations in the atomic model of the S. typhimurium SJW1655 flagellar filament. Residue 449, located in the C-terminal
-helix of domain D1 (CD1), and responsible for the R-type straight filament in the A to V mutation, is surrounded by F53 and F131 located on domain ND1a, with the mutation F53V destabilizing the R-type filament in favour of an L-type straight filament through a reduction in hydrophobic interactions.
While exact homologues of these S. typhimurium FliC amino acid changes are not apparent in FlaAB, an alignment of S. typhimurium FliC, P. mirabilis FlaA and FlaAB (Fig. 6) does reveals at least one amino acid substitution in the FlaAB protein that may play a role in the morphological changes observed in the different environmental conditions. This substitution is in a homologous site to one of the residues identified by Kanto et al. (1991)
as responsible for flagellar shape in S. typhimurium FliC. While it is not the same substitution (A414V in S. typhimurium, K414N in P. mirabilis), the replacement amino acid residue (N) in the FlaAB protein has an acidic and less bulky side chain, which may contribute to its ability to maintain its shape in acidic environments.
Moreover, larger deletions can also affect flagellin filament structure through changes in intersubunit interactions. Two spontaneous mutant strains arising from SJW1103, SJW46 and SJW61 have deletions of 88 and 96 aa, respectively, in regions of the outermost subdomain, possibly contributing to the mechanical instability and high level of polymorphism in this strain (Mimori-Kiyosue et al., 1998). The absence of whole flagellin domains can also positively affect motility. Cohen-Krausz & Trachtenberg (2003)
found that the largest and most variable domain, D3, is totally absent in the flagellin subunits forming the complex filament of Rhizobium lupini and suggest that the consequence of this may be a better hydrodynamic performance in viscous environments (Cohen-Krausz & Trachtenberg, 2003
). While not strictly in the conserved terminal ends of FlaAB, several other substitutions (A191D, T194D, A196N, K198L, N200I and S210I) render the FlaAB protein more acidic and hydrophobic overall and may also play a role in retention of the helical structure in the FlaAB+ strain under high salt and acidic pH conditions.
The prevailing view of bacterial flagella has been one of a highly immunogenic structure that plays a substantial role in evasion of the host response during infection through either phase variation or switching. While this stance is well supported by research, the results of the current study suggest that flagellar antigenic variation may also result in mechanistic changes in the flagellar filament's helicity that aid in bacterial survival by improving the efficiency of the flagellar filament propeller.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 August 2003;
revised 1 December 2003;
accepted 26 January 2004.
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