Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 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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Our laboratory previously characterized (Belas & Flaherty, 1994) two tandemly arranged and highly homologous flagellin-encoding genes of P. mirabilis, flaA and flaB (Fig. 1
). While flaA and flaB have an overall DNA sequence identity of about 80 %, domains adjacent to the 5' and 3' ends contain regions of nearly 100 % identity, suggesting that they may be sites of homologous recombination between the two genes. Measurements of transcription and primer extension analysis have identified a
28 promoter (Arnosti & Chamberlin, 1989
; Helmann, 1991
) upstream of flaA from which the gene is expressed (Belas, 1994
). These studies also showed that flaB is not transcribed, i.e. it is a silent allele. In concert with the transcriptional data, FlaA mutants (flaA' : : cam : : 'flaA) are non-motile, while disruption of flaB (flaB' : : cam : : 'flaB) has no effect on motility (Belas, 1994
). One of the interesting findings from these studies is that flaA' : : cam : : 'flaA mutations are unstable and occasionally revert to a motile phenotype that displays wild-type swimming and swarming behaviour. The motile revertants possess flagella that are antigenically distinct from wild-type flagella and fail to bind anti-FlaA antisera. N-terminal amino acid sequencing of the flagellin obtained from these motile revertants confirmed the occurrence of changes in the amino acid sequence. These revertants are genotypically different from the parent and contain an in-frame fusion of the 5' end of flaA (
28 promoter region plus coding region of flaA) and the 3' coding region of flaB (Belas, 1994
). Murphy & Belas (1999)
characterized several different revertants and found that the formation of the resulting hybrid flaAB gene was the result of a conservative loss of a 1410 bp segment of intervening DNA containing the 3' end of the flaA coding region through to a site in the 5' end of flaB. Interestingly, the placement of the ends of the 1410 bp segment varied from revertant to revertant, yielding multiple flaAB variants and a potential suite of FlaAB proteins. Hybrid flagellin DNAs were also found in wild-type cells obtained from both laboratory cultures and experimental mouse urinary tract infections. An analysis of many such hybrid flagellin genes also confirmed the presence of numerous flaAB gene variants.
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METHODS |
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DNA manipulation.
Genomic DNA was extracted and PCR amplified using standard methods (Ausubel et al., 1987) and previously described conditions (Belas, 1994
; Murphy & Belas, 1999
). Agarose gel electrophoresis was used to separate DNA by standard methods (Ausubel et al., 1987
). Gels were stained with SYBR Gold (Molecular Probes) or ethidium bromide (Sigma-Aldrich) and the DNA fluorescence was visualized using a Fluor Imager model 575 and the ImageQuant (version 4.1) analysis software (Molecular Dynamics). PCR products were inserted into the TA vector pCR2.1 (Invitrogen), which was then transformed into competent Escherichia coli DH5
using established procedures (Ausubel et al., 1987
). Sequence editing and detection of ORFs was carried out using the computer programs Chromas (version 1.42) and DNAMAN (Lynnon BioSoft).
RNA dot-blots.
Using the hot phenol technique (von Gabain et al., 1983), total RNA was extracted from 50 ml LB cultures grown at 37 °C for 4 h, according to the method of Belas (1994)
. The extracts were checked for purity and concentration by formaldehyde gel electrophoresis (Ausubel et al., 1987
; Belas, 1994
) and spectrophotometric measurements at 260 and 280 nm (Ausubel et al., 1987
). Samples of total RNA or chromosomal DNA were diluted tenfold and applied to consecutive slots in a dot-blot manifold (model SRC-96, Schleicher and Schuell). Labelled probes were added to the hybridization solution at a specific activity of 0·1 µCi ml1 (3·7x109 Bq ml1) of solution, and hybridization was carried out at 42 °C, followed by washes by established methods. Oligonucleotides were end-labelled with 6000 Ci [
-32P]dATP mmol1 (2·2x1014 Bq mmol1; Amersham Pharmacia Biotech) using the KinAce-It kinasing kit (Stratagene) according to the manufacturers' instructions. Phospho-imaging analysis (Storm 840; Molecular Dynamics) was used to detect hybridization and the dot intensity was determined by the mean pixel intensity in equal-sized squares (ImageQuant version 1.2; Molecular Dynamics).
RT-PCR.
RNA for RT-PCR was extracted from 2 ml overnight LB cultures that were subsequently diluted 1 : 100 and grown for 4 h to mid-exponential phase. Extraction was carried out using the RiboPure kit for bacterial RNA extraction (Ambion), with the following modifications. After initial chloroform extraction, the partially purified RNA was incubated with 10 U RNase-free DNase I (Roche Diagnostics) for 45 min at 37 °C to digest genomic DNA, before completion of final purification. The RNA was checked for purity and concentration as described above. Reverse transcription was carried out with 500 ng purified RNA in a 20 µl reaction using the GeneAmp Gold RNA PCR kit, according to the manufacturer's instructions (Applied Biosystems). flaAB was detected using 0·4 µM of the flaAB-specific primer fla3005R (5'-CCAGAGCGTTTGCATCGAT-3'), while flaA was detected with flaA-specific primer fla1768R (5'-GATGCTTTTAATCCAAGTTTAGTTTAGTACCT-3'). A 3 µl aliquot of the reverse transcriptase mix was used as template in a 50 µl PCR amplification reaction according to the manufacturer's instructions (Applied Biosystems), with 0·15 µM each of the 5' primer fla1072F (5'-CTTTAGGAAGTGCAATCGAGC-3') and the respective 3' primer. PCR cycling conditions were as follows: 10 min at 95 °C (for hot start enzyme AmpliTaq Gold DNA polymerase; Applied Biosystems), 34 cycles of 94 °C for 1 min, 62 °C for 2 min (a combined annealing/extension step), 1 cycle of 72 °C for 10 min. Gel analysis of products was carried out on a 5 µl sample diluted 1 : 4 in deionized H2O.
To confirm that the RNA preparations were free of contaminating DNA, control PCR amplifications with RNA samples plus oligonucleotide primers fla1072F and fla3005R were performed. Amplifications that produced a unique 1933 bp fragment, in addition to the expected 523 bp fragment, indicated the presence of genomic DNA and were not used.
Phenotypic switching frequency of FlaAB.
The phenotypic switching frequency and relative abundance of FlaAB-producing cells in the population were determined using a lacZ transcriptional fusion strain that produces -galactosidase when FlaAB is expressed. This strain, JM3006, was constructed as follows. A plasmid, pflaDAB (Belas, 1994
), carrying the intact flaAflaB genes was modified using site-directed mutagenesis (QuikChange, Stratagene) to introduce a BamHI site between the flaB stop codon and the putative flaB terminator. A promoterless lacZ/kan cassette (Barcak et al., 1991
) was then inserted at the BamHI site to produce the 'flaB : : lacZ plasmid, pJM104. pJM104 was digested with EcoRV and the 'flaB : : lacZ fragment was cloned into the suicide vector pGP704 (Miller & Mekalanos, 1988
) in E. coli SM10
pir and conjugally transferred to P. mirabilis wild-type (Belas et al., 1991b
). Colonies containing the mutated flaB : : lacZ locus were identified by the acquired kanamycin resistance and verified by EcoRI restriction digests and sequence analysis of the mutated (flaB : : lacZ) genomic locus. Swimming and swarming motility of six such kanamycin-resistant clones was measured as previously described (Belas, 1994
). Stability of the mutant was determined by measuring kanamycin resistance after overnight growth of the cells in LB lacking the antibiotic, as well as by PCR analysis of the flaAflaB locus. The frequency of FlaAB expression was measured by incubating JM3006 in LB plus kanamycin for 4 h at 37 °C and spreading a diluted sample of the cells on LSW agar containing 40 µg X-Gal ml1. Following overnight incubation at 37 °C, the numbers of Lac+ (FlaAB; blue) and Lac (FlaA; white) colonies were counted (n=1000) as follows. Briefly, a digital image of the colonies was obtained and analysed using Adobe Photoshop, by selecting a blue-colour threshold value of R(ed) 115, G(reen) 222 and B(lue) 230, which is a pale-blue colour. All colonies having this tint or a darker blue were identified using the Photoshop Replace Colour tool, with the settings of hue 180, saturation 100, lightness 28 and fuzziness 100. The FlaAB colonies in the digital image were thereby assigned a red colour, distinguishing them from FlaA colonies and allowing for quick manual enumeration. An estimate was thus generated of the percentage of the population expressing FlaAB.
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RESULTS |
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An estimate of the ratio of flaAB mRNA to flaA transcript in the population of wild-type cells was obtained using densitometry analysis of the RNA dot-blot data presented in Fig. 2. The density (51±3 pixels mm2) of the 1 : 1 dilution of wild-type RNA hybridized to the flaAB-specific probe (Fig. 2
, row 4, column 1) was nearly equal to the density, (49±5 pixels mm2) of the 1 : 64 dilution of wild-type RNA hybridized to the flaA oligonucleotide (Fig. 2
, row 2, column 7). This means that there was about 64 times more flaA mRNA than flaAB mRNA present in the wild-type population, or put another way, flaAB mRNA constituted about 1·5 % of the total flagellin mRNA present in the population. Since the same
28 promoter driving flaA transcription was presumably used to transcribe the hybrid flaAB gene, the difference in concentration between flaA and flaAB mRNA was due mainly to differences in transcript abundance and not differential promoter efficiency.
Analysis of flaAB mRNA
Confirmatory evidence of flaAB transcription in wild-type cells was also obtained by RT-PCR. A 500 ng quantity of DNaseI-treated total RNA extracted from the wild-type and the flaAB-locked strain (DF1003) was reverse-transcribed and the cDNA product was used as a PCR template. These results are shown in Fig. 3(a). Under the conditions of the assay, the RT-PCR reaction of wild-type RNA plus the flaA-specific primer fla1768R (Fig. 3a
, left lane) produced the predicted flaA product (0·7 kbp), while RNA extracted from the flaAB-locked strain produced an RT-PCR product of 0·50 kbp (Fig. 3a
, middle lane) when primed with the flaAB-specific primer. Significantly, RT-PCR of wild-type RNA plus the flaAB-specific oligonucleotide (Fig. 3a
, right lane) resulted in a product that was an identical size (0·50 kbp) to that produced by the flaAB-locked RNA, strongly implying that it too is produced from flaAB mRNA. As can be seen in Fig. 3(a)
, much less flaAB product was obtained from wild-type cells compared to flaA product from the wild-type, or compared to flaAB product from the phase-locked strain. Although the percentage of flaAB to flaA in wild-type cells was not determined by RT-PCR, visual examination of the bands in Fig. 3(a)
suggests that the difference in amount parallels the RNA dot-blot frequency of about 1·5 %. The presence of contaminating DNA in all RNA samples was assessed by PCR amplification using fla1072F with fla3005R, fla2333R (5'-ACACTATCGTCATTAAATCGAAGGTA-3') or fla1768R. None of these reactions produced a positive result (data not shown), indicating that the RNA samples were free of contaminating DNA. Therefore, the simplest interpretation of the RT-PCR data is that flaAB is formed and transcribed in wild-type P. mirabilis populations.
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Phenotypic analysis suggests FlaAB is expressed in approximately 1 % of the population
To corroborate the mRNA data, the percentage of bacterial colonies expressing FlaAB was measured through the use of a strain (JM3006) harbouring a stable flaB : : lacZ transcriptional fusion. This strain was constructed so as to allow -galactosidase to be produced only if flaAB was transcribed and translated. Indeed, JM3006 possesses wild-type swimming and swarming motility, suggesting that the flaB : : lacZ fusion does not impair flagellar synthesis or function (data not shown). Fig. 4
shows a photograph of colonies of this FlaAB-expression-detection strain growing on XGal-containing agar. In this assay, blue colonies express varying levels of FlaAB, while white colonies express FlaA. Using Adobe Photoshop, a cut-off threshold of blue was chosen to distinguish FlaAB+ from FlaA+ colonies, and an analysis of 1000 colonies from six separate experimental replicates was performed. The frequency of FlaAB expression was estimated to be approximately 1 %, which agrees with the estimated value (1/64 or 1·5 %) obtained by RNA dot-blot quantification.
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DISCUSSION |
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In interpreting these results, it is important to be mindful of the limitations associated with each method used. Indeed, it is due to these limitations that multiple means were sought to measure flaAB transcription and expression. One factor that could alter the interpretation of the results is the potential for selective amplification of templates during PCR. This could lead to a distortion in the amount of product generated. In control PCR amplifications using the flaA- and flaAB-specific oligonucleotides, with purified flaA or flaAB DNA alone, or in varying ratios mixed together, we found no evidence for selective amplification of either template DNA over a 10 000-fold range in concentration (data not shown). This suggests that selective amplification is not a complicating factor in the interpretation of the results. Similarly, the difference in wild-type flaAB mRNA abundance observed in RT-PCR analysis compared to the RNA dot-blot method may have been affected by the different RNA extraction methods used for each procedure. The estimate of FlaAB frequency in the phenotypic analysis may also have been affected by the blue cut-off threshold value used to distinguish Lac+ from Lac colonies (see Methods). This conservative threshold may have resulted in an underestimate of the frequency of FlaAB-expressing cells.
The abundance of flaAB mRNA estimated from these data is relatively high when compared to the abundance of alternative flagellins produced by other known flagellar switches (Gillen & Hughes, 1991; Harris et al., 1987
). The possibility exists that selective degradation of either flaA or flaAB mRNA could affect the outcome of these experiments and skew the abundance estimate. In an earlier study (Belas, 1994
), we found that P. mirabilis flagellin mRNA was quite stable in vitro, suggesting that flaA mRNA, which would presumably represent the majority of flagellin transcript in the earlier work, is not readily degraded. The stability of flaAB mRNA is not known, and it remains a possibility that this factor could influence interpretation of the current estimate of flaAB abundance.
Another potential complication in estimating the abundance of flaAB mRNA may arise if the oligonucleotide primers used to detect flaA and flaAB respectively had different affinities for their respective targets. Indeed, the flaAB primer does show about 1·5- to 2-fold lower affinity for its target than does flaA for its cognate site, and this difference may lead to an overestimation of the abundance of flaAB by an equivalent factor. Given this, the estimated abundance of flaAB is best used as a general measurement and not a strict value. However, it is important to be mindful that none of these limitations directly affects the conclusion that flaAB is transcribed.
While the molecular mechanism responsible for generating P. mirabilis flaAB transcripts is not known, the existence and relative abundance of flaAB mRNA suggest that the mechanism used to produce it is not likely to be similar to the well-studied Hin recombinase switch responsible for Salmonella enterica serovar Typhimurium flagellar antigenic switching (Gillen & Hughes, 1991). Nonetheless, it is reasonable to speculate that the production of FlaAB flagellin must benefit P. mirabilis in some manner to warrant the large expenditure of energy and carbon required to produce the hybrid flagella.
What role might flaAB play? One possible role for flaAB could be as a generator of flagellar (H) antigen variation, as was originally suggested by us (Belas, 1994). FlaAB flagellar filaments are likely to have different amino acid residues exposed to the external environment, as compared to FlaA flagellin. Thus, the transcription of flaAB and expression of FlaAB protein during urinary tract infections may be an effective means used by the cells to evade the host immune defences.
Alternatively, transcription and expression of flaAB may serve another function: production of a flagellar filament whose morphology yields a more efficient propeller for swimming and swarming motility in extreme conditions. This scenario is entirely plausible, since FlaAB flagellin is different from FlaA flagellin (Murphy & Belas, 1999), and even small changes in the amino acid residues composing the filament could lead to significant alterations in the quaternary structure of the resulting flagellar filament. We have recently analysed the swimming and swarming motility of the wild-type and DF1003 (FlaAB-locked) strains by cell motion analysis, and observed significant differences in the motility of DF1003 (compared to wild-type) at extremes of pH, salinity and viscosity, suggesting that in such conditions expression of FlaAB flagella offers the bacterium an advantage (Manos et al., 2004
). The persistence of P. mirabilis in human urinary tract infection may be due in part to the expression of FlaAB flagella. High salinity, alkaline pH and increased viscosity are environmental features of the urinary tract, while acidic pH favours urinary tract infection (Raz & Stamm, 1993
). The ability to survive in or rapidly move through such environments may offer the P. mirabilis flaAB phenotype an advantage during colonization and pathogenesis, and enhance its prospects for survival and increased virulence.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 April 2004;
revised 19 May 2004;
accepted 1 June 2004.
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