Department of Biological Sciences, University of Wisconsin, PO Box 413, Milwaukee, WI 53301, USA
Correspondence
Steven Forst
sforst{at}uwm.edu
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AF525420 (mrxAJ).
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INTRODUCTION |
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Hair-like appendages called fimbriae participate in the specific attachment of pathogenic bacteria to host cells (Low et al., 1996). This facilitated attachment is a prerequisite for bacterial invasion and colonization, important early steps in pathogenesis (Stentebjerg-Olesen et al., 1999
). The well-characterized Pap fimbriae of Escherichia coli (Tullus et al., 1992
) and type 1 fimbriae of E. coli (Connell et al., 1996
), Salmonella (Tinker et al., 2001
) and Proteus (Zhao et al., 1997
) are important factors in the colonization of the human intestinal and urogenital tracts. Fimbriae also play a role in the symbiotic interaction between Vibrio fischeri and the Hawaiian squid, Euprymna scolopes (Stabb & Ruby, 2003
).
Pap and type 1 fimbriae are assembled by a chaperone-usher pathway (Soto & Hultgren, 1999). The operons encoding these fimbriae generally consist of between eight and eleven genes, which include regulatory, assembly and structural components. While the assembly and structural proteins of the various fimbriae share similarities, the regulation of the operons can vary considerably (Blomfield, 2001
). The pap operon is regulated by a complex mechanism involving local regulatory proteins (PapB, PapI), the leucine-responsive regulatory protein (Lrp), cyclic AMP receptor protein (CRP), and the differential site-specific methylation of two GATC sites situated upstream of the promoter (Hernday et al., 2002
). The fim operon of E. coli and the mrp operon of Proteus mirabilis are regulated by a recombinase-mediated inversion of a promoter-bearing invertible element. The recombinase genes are linked upstream of the major fimbrial subunit gene. Lrp stimulates recombination of the fim invertible element, possibly by altering the local spatial organization of the DNA in this region (Blomfield, 2001
). Both Pap and type 1 fimbriae are produced under prolonged static growth conditions, but generally are not produced on solid agar media (Old & Duguid, 1970
; Gally et al., 1993
).
X. nematophila produces fimbriae on nutrient agar, but not when grown under aerated conditions in LB broth (Binnington & Brooks, 1993; Moureaux et al., 1995
). The X. nematophila fimbriae are 7 nm in diameter and up to 3 µm in length, consisting primarily of a 16 kDa major subunit protein (Moureaux et al., 1995
). Purified fimbriae agglutinate erythrocytes of sheep and rabbit in a mannose-resistant pattern. Immunogold labelling indicates that fimbriae are present on X. nematophila inhabiting the nematode gut sac (Binnington & Brooks, 1993
). Although there has been speculation that the fimbriae in X. nematophila are involved in the species-specific symbiotic association with Steinernema carpocapsae (Binnington & Brooks, 1993
; Forst & Nealson, 1996
; Moureaux et al., 1995
), the role they play in the complex life cycle of the bacterium has not been established. To begin to understand the function of fimbriae in Xenorhabdus, we have characterized the mannose-resistant fimbrial operon (mrx) of a X. nematophila strain.
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METHODS |
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Purification of DNA and RNA.
Genomic DNA was extracted using the Edge BioSystems purification kit. Total RNA was extracted with the RNeasy mini kit (Qiagen) or Trireagent (Sigma). RNA samples used for RT-PCR were predigested with the RNase-free DNase reagent (Qiagen).
Sequence analysis of the mrx operon.
To sequence the mrx operon, three separate partial libraries were constructed in pBluescript SK(+). mrxA and mrxD PCR probes based on partial genomic sequence information of Xenorhabdus strain 85816 (kindly provided by B. Goldman, Monsanto) were used to screen a X. nematophila AN6 library. The mrxA-containing clone, pBS4, carried a 9·3 kb EcoRIHindIII fragment, which contained pnp, mrxA, and 212 bp downstream of the stop codon. The mrxD-containing clone, pBSD21, carried a 5 kb HindIII insert, containing mrxC, mrxD, mrxG and 687 bp of the 5' region of mrxH. To isolate the clone pBSH1, a probe derived from the mrxH sequence of AN6 was used, which carried a 4·5 kb EcoRI insert containing mrxG, mrxH, mrxJ and the downstream gene encoding the large subunit of carbamyl phosphate synthase. To complete the sequence between mrxA and mrxC, a 1245 bp PCR product was amplified from the beginning of mrxA to an internal sequence in mrxC. This PCR fragment contained a 439 bp overlap with mrxA and a 241 bp overlap of mrxC. The overlapping sequences of the PCR fragment were identical to that derived above and provided the sequence between the 3' end of pBS4 and the 5' end of pBSD21. The organization of this region of the mrx operon was confirmed by overlapping PCR analysis. Automated nucleotide sequence analysis was performed at the Core DNA Facility at the University of Wisconsin-Milwaukee. Sequences were assembled with MacVector 5.0. Amino acid identity searches were performed using the NCBI BLAST website, and identity values were calculated using pairwise sequence alignment (http://genome.cs.mtu.edu/align/align.html). To search for putative Lrp-binding sites upstream of mrxA, the MEME (Bailey & Elkan, 1994) probability matrices algorithm was used (http://meme.sdsc.edu/meme/website/intro.html). This analysis was performed using individual Lrp-binding sequences of the papBA promoter.
Construction of mrxA-null strain (AHA1).
An internal mrxA fragment was PCR amplified with the primer pair 5'-CGCAGCAACTGCAGCAC-3' (forward) and 5'-CAGGTAGTTCAAAGTAAAGTATGCG-3' (reverse). A PstI site (underlined) was engineered into the foward primer. A natural EcoRV site exists within mrxA. The PCR product was ligated into pSTBlue-1 (Novagene), and a PstIEcoRV fragment (418 bp) from the resultant plasmid was ligated into the conjugal suicide vector pKnock-Cm (Alexeyev, 1999). This was followed by electroporation into E. coli S17-1
pir and conjugal transfer into X. nematophila AN6. The desired mutant (AHA1) was selected on LB agar containing 50 µg ampicillin ml1 and 25 µg chloramphenicol ml1, and confirmed by Southern analysis.
PCR and RT-PCR reactions.
PCR reactions (50 µl) contained 40 ng genomic DNA, 20 pmol primers, 2 mM dNTPs, 2·5 mM MgCl2 and 2·5 units Taq DNA polymerase (Promega). Standard reactions were performed for 30 cycles. RT-PCR reactions (50 µl), using the AccessQuick RT-PCR system (Promega), contained 100 ng DNA-free RNA, 20 pmol primers and 1 unit reverse transcriptase. The reverse transcription reaction was conducted at 52 °C for 45 min. To obtain semi-quantitative results, the PCR reaction was carried out for 25 cycles.
Primer extension analysis.
Three primers were used in primer extension reactions to determine the start of transcription of mrxA and examine the potential existence of a promoter within the intergenic region between mrxA and mrxC. Primer 1 (5'-GTGTTCAGGTTTTGA-TTCACCACC-3') is internal to the start codon of mrxA. Primer 2 (5'-CGGGGAATAGAGG-AAATATGTCAC-3') is located in the middle of the intergenic region. Primer 3 (5'-GCCGATAATGCAAACAGCACC-3') is internal to the start codon of mrxC. These primers were end-labelled with [32P]ATP and T4 polynucleotide kinase. The Primer Extension System-AMV Reverse Transcriptase (Promega) was used for the primer extension reaction. The primer was annealed to total RNA by incubating at 52 °C for 20 min and cooling at room temperature for 10 min. The extension reaction was conducted at 42 °C for 30 min. The extension product was separated on an 8 % polyacrylamide denaturing gel and sized using a radiolabelled
X174 DNA marker ladder.
Northern blot analysis.
Total cellular RNA was denatured with formamide and formaldehyde, and resolved by gel electrophoresis on 1·25 % Seakem Gold agarose gel (BioWhittaker Molecular Application). The level of RNA in each lane was examined by staining the gel with SYBR Green II RNA gel stain (BioWhittaker Molecular Application). RNA was transferred to a nitrocellulose membrane, which was cut into three sections and hybridized with probes spanning mrxA, the intergenic region between mrxA and mrxC, or mrxC.
Extraction of surface molecules.
For preparation of fimbriae, overnight cultures (100 µl) were spread on nutrient agar plates and incubated at 30 °C for 48 h. Cells were harvested by repeated resuspension in 0·5 ml PBS (1 ml total). The cell suspension was vortexed vigorously for 30 s and centrifuged at 15 000 g for 10 min at 24 °C. The supernatants containing released fimbriae were spun for 14 min at 353 000 g at 4 °C. The resulting fimbrial pellet was resuspended in 20 µl SDS loading buffer, incubated at 24 °C for over 1 h and loaded onto a 15 % SDS-PAGE gel. For N-terminal sequence analysis of MrxA, the protein was transferred onto Immobilon-P membranes (Millipore). Amino acid sequence analysis was carried out at the Protein and Nucleic Acid Facility of the Medical College of Wisconsin. To examine production of fimbriae and flagella under broth conditions, cultures were grown in LB broth. Cells were pelleted and processed as described above.
Examination of fimbrial production by electron microscopy.
A 10 µl sample of bacterial suspension (see above) was placed on a 400-mesh copper-coated grid, and cells were allowed to settle for 30 s. After removing the liquid, one drop 0·8 % phosphotungstic acid (pH 6·5) was placed on the grid, which was incubated for 30 s, and then processed for electron microscopy.
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RESULTS |
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Processing of mrx transcripts
To analyse further the regulation of the mrx operon, Northern blot analysis was carried out with total cellular RNA prepared from cells grown on nutrient agar. Probes directed to mrxA, the mrxAC intergenic region and to mrxC were used in the analysis (Fig. 7). A major RNA transcript of approximately 940 nt was detected with the mrxA probe (Fig. 7
, lane a). The size of this transcript was consistent with the production of an mRNA initiating at the
70 promoter upstream of mrxA and terminating in the mrxAC intergenic region. In contrast, RNA transcripts were not detected with either the mrxAC (lane b) or the mrxC (lane c) probes. These results indicated that mrxA RNA was expressed at high levels in cells growing on agar, while polycistronic mRNAs were not present at levels that could be detected by Northern blot analysis.
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DISCUSSION |
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Results from Northern blot analysis and RT-PCR indicated that mrxA mRNA was highly abundant in cells grown on agar and that a polycistronic mrx mRNA was present at significantly lower levels. Tandem inverted repeats were identified downstream of mrxA. Transcription initiation at the mrxA promoter and partial transcription termination in the inverted repeat region could result in high levels of mrxA mRNA production relative to the downstream genes. Besides functioning in transcription termination, the inverted repeats may form stemloop structures at the 3' end of the mrxA RNA, thereby protecting against mRNA degradation by exonucleases. In the pap and sfa fimbrial operons of E. coli, mRNA transcripts for the major subunit genes are abundant, while the transcripts of the downstream genes are detected at lower levels. The high ratio of major to minor subunit transcripts is achieved by the combination of early transcription termination, post-transcriptional RNA processing and differential stabilization of cleaved RNA (Bäga et al., 1987, 1988
; Hacker & Morschhäuser, 1994
). The high abundance of mrxA transcripts and low level of polycistronic mrx mRNA may be achieved by similar mechanisms.
We have shown that the mrx operon is not regulated by promoter inversion or by regulatory genes linked to the structural operon. The finding that fimbriae are not produced in an lrp mutant strain, and that putative Lrp-binding sites are present upstream of mrxA, suggests that Lrp may function as a positive regulator of the mrx operon. Whether mrx transcription is directly controlled by Lrp and/or other regulatory proteins remains to be determined. Unlike the pap, fim and mrp operons, mrx is highly expressed in cells grown on solid agar surfaces. In P. mirabilis, the switching of the mrpA promoter to the OFF position accounts for the lack of fimbrial production in cells grown on agar (Zhao et al., 1997). In Salmonella typhimurium, the expression of fimW, located at the 3' end of the fim operon, increases in cells grown on agar, and a fimW mutant produced higher levels of fimbriae than the wild-type (Tinker et al., 2001
). These results indicate that FimW functions as a negative regulator of the fim operon in cells grown on agar. Sal. typhimurium and E. coli can produce fimbrial structures called curli fibres when grown on agar surfaces at lower temperature (Römling et al., 1998
). The mechanism by which curli is regulated is complex (Prigent-Combaret et al., 2001
), and the environmental signals that stimulate production on agar surfaces are not well understood. The Cpx two-component signal transduction system has recently been shown to play a key role in surface sensing and adhesion (Otto & Silhavy, 2002
). It is conceivable that Mrx fimbrial production involves a surface-sensing regulatory mechanism that is activated by growth on solid surfaces.
Xenorhabdus cells produce fimbriae when they inhabit the nematode intestinal vesicle (Binnington & Brooks, 1993), but are motile within the insect cadaver (Forst & Nealson, 1996
). Motility can be initiated in vitro during late exponential phase growth (Forst & Boylan, 2002
; Kim et al., 2003
). These findings suggest that the differential regulation of cell surface adhesion and swimming motility plays an integral role in the life cycle of Xenorhabdus. MrxJ may be involved in the coordinate regulation of fimbrial and flagellar synthesis, since it shares significant sequence identity with MrpJ, which functions to repress flagellar synthesis in P. mirabilis when Mrp fimbriae are produced (Li et al., 2001
). We envision that fimbriae are produced late in the infectious cycle and are present on the bacterial surface when the dauer juvenile stage of the nematode is being colonized by Xenorhabdus. Future studies on the Mrx fimbriae will be directed at addressing the following questions: what is the mechanism by which the mrx operon is regulated, and what role do Mrx fimbriae play in the colonization of the nematode partner? These studies should provide novel ideas on the function of fimbriae and their regulation in pathogenic and symbiotic bacteria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bäga, M., Norgren, M. & Normark, S. (1987). Biogenesis of E. coli Pap pili: PapH, a minor pilin subunit involved in cell anchoring and length modulation. Cell 49, 241251.[Medline]
Bäga, M., Göransson, M., Normark, S. & Uhlin, B. E. (1988). Processed mRNA with differential stability in the regulation of E. coli pilin gene expression. Cell 52, 197206.[CrossRef][Medline]
Bailey, T. & Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. In Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 2836. Menlo Park, California: AAAI Press.
Binnington, K. C. & Brooks, L. (1993). Fimbrial attachment of Xenorhabdus nematophilus to the intestine of Steinernema carpocapsae. In Nematodes and the Biological Control of Insect Pests, pp. 147155. Edited by R. Bedding, R. Akhurst & H. Kaya. Melbourne, Australia: CSIRO Publications.
Blomfield, I. C. (2001). The regulation of Pap and type 1 fimbriation in Escherichia coli. Adv Microb Physiol 45, 149.[Medline]
Connell, H., Agace, W., Klemm, P., Schembri, M., Mårild, S. & Svanborg, C. (1996). Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract. Proc Natl Acad Sci U S A 93, 98279832.
Forst, S. & Nealson, K. (1996). Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol Rev 60, 2143.[Medline]
Forst, S. & Boylan, B. (2002). Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila. Antonie van Leeuwenhoek 81, 4349.[CrossRef][Medline]
Forst, S. & Clarke, D. (2002). Bacteria-nematodes symbiosis. In Entomopathogenic Nematology, pp. 5777. Edited by R. Gaugler. London: CABI Publishing.
Forst, S., Dowds, B., Boemare, N. & Stackebrandt, E. (1997). Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu Rev Microbiol 51, 4772.[CrossRef][Medline]
Gally, D. L., Bogan, J. A., Eisenstein, B. I. & Blomfield, I. C. (1993). Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media. J Bacteriol 175, 61866193.[Abstract]
Girardeau, J. P., Bertin, Y. & Callebaut, I. (2000). Conserved structural features in Class I major fimbrial subunits (pilin) in gram-negative bacteria. Molecular basis of classification in seven subfamilies and identification of intrasubfamily sequence signature motifs which might be implicated in quaternary structure. J Mol Evol 50, 424442.[Medline]
Hacker, J. & Morschhäuser, J. (1994). S and F1C fimbriae. In Fimbriae: Adhesion, Genetics, Biogenesis and Vaccines, pp. 2736. Edited by P. Klemm. Boca Raton, FL: CRC Press.
He, H. (2002). Functional analysis of the mannose resistant fimbrial operon, mrx, in Xenorhabdus nematophila. PhD thesis, University of Wisconsin-Milwaukee.
Hernday, A., Krabbe, M., Braaten, B. & Low, D. (2002). Self-perpetuating epigenic pili switches in bacteria. Proc Natl Acad Sci U S A 99, 1647016476.
Heungens, K., Cowles, C. E. & Goodrich-Blair, H. (2002). Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes. Mol Microbiol 45, 13371353.[CrossRef][Medline]
Kim, D., Boylan, B., George, N. & Forst, S. (2003). Inactivation of ompR promotes precocious swarming and flhDC expression in Xenorhabdus nematophila. J Bacteriol 185, 52905294.
Li, X. & Mobley, H. L. T. (1998). MrpB functions as the terminator for the assembly of Proteus mirabilis mannose-resistant Proteus-like fimbriae. Infect Immun 66, 17591763.
Li, X., Rasko, D. A., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. T. (2001). Repression of bacterial motility by a novel fimbrial gene product. EMBO J 20, 48544862.
Low, D., Braaten, B. & Woude, M. V. D. (1996). Fimbriae. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 146157. Edited by C. N. Frederick. Washington, DC: American Society for Microbiology.
Meslet-Cladiere, L. M., Pimenta, A., Duchaud, E., Holland, I. B. & Blight, M. A. (2004). In vivo expression of mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection. J Bacteriol 186, 611622.
Mol, O. & Oudega, B. (1996). Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli. FEMS Microbiol Rev 19, 2552.[CrossRef][Medline]
Moureaux, N., Karjalainen, T., Givaudan, A., Bourlioux, P. & Boemare, N. (1995). Biochemical characterization and agglutinating properties of Xenorhabdus nematophilus F1 fimbriae. Appl Environ Microbiol 61, 27072712.[Abstract]
Old, D. C. & Duguid, J. P. (1970). Selective outgrowth of fimbriate bacteria in static liquid medium. J Bacteriol 103, 447456.[Medline]
Otto, K. & Silhavy, T. (2002). Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci U S A 99, 22872292.
Prigent-Combaret, C., Brombacher, E., Vidal, O., Ambert, A., Lejeune, P., Landini, P. & Dorel, C. (2001). Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J Bacteriol 183, 72137223.
Römling, U., Bian, Z., Hammer, M., Sierralta, W. D. & Normark, S. (1998). Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation. J Bacteriol 180, 722731.
Soto, G. E. & Hultgren, S. J. (1999). Bacterial adhesins: common themes and variations in architecture and assembly. J Bacteriol 181, 10591071.
Stabb, E. V. & Ruby, E. G. (2003). Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri. Appl Environ Microbiol 69, 820826.
Stentebjerg-Olesen, B., Chakraborty, T. & Klemm, P. (1999). Type 1 fimbriation and phase switching in a natural Escherichia coli fimB null strain, Nissle 1917. J Bacteriol 181, 74707478.
Tinker, J. K., Hancox, L. S. & Clegg, S. (2001). FimW is a negative regulator affecting type 1 fimbrial expression in Salmonella enterica serovar typhimurium. J Bacteriol 183, 435442.
Tullus, K., Kuhn, I., Orskov, I., Orskov, F. & Mollby, R. (1992). The importance of P and type 1 fimbriae for the persistence of Escherichia coli in the human gut. Epidemiol Infect 108, 415421.[Medline]
Vivas, E. I. & Goodrich-Blair, H. (2001). Xenorhabdus nematophilus as a model for host-bacterium interactions: rpoS is necessary for mutualism with nematodes. J Bacteriol 183, 46874693.
Webster, J. M., Chen, G., Hu, K. & Li, J. (2002). Bacterial metabolites. In Entomopathogenic Nematology, pp. 99114. Edited by R. Gaugler. London: CABI Publishing.
Zhao, H., Li, X., Johnson, D. E., Blomfield, I. & Mobley, H. L. T. (1997). In vivo phase variation of MR/P fimbrial gene expression in Proteus mirabilis infecting the urinary tract. Mol Microbiol 23, 10091019.[Medline]
Zhou, X., Kaya, H., Heungens, K. & Goodrich-Blair, H. (2002). Response of ants to a deterrent factor(s) produced by the symbiotic bacteria of entomopathogenic nematodes. Appl Environ Microbiol 68, 62026209.
Received 21 October 2003;
accepted 23 December 2003.
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