A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface

G. W. P. Joshua1, A. V. Karlyshev1, M. P. Smith2, K. E. Isherwood2, R. W. Titball1,2 and B. W. Wren1

1 London School of Hygiene and Tropical Medicine, Dept Infectious and Tropical Diseases, Keppel St, London WC1E 7HT, UK
2 DSTL, Porton Down, Salisbury SP4 0JQ, UK

Correspondence
B. W. Wren
Brendan.wren{at}lshtm.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9·5 % an intermediate infection, and 9·5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.


Abbreviations: EM, electron microscopy


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The outcome of infection by pathogenic bacteria depends on virulence factors expressed by the bacteria, the existence of corresponding host targets and host responses to these factors. The usual models for studying these interactions (mouse, mammalian cell culture) are genetically unwieldy and do not lend themselves to experimental analysis. Also, there are cost and ethical restraints on the use of laboratory mammals. The idea of using a non-mammalian and genetically tractable host organism is therefore attractive. It has been demonstrated that yeast (Saccharomyces cerevisiae), slime moulds (Dictyostelium discoideum), plants (Arabidopsis thaliana), fruit flies (Drosophila melanogaster) and the nematode Caenorhabditis elegans can be infected by human-pathogenic and other bacteria (reviewed by Strauss, 2000). Moreover, the mechanisms of invasion and host responses may be paralleled in mammalian cells.

In the wild, C. elegans is a free-living, soil-dwelling nematode feeding on micro-organisms. In the laboratory it is maintained on a uracil auxotrophic strain (OP50) of Escherichia coli, which has limited growth on NGM plates (Lewis & Fleming, 1995). A number of bacteria infect C. elegans, with various infection phenotypes. These include a variety of plant and animal pathogens such as Erwinia chrysanthemi, Agrobacterium tumefaciens, Shewanella frigidimarina, Photorhabdus luminescens, Xenorhabdus nematophilus (Couillault & Ewbank, 2002) and ‘Microbacterium nematophilum (Hodgkin et al., 2000). They also include bacteria pathogenic to humans, including the Gram-negative bacteria Pseudomonas aeruginosa, which kills the worm by one of three mechanisms – fast, slow or neurotoxin-mediated (Tan et al., 1999; Darby et al., 1999), Burkholderia pseudomallei, which kills via a neuromuscular endotoxin (O'Quinn et al., 2001), and Serratia marcescens, which also kills via a (different) toxin (Kurz & Ewbank, 2000); and the Gram-positive bacteria Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus (Garsin et al., 2001).

We have investigated the effect of Yersinia spp. on C. elegans. The pathogenic yersiniae are an important cause of human disease, Yersinia pestis causing bubonic and pneumonic plague (Perry & Fetherston, 1997), and Yersinia enterocolitica and Yersinia pseudotuberculosis causing food-borne enteric disease. Y. enterocolitica causes gastrointestinal syndromes ranging from an acute enteritis to mesenteric lymphadenitis and Y. pseudotuberculosis causes mesenteric lymphadenitis and septicaemia (Naktin & Beavis, 1999). During bubonic plague Y. pestis is transmitted via an insect (flea) vector; in contrast, the enteropathogenic yersiniae are transmitted by the faecal/oral route and survive in water and soil. The yersiniae are model organisms to study bacterial pathogenesis. There are well-developed systems for genetic manipulation (e.g. transposon mutagenesis) and animal models of infection (e.g. the murine yersiniosis model). The full genome sequences of strains belonging to Y. pestis biovars Orientalis and Medievalis have been completed (Parkhill et al., 2001; Deng et al., 2002), and the genome sequences of Y. pseudotuberculosis and Y. enterocolitica O8 are near completion (http://bbrp.llnl.gov/bbrp/html/microbe.html and http://www.sanger.ac.uk/Projects/Y_enterocolitica). Moreover, Y. pseudotuberculosis and Y. pestis share near-identical sequence similarity but cause remarkably different diseases and have different routes of transmission, whereas Y. enterocolitica shares only 70 % sequence similarity with Y. pseudotuberculosis and Y. pestis but exhibits a disease phenotype similar to that of Y. pseudotuberculosis.

All of the human-pathogenic yersiniae possess plasmid pYV and most of the pathogenicity studies on yersiniae have largely focused on determinants found on this plasmid, which encodes the type III secretion system essential for virulence. Y. pestis possesses two additional plasmids (pMT1 and pPCP1) which also encode key virulence determinants. In addition Y. pestis possesses a number of inactivated genes, which may no longer be required for its lifestyle (Parkhill et al., 2001). Achtman et al. (1999) have suggested that Y. pestis is a recently emerged clone of Y. pseudotuberculosis, evolving between an estimated 1500 and 20 000 years ago. Studies on the pathogenic yersiniae may, therefore, shed light on the evolutionary relationships between these three species and the mechanisms by which they survive in the environment, occupy new niches and cause disease.

In a recent report Darby et al. (2002) observed that both Y. pestis and Y. pseudotuberculosis infected C. elegans, colonizing the head of the worm and blocking feeding. It was suggested that this was due to the formation of a biofilm on the head/mouth of the worm as a defence against predation by invertebrates. Biofilms are matrix-encased bacterial communities that are specialized for surface persistence. They are more resistant to antibiotics, chlorine and components of the host innate immune system (Drenkard & Ausubel, 2002; Singh et al., 2002; Yildiz & Schoolnik, 1999). Additionally, biofilm formation has been shown to be important in bacterial–fungal interactions, where virulence factors studied in the context of human infection may also have a role during interactions in the bacterial–fungal ecological niche (Hogan & Kolter, 2002; Mendoza De Gives et al., 1999).

In this study we sought to characterize the C. elegans infection caused by the three human-pathogenic Yersinia species and to identify genetic features of both the pathogen and the host which contribute to the establishment of disease in C. elegans.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial and C. elegans strains and mutants.
Y. pseudotuberculosis YPIII pIB1 was maintained at LSHTM; YPIII pIB1 strains plus (red) and minus (white) the pYV plasmid were isolated from Congo red-magnesium oxalate agar (CRMOX) plates (Riley & Toma, 1989). The presence of the plasmid in red colonies was confirmed by plasmid preparation and restriction endonuclease digestion; absence of pYV in white colonies was also confirmed. Presence or absence of pYV in all strains was also ascertained by examination of growth on CRMOX plates and by PCR analysis using primers yscP-1 (5'-ATTAGAACCTGAGTATCAACC-3') and yscP-2 (5'-AACAAATAACTCATCATGTCC-3') to amplify a 466 bp internal fragment from the yscP gene (Table 1). YPIP32637 and a derivative lacking the haemin storage locus, YPIP32637 hms-, were from Dr M. Prentice (St Bartholomew's and the London Medical School, UK); other Y. pseudotuberculosis strains were from Dr H. Fukushima (The Shimane Prefectural Institute of Public Health and Environmental Science, 5821-1 Nishihamasada, Shimane 699-0122, Japan). Y. enterocolitica 8081 and WA314 were maintained at LSHTM. Other strains were from Dr T. Cheasty (Central Public Health Laboratories, Colindale, UK): E97125 (serogroup O3/biotype 4); E99007 (serogroup O3/biotype 4); E94840 (serogroup O5/biotype 1a); E99191 (serogroup O9/biotype 3); E105689 (serogroup O9/biotype 3); E107919 (serogroup O9/biotype 3); E108325 (serogroup O9/biotype 3); E109997 (serogroup O9/biotype 3); E110811 (serogroup O9/biotype 3); E96431 (serogroup O19/biotype 1b); E93482 (serogroup untypable/biotype 1a); and 10938 and 11175 (of unknown serogroup and biotype). Y. pseudotuberculosis and Y. enterocolitica were grown in Luria–Bertani (LB) medium at 28 °C. Y. pestis strains were grown in blood agar base (BAB) medium at 28 °C. Signature-tagged mutants were described in a previous study (Karlyshev et al., 2001), and were grown with kanamycin (50 µg ml-1). Overnight cultures of bacteria were seeded on NGM plates (Lewis & Fleming, 1995) and, for infection assays, 20 young adult C. elegans were aliquoted onto seeded plates and grown at 20 °C. Wild-type and mutant strains of C. elegans were maintained in the laboratory on E. coli OP50. Biofilm formation on polystyrene, that is on an abiotic surface, was measured as described by Nesper et al. (2001) with minor modifications. Briefly, bacteria were grown overnight in LB, diluted 1 : 10 in fresh LB, and 100 µl aliquots grown overnight in a round-bottom 96-well microtitre plate (Nunc) (in triplicate). Bacterial cultures were poured out, and bacteria attached to the wells were washed three times with water, fixed in 2·5 % glutaraldehyde, washed twice with water and stained with 0·25 % crystal violet. After washing three times, adherent stained bacteria were solubilized with 2x200 µl ethanol/acetone (80 : 20, v/v) and the A570 read after making up the volume to 1 ml with water.


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Table 1. Y. pseudotuberculosis (designated YP) and Y. pestis strains tested in C. elegans

 
Mutant genes in the signature-tagged Y. pseudotuberculosis library were identified by amplifying DNA flanking the transposon using the single-primer PCR method described by Karlyshev et al. (2000). The amplified DNA was sequenced using internal primers P6M (5'-GCCAGATCTGATCAAGAGAC-3') and P7M (5'-GCCGAACTTGTGTATAAGAGTC-3').

Microscopy.
For EM (Fig. 2A, B, C), C. elegans were taken from plates and bisected under fixative (3 % glutaraldehyde/0·075 M cacodylate buffer; pH 7·4). Specimens were post-fixed in 1 % osmium tetroxide, dehydrated through an alcohol series and embedded in TAAB resin (TAAB Laboratories). Specimens in Fig. 2(D, E) were fixed in 2 % paraformaldehyde/1 % glutaraldehyde and cryosectioned. For light microscopy (Fig. 2F, G), C. elegans were fixed in 4 % paraformaldehyde, stained with 0·1 % crystal violet for 3 min and viewed with epi-illumination on 5 % agarose pads (Sulston & Horvitz, 1977).



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Fig. 2. (A) Bacterial mass (Yp) on head of C. elegans (b, buccal cavity; ph, pharynx). (B) Low-power electron micrograph of a transverse section of gut (g) containing Y. pseudotuberculosis, shown at higher magnification (Yp) in (C). (D) A transverse section of C. elegans (Ce) with bacteria (Yp) in an extracellular matrix (m) closely associated with the cuticle (c), shown at higher magnification in (E). (F) Bacterial mass and extracellular matrix stained with crystal violet (Yp/m) compared to C. elegans grown on E. coli (G). Scale bars in µm.

 

   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
C. elegans infection phenotype after infection with human-pathogenic yersinia
When C. elegans were fed with Y. pseudotuberculosis YPIII pIB1 on NGM plates, the bacteria colonized the worm, forming a discrete mass surrounding the mouth and anterior third of the worm. The worms also moved aberrantly, with an increase in body flexion similar to the movement of goa-1 mutants (Darby & Falkow, 2001) (Fig. 1). The worms were able to flex but became progressively unable to translocate from a stationary position. EM analysis of infected worms showed a dense mass of bacteria (Fig. 2A) enclosed in an extracellular matrix (Fig. 2D, E). The bacterial mass was stained with crystal violet as shown by light microscopy (Fig. 2F, G). The presence of intact bacteria in the gut (Fig. 2B, C), the survival of C. elegans when grown on the majority of strains of Y. pseudotuberculosis (Table 1), and the observation that when the C. elegans : Y. pseudotuberculosis ratio was high (large numbers of worms or small number of bacteria) bacteria were ingested but the nematodes remained unaffected (data not shown), provide evidence that C. elegans can utilize Y. pseudotuberculosis as a food source prior to the onset of infection. When a selection of 41 Y. pseudotuberculosis strains, representing 21 serotypes, were tested for their ability to infect C. elegans, it was found that most strains (76 %) did not infect (Table 1). In strains which did infect, the infection could be differentiated into three phenotypes: severe (9·5 %), when worms developed the anterior biofilm, showed initial aberrant motion but were subsequently unable to translocate (that is, move around the plate; as shown by Y. pseudotuberculosis YPIII pIB1, used in our initial studies); intermediate (9·5 %), when worms developed the anterior bacterial mass but were able to move around the plate in an aberrant manner; or weak (5 %), when worms did not develop a visible biofilm but exhibited aberrant movement (Table 1). Whereas the hms- mutant of strain YPIP32637 did not infect C. elegans, wild-type YPIP32637 caused a weak infection. There was no consistent relationship between serotype and presence or severity of infection.



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Fig. 1. (A) C. elegans fed on Y. pseudotuberculosis YPIII pIB1with colonization by the bacteria (Yp) over the head and anterior end; (B) C. elegans grown on E. coli OP50. (C) Aberrant movement by infected C. elegans is shown by tight turns in tracks (tr), compared to the normal sinusoidal movement of worms on E. coli OP50 (D). Scale bars in µm.

 
We also tested the same strains for their ability to form a biofilm on an abiotic (polystyrene) surface. Twenty-one per cent were able to do so (A570>0·1; see Table 1), to varying degrees. Similarly, there was no consistent relationship between strains causing biofilm formation on biotic or abiotic surfaces. The pYV plasmid had no effect on biofilm formation either on C. elegans or on polystyrene. C. elegans was equally infected with a severe infection phenotype by YPIII pIB1 plus or minus the pYV plasmid or prototype YPIII pIB1, cultures of which contained a proportion of bacteria minus pYV. Similarly, YPIII pIB1 plus or minus pYV or prototype YPIII pIB1 formed a minimal biofilm (A570 0·066–0·092) on polystyrene (Table 1).

Three strains of Y. pestis (virulent biovar Orientalis strains GB and CO92, and the biovar Orientalis strain Java-9, which lacks plasmid pMT1) caused a severe infection. In contrast, the hms- Y. pestis F361-66 strain (which has lost the 102 kb hms/yersinabactin locus and is attenuated in the mouse) did not infect C. elegans (Table 1). All 15 of the Y. enterocolitica strains tested (see Methods) had no effect on C. elegans.

Bacterial virulence factors
Karlyshev et al. (2001) recently tested 603 signature-tagged Y. pseudotuberculosis YPIII pIB1 transposon mutants in the murine yersiniosis model and identified 39 attenuated mutants. Of the 39 strains which were attenuated in the mouse, six showed altered pathogenicity in C. elegans, with two showing complete attenuation and four showing a change from a severe to a weak infection (see Table 2). Additionally, a defined phoP-negative Y. pseudotuberculosis YPIII pIB1 strain was tested; it caused a severe infection phenotype, suggesting that the PhoP/Q regulatory locus (Groisman, 2001) is not important in biofilm formation (data not shown). Two hundred randomly selected Y. pseudotuberculosis YPIII pIB1 transposon mutants were also tested in C. elegans, and a single mutant was identified which showed complete attenuation. Sequencing the DNA flanking the transposon of this mutant (Karlyshev et al., 2000) showed that the hmsF gene had been interrupted.


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Table 2. Genes disrupted in Y. pseudotuberculosis mutants with altered pathogenicity

 
Host resistance factors
As well as the N2 wild-type we also tested seven other C. elegans wild-type strains (AB1, CB4852, CB4855, CB4932, DH424, KR314, RW7000; for strain descriptions see the Caenorhabditis Genetics Center website: http://biosci.umn.edu/CGC/CGChomepage.htm) and 15 defined C. elegans mutants; mutations in genes related to feeding, movement and surface recognition were selected (lin-4, lin-12, eat-1, eat-2, eat-10, lon-1, egl-9, mab-1, srf-4, srf-8, srf-9, srf-2, srf-3, srf-5 and daf-1; Hodgkin et al., 1988, 2000). All wild-type strains of C. elegans were infected with Y. pseudotuberculosis YPIII pIB1, with a severe infection phenotype. When the defined mutants were tested with Y. pseudotuberculosis YPIII pIB1, 11 showed a severe infection phenotype. However, exposure of C. elegans srf-2, srf-3, srf-5 or daf-1 mutants to Y. pseudotuberculosis YPIII pIB1 did not result in the appearance of any signs of infection; no biofilm formed on the worms, and they moved and fed as normal.


   DISCUSSION
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INTRODUCTION
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Darby et al. (2002) recently reported the blockage of feeding of C. elegans feeding on Y. pseudotuberculosis or Y. pestis and postulated that this was the result of the formation of a biofilm over the anterior of the worm. We provide evidence supporting this finding and present additional data on the mechanism of biofilm formation, on the infection phenotype and on the genetic control of biofilm formation.

Bacterial biofilms are aggregates of bacteria in a hydrated polymeric matrix of their own synthesis (Costerton et al., 1999). They may form on abiotic or biotic surfaces and such biofilms may influence the properties of pathogens. This is exemplified by P. aeruginosa, as the biofilm mode of growth confers antibiotic resistance within the lungs of patients with cystic fibrosis (Costerton et al., 1995). A frequently used assay for biofilm formation on abiotic surfaces is to grow the bacteria on polystyrene and then measure the absorbance after staining with crystal violet (Nesper et al., 2001; Solano et al., 2002); this assay is not appropriate for measurement of biofilm formation on a biotic surface such as C. elegans. We have shown the presence of an extracellular matrix containing bacteria on C. elegans infected with Y. pseudotuberculosis by EM (Fig. 2D, E) and stained the bacterial mass with crystal violet (Fig. 2F, G), and periodic acid–Schiff (data not shown). These findings strongly suggest the formation of a biofilm around C. elegans infected with Y. pestis or Y. pseudotuberculosis.

Neither Y. pestis nor Y. pseudotuberculosis has previously been reported to form biofilms on abiotic surfaces; we found that in repeated experiments in triplicate, a subset of Y. pseudotuberculosis strains are able to form a biofilm to varying degrees on polystyrene (Table 1). It is noteworthy that there is no relationship between strains forming a biofilm on polystyrene and on C. elegans; the formation of the biofilm on C. elegans appears to be an interactive process involving both bacterial and host recognition molecules. We have identified a number of bacterial genes involved in the colonization of C. elegans, and a number of C. elegans mutants refractory to infection. However, although the srf genes conferring the resistance phenotype have been mapped, they have not yet been assigned to a sequence or cloned. We are currently investigating the identity of those genes.

The C. elegans srf-2, srf-3 and srf-5 mutants, which were resistant to infection, that is in which biofilm formation did not occur, show ectopic binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA) (Link et al., 1992; Politz et al., 1990) and their role in the formation of the biofilm remains to be determined. These mutants are also resistant to infection by ‘M. nematophilum’, in which the normal infection is a localized raised lesion (Hodgkin et al., 2000); it does not form a biofilm. It is likely that Y. pestis, Y. pseudotuberculosis and ‘M. nematophilum’ attach via related molecular targets on the nematode surface. It is noteworthy that srf-2, srf-3 and srf-5 are otherwise wild-type, whereas srf-4, srf-8 and srf-9, which are not resistant to infection by either Y. pseudotuberculosis or ‘M. nematophilum’ (Hodgkin et al., 2000) have other defects including defective copulatory bursae development, uncoordinated movement and abnormal egg laying. daf-1, mutants of which were also resistant to Y. pseudotuberculosis infection, encodes a receptor tyrosine kinase forming part of the dauer-formation pathway which includes the srf-6 gene (Grenache et al., 1996).

In several bacterial species, biofilm formation has been shown to be under genetic control. A microarray analysis of free-living and sessile P. aeruginosa showed 34 genes activated and 39 genes repressed in biofilm populations (Whiteley et al., 2001). Pseudomonas fluorescens biofilm formation was abrogated in strains with mutations in the clpP gene, which encodes a subunit of the cytoplasmic Clp protease (O'Toole & Kolter, 1998). In Salmonella enteritidis, mutation of the bcsABZC or bcsEFG gene clusters, which are required for the synthesis of an exopolysaccharide, prevents biofilm formation. In Y. pseudotuberculosis, mutation of the hmsT gene similarly prevents biofilm formation on C. elegans (Darby et al., 2002). It is known that HmsT is a positive regulator of the hmsHFRS operon in Y. pestis, and homologues of HmsF and HmsR are required for the synthesis of polysaccharide components of biofilms of Staphylococcus epidermidis (Gerke et al., 1998). We have also identified a Y. pseudotuberculosis YPIII pIB1 hms gene (hmsF; Table 2) as necessary for biofilm formation. Biofilm formation was also absent in a Y. pseudotuberculosis IP32637 defined hms-negative mutant. In addition, we have shown that a pigmentation-negative strain of Y. pestis (F361-66), which lacks the hms locus, is unable to cause biofilm formation in C. elegans. It may be significant that the blockage of fleas by Y. pestis is also dependent on the hms locus.

The murine lethal toxin (phospholipase D) encoded on the 100 kb plasmid pMT1 in Y. pestis also plays a role in the survival of Y. pestis in the midgut of fleas (Hinnebusch et al., 2002). However, pMT1 does not appear to be important for infection of C. elegans since the Y. pestis Java-9 strain that lacks this plasmid (confirmed by DNA microarray analysis; S. J. Hinchliffe & B. W. Wren, unpublished data) readily caused biofilms. By contrast, Y. pestis F361-66, which lacks the hms locus, failed to block C. elegans and would, presumably, be unable to block fleas.

We have identified six further genes in which disruption prevents or reduces biofilm formation (Table 2); two of these relate to lipopolysaccharide (LPS) biosynthesis, although the mutant 4H9 has reduced viability and lack of biofilm formation may not be solely due to reduced LPS biosynthesis. Mutation of galU and galE in Vibrio cholerae causes production of an altered LPS core oligosaccharide; mutants are unable to synthesize exopolysaccharide (VPS) or to form a biofilm (Nesper et al., 2001). Clearly bacterial polysaccharides are intrinsic to the formation of the biofilm in the Y. pseudotuberculosis/C. elegans infection model, as they are in biofilms formed by other bacterial species.

We do not know, as yet, the relevance of the sensory transduction gene (1G6-1) in biofilm formation in Y. pseudotuberculosis YPIII pIB1, nor of the two genes (2H10 and 1C9) with no similarity at the protein level to published sequences.

The C. elegans/Y. pseudotuberculosis and C. elegans/Y. pestis infection models may be useful in the investigation of two areas of nematode/bacterial biology. As discussed above, Y. pseudotuberculosis and Y. pestis share 97 % sequence similarity yet cause markedly different disease in humans. Although the virulence of Y. pestis is conferred largely by the pYV, pPCP1 and pMT1 plasmids, the mechanism by which Y. pestis causes blockage in the flea following formation of a biofilm while Y. pseudotuberculosis can form a biofilm (on C. elegans), but cannot block the flea remains to be determined. It may relate to the second area of interest: host interaction. Acylhomoserine lactones (AHLs), which mediate quorum sensing in Gram-negative bacteria, are constituitively produced by Y. pseudotuberculosis (Atkinson et al., 1999; S. Atkinson, personal communication) but biofilms are not normally formed. It is a tenable hypothesis that quorum-sensing processes followed by biofilm formation come into operation when bacteria are co-localized by receptor-mediated binding on the worm surface. As such, the identities of srf-2, srf-3, srf-5 and daf-1 are of interest. Human orthologues may be relevant to clinical disease where bioflms form, e.g. P. aeruginosa in cystic fibrosis patients; and flea orthologues may be important in flea blockage. The C. elegans/Yersina spp. model therefore represents a useful experimental system to investigate the genetic and biochemical basis of biofilm formation by bacteria on a biotic surface, and the evolution of Y. pestis from Y. pseudotuberculosis.


   ACKNOWLEDGEMENTS
 
We thank Dr H. Fukushima (The Shimane Prefectural Institute of Public Health and Environmental Science, 5821-1 Nishihamasada, Shimane 699-0122, Japan) and Dr M. Prentice (St Bartholomew's and the London Medical School, UK) for Y. pseudotuberculosis strains; and Dr T. Cheasty (Central Public Health Laboratories, Colindale, UK) for Y. enterocolitica strains. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center (1445 Gortner Ave, St Paul, MN 55108-1095), which is funded by the NIH National Centre for Research Resources. We acknowledge financial support from DSTL.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 9 May 2003; revised 22 July 2003; accepted 28 July 2003.