Vol. 174, No. 6

JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 2047-2051 0021-9193/92/062047-05$02.00/0 Copyright © 1992, American Society for Microbiology

LcrF Is the Temperature-Regulated Activator of the yadA Gene of Yersinia enterocolitica and Yersinia pseudotuberculosis M. SKURNIK* AND P. TOIVANEN Department of Medical Microbiology, Turku University, Kiinamyllynkatu 13, 20520 Turku, Finland Received 15 November 1991/Accepted 17 January 1992

The virulence plasmid of human pathogenic Yersinia species, pYV, encodes secreted proteins, Yop proteins, and an outer membrane protein, YadA. YadA has been associated with binding to a variety of substrates and with interference with host defense. YadA is regulated by temperature and is expressed only at 37°C. Unlike the yop regulon, the yadA gene is not under Ca2" regulation. Here, we show that LcrF (VirF), the temperature-regulated activator of the yop regulon, also acts as an activator for yadA.

The virulence plasmid of human pathogenic Yersinia species, pYV, encodes an outer membrane protein, YadA (for Yersinia adhesin, previously known as Yopl or P1), associated with several binding phenomena. YadA has been shown to mediate (i) autoagglutination (3, 29), (ii) mannose-resistant guinea pig hemagglutination (16), (iii) binding of bacteria onto epithelial cells (8, 15, 24), (iv) binding of bacteria to soluble collagen (11), (v) binding of bacteria to immobilized fibronectin (32), (vi) resistance to normal serum (3, 18, 28), (vii) binding of bacteria to intestinal brush border vesicles (21), (viii) binding to mucin (22), and (ix) colonization of mouse intestines (17). The YadA protein is expressed by Y. enterocolitica and Y. pseudotuberculosis grown at 37°C but not at 25°C (5, 16). Because of a 1-bp deletion early in the yadA gene, it is not expressed in Y. pestis (31). In addition, YadA expression is affected by the growth medium such that highest expression is achieved in a poor medium (16). The yadA gene has been sequenced, and the transcription start point has been determined (31). The open reading frame and transcription start are separated by a 270-nucleotide-long leader sequence (31), which may play a role in the temperature regulation of YadA expression. Moreover, the promoter region of theyadA gene is very A+T rich, a phenomenon typical of DNA regions where regulatory proteins bind after temperature-induced topological changes have taken place. YadA expression responds very rapidly to temperature shift to 37°C, so that as quickly as 2 min after a 22 to 37°C shift, YadA expression can be detected by pulse-labeling (5). The cloned yadA gene is poorly or not at all expressed in E. coli (3, 6), suggesting that some Yersinia-specific factor(s) regulates expression. In this report, we describe a set of bacterial strains, the construction of which allowed the investigation of interactions of the yadA gene with other factors. Standard methods for manipulating DNA were used (2). Bacterial strains and plasmids used in this work are described in Table 1. Detection of YadA by the autoagglutination (AA) test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done as follows. One bacterial colony of the strain to be tested was suspended into 300 to 500 ,ul of distilled water, after which 50-,u samples were inoculated into two tubes containing 2 ml of MedECa (0.1 g of MgSO4. 7H20, 2 g of citric acid, 10 g of K2HPO4,

and 3.5 g of NaNH4HPO4 4H20 per liter, containing 0.2% glucose, 0.2% Casamino Acids, 1 mg of vitamin B12 per liter, and 5 mM CaCl2) each (27). A higher dose of the Y. pseudotuberculosis strains was inoculated to achieve growth in MedECa. The paired tubes were incubated stationary overnight, one at 37°C and the other at 22°C. The AA test was regarded as positive when bacteria in the 37°C tube formed a flocculate pellet with a clear supernatant, and when, simultaneously, the medium in the 22°C tube was turbid throughout. Bacteria in the tubes were centrifuged and solubilized into SDS-PAGE sample buffer at 37°C (> 1 h) to maintain most of YadA in its polymerized form (29), TABLE 1. Bacteria and plasmids used in this study Name of strain or plasmid

Bacteria Y enterocolitica 6471/76 (YeO3) 6471/76-c (YeO3-c)

Reference or source

26 Serotype 0:3, patient isolate Virulence plasmid-cured deriva- 26 tive of 6471/76

Y pseudotuberculosis

YPIII/pIBl YPIII E. coli S17-1 C600

5, 13 Serotype III, patient isolate Virulence plasmid-cured deriva- 5 tive of YPIII/pIB1 Mobilization host Cloning host

25 1

Virulence plasmid of YeO3 Virulence plasmid of YPIII/

27 5

Plasmids

pYV03 pIBl pIB102 pYMS4418

pYMS4448 pYMS4232 pYMS4514 pBR322 pTM100

*

Comments

Corresponding author. 2047

pIBl 6 pIBl-yad4::TnS 4.5-kb SphI fragment of pYV03 This work containingyadA cloned into pBR322 This work 4.5-kb SphI fragment of pYMS4418 cloned into pTM100 8.7-kb ClaI fragment of pYV03 This work containing AlcrF, IcrLMC, and lcrK This work IcrF of pYV03 cloned into pYMS4448 7 Cloning vector 19 Mobilizable derivative of pACYC184

2048

NOTES

J. BACTERIOL.

(pM1u~

Yxp

c

BTH-fekd

BamXnH-floroked)

,

SphLlS sc-reenfor clmR. teS

N

FIG. 1. Cloning and construction strategy for plasmids. The YeO3 yadA gene in different plasmids is indicated by filled lines. EcoRI fragments 7 and 8 of pYV03, used as probes to screen the ClaI libraries (see text), are located to the right half of pYMS4232 containing the 8.7-kb ClaI fragment of pYV03. Abbreviations used in this figure: B, BamHI; BAP, bacterial alkaline phosphatase treatment; bla, P-lactamase gene; C, ClaI; clm, chloramphenicol resistance gene; E, EcoRI; EV, EcoRV; H, HindIII; 1cr, low calcium response; N, NsiI; oriT, onT of plasmid RK2; Ps, PstI; Pv, PvuI; Sa, Sall; Sc, Scal; Sp, SphI; tet, tetracycline resistance gene; Xb, XbaI.

because of easier detection by SDS-PAGE. The SDS-PAGE procedure was performed by standard methods (2), and the gel was stained with Coomassie brilliant blue. For mobilization of plasmids, 25 VI each of overnight cultures of the donor and recipient strains was mixed and spread on an antibiotic-free Luria agar plate. The plate was then incubated overnight either at 22°C (for Yersinia strains) or at 37°C (for Escherichia coli strains). Bacteria from the plate were thereafter diluted serially and plated on selective agar. Successful mobilization was verified by plasmid isolation and restriction analysis. The mobilizable plasmid, pYMS4448, carrying the YeO3 yadA gene and its promoter region, was constructed as outlined in Fig. 1. The yadA gene was first cloned into pBR322 in a 4.5-kb SphI fragment to obtain plasmid pYMS4418. The same fragment was subsequently cloned

into pTM100, which is a mobilizable derivative of pACYC184 (19), to obtain plasmid pYMS4448 (Fig. 1). The YadA protein was not expressed by E. coli C600/pYMS4448 (Fig. 2) or by E. coli S17-1/pYMS4448 (Table 2). After negative results in both E. coli strains, pYMS4448 was mobilized from S17-1/pYMS4448 into a Yersinia background, i.e., into the pYV- Y. pseudotuberculosis YPIII, to test the possibility that this background allows YadA expression. However, YPIII/pYMS4448 was negative for YadA expression at both temperatures (Table 2). Since pYMS4448 carries the YeO3-derived yadA gene, an identical construct of the Y pseudotuberculosis yadA gene was tested, with identical results (data not shown). These observations suggested that the regulatory genes for the yadA gene expression are located on pYV, the Yersinia virulence plasmid. To assess this hypothesis, we mobilized pYMS4448 into

NOTES

VOL. 174, 1992 1

2

3

4

5

6

YadA _p

FIG. 2. Coomassie brilliant blue-stained SDS-polyacrylamide gel. Whole bacterial lysates were solubilized at 37°C and applied to the gel. Bacteria for lanes 1, 3, and 5 were grown at 22°C, and those for lanes 2, 4, and 6 were grown at 37°C. Lanes 1 and 2, C600/ pYMS4514; lanes 3 and 4, C600/pYMS4232/pYMS4448; lanes 5 and 6, C600/pYMS4448. The YadA band is indicated.

E. coli S17-1/pIB102 and Y. pseudotuberculosis YPIII/ pIB102. The pIB102 plasmid is a derivative of pIB1, in which TnS is inserted in the 5' end of the yadA gene (6). Thus, strains carrying pIB102 alone do not express YadA (Table 2). The S17-1/pIB102 construct was obtained by transforming S17-1 with pIB102. Both S17-1/pIB102/pYMS4448 and YPIII/pIB102/pYMS4448 expressed YadA in a temperatureregulated manner (Table 2), strongly indicating that the gene(s) encoding the transactingyadA-regulating factor(s) is located in pYV. TABLE 2. Detection of YadA by SDS-PAGE and AA test from different bacterial constructs

gene(s) Bacteria strainRelevant Bacterial strain present

YadA in SDS-

PAGE

AA

test

22°C 37°C E. coli

S17-1/pYMS4448 S17-1/pIB102 S17-1/pIB102/pYMS4448

C600/pYMS4232IpYMS4448 C600/pYMS4514

yadA yadA::TnS IcrLMC

lcrF yadA::TnS lcrLMC lcrFyadA yadA IcrLMC AlcrF yadA IcrF

Y pseudotuberculosis YPIII

YPIII/pIBl YPIII/pYMS4448

YPIII/pIB102 YPIII/pIB102/pYMS4448

-

-

-

-

-

-

-

+

+

-

+a +a

-

+

+

-

+

+

-

-

-

-

-

-

-

+

+

-

+ +

+ +

-

yadA IcrLMC IcrF yadA yadA::TnS lcrLMC IcrF yadA::TnS IcrLMC

lcrFyadA Y enterocolitica YeO3-c YeO3

yadA lcrLMC IcrF yadA lcrF YeO3-c/pYMS4514 a +, very weak positive; see text for details.

2049

To determine whether the IcrLMC locus of pYV (14) contains the regulatory genes of yadA expression, we constructed a strain carrying the yadA4 gene and the lcrLMC locus in trans. Plasmids carrying the lcrLMC locus were isolated from the ClaI libraries of pYV8081, pIB1, and pYVO3 (4, 31). From the known location of lcrLMC and the restriction map of pYVO3 (23) (Fig. 1), we deduced that EcoRI fragments 7 and 8 should be used as probes to screen the libraries. From each library, several hybridization-positive clones were obtained. Restriction analysis of the recovered plasmids showed that each carried an 8.7-kb ClaI fragment. The restriction map of one of these, pYMS4232 originating from YeO3, is shown in Fig. 1. Into these clones, pYMS4448 was mobilized. The result of one of the constructed strains, C600/pYMS4232/pYMS4448, is shown in Table 2; others were identical. The AA test was weakly positive; the 37°C AA test tube was turbid; however, faint agglutination was visible, indicating that a small amount of YadA might be expressed in this strain. Indeed, examination of the Coomassie brilliant blue-stained gel revealed a faint band at the position of YadA (not detectable in Fig. 2, lane 4). This and the fact that pYMS4232 contains lcrLMC and also an almost complete IcrF gene suggested that LcrF, the temperature-regulated activator of the Yop regulon of pYV (9, 33), also regulates the expression of YadA. The experiments described below were done to ascertain this. We cloned the IcrF gene into pYMS4448. In doing so, we avoided possible interference from other pYV genes. Polymerase chain reaction was then used to amplify the IcrF gene of YeO3. The primers (MS-25, 5'-CGCGG ATCCA ATCCA GCTCA ACGCG GG-3', and MS-26, 5'-CGCGG ATCCG TGACA TAAGG TTTCG CGG-3') for polymerase chain reaction were selected from the published sequence of the Y enterocolitica gene (positions 34 to 51 and 1476 to 1458, respectively, in reference 10, boldface above). Both primers were constructed with a BamHI site in the 5' ends. The IcrF gene of YeO3 was amplified (30 cycles) with the following parameters: denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min, using the Perkin-Elmer Cetus thermal cycler and AmpliTaq reaction mixtures as instructed by the manufacturer. The amplified 1,460-bp DNA fragment was purified from an agarose gel, and a portion of the purified fragment was digested with BamHI and ligated into BamHI-digested pYMS4448 (Fig. 1). Transformants harboring pYMS4448 with insertion of IcrF were identified by hybridization, using the polymerase chain reaction product as a probe. Restriction digestions confirmed the correct insertion of IcrF into pYMS4448. One of the recovered hybrid plasmids was designated pYMS4514. The pYMS4514 plasmid was transformed into S17-1 and mobilized into appropriate bacterial strains. These strains were then tested for YadA expression. The results (Table 2; Fig. 2) showed that YadA is expressed by pYMS4514 in all different test hosts in a temperature-inducible manner comparable to the wild-type situation. This confirms that LcrF functions as a temperature-inducible activator of the yadA gene. Recently, Michiels and coworkers (20) analyzed transposon mutants of pYV and showed that IcrF mutants completely, and lcrB mutants partly, failed to synthesize YadA. These and our data suggest that the regulation of yadA expression takes place through a temperature-regulated activator(s) and that the expression of the activator(s) is modulated by other factors. Our present hypothesis foryadA regulation is that transcription of the yadA gene would require only the LcrF protein as an activator. In line with

2050

NOTES

this, the factors modulating the expression of the IcrF gene or interacting with the LcrF protein would also influence yadA gene expression. In our hypothesis, the role of the lcrB locus in yadA expression would be modulation of lcrF expression, although we cannot, at present, rule out the possibility that lcrB has a direct effect onyadA gene expression. We hypothesize that a functional IcrB locus enhances the transcription of the IcrF gene from its own promoter. Thus, the low-level expression of YadA and the Yops by the lcrB mutants (20) would reflect decreased LcrF production by the mutants. Unfortunately, this point was not addressed by Michiels and coworkers (20). The LcrF-mediated activation takes place, most likely, by direct binding of LcrF to theyadA promoter region, although this remains to be demonstrated. The extremely rapid expression of YadA after only 2 min of temperature shift from 25 to 37°C (5) suggests that an amount of LcrF sufficient to initiate yadA expression must be constitutively available in the bacterial cell. For example, LcrF probably exists in inactive form at lower temperatures and is activated, perhaps by a conformational change, after upward temperature shift. The rapid expression of YadA differs from that seen for the Yops, the expression of which starts only after a lag period of 30 to 45 min (12). This difference may be due to the calcium concentration-dependent negative regulation influencing the yop genes but not theyad4 gene. Since LcrF also activates the yop genes, the 5' regions of several genes were compared, but no obvious similarities were detected (data not shown). Further work will be needed to establish the molecular mechanism of this regulation. (A preliminary report of this work was presented in reference 30.) We thank Reija Venho for excellent technical assistance. The language was revised by Jeri Hill. This work was supported by The Sigrid Juselius Foundation. REFERENCES 1. Appleyard, R. K. 1954. Segregation of new lysogenic types during growth of doubly lysogenic strain derived from Escherichia coli K12. Genetics 39:440-452. 2. Ausubel, F. M., R. Brent, R. E. Kingston, 0. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current protocols in molecular biology. Green Publishing Associates and John Wiley & Sons, Inc., New York. 3. Balligand, G., Y. Laroche, and G. Cornelis. 1985. Genetic analysis of a virulence plasmid from a serogroup 9 Yersinia

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VOL. 174, 1992 28. Skurnik, M., A. Al-Hendy, and P. Toivanen. Submitted for publication. 29. Skurnik, M., I. Bilin, H. Heikkinen, S. Piha, and H. Wolf-Watz. 1984. Virulence plasmid-associated autoagglutination in Yersinia spp. J. Bacteriol. 158:1033-1036. 30. Skurnik, M., and P. Toivanen. 1991. LcrF is the temperatureregulated activator of the yadA gene of Yersinia enterocolitica and Yersinia pseudotuberculosis, abstr. B-6, p. 26. Abstr. 91st Annu. Meet. Am. Soc. Microbiol. 1991. American Society for

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Microbiology, Washington, D.C. 31. Skurnik, M., and H. Wolf-Watz. 1989. Analysis of the yopA gene encoding the Yopl virulence determinants of Yersinia spp. Mol. Microbiol. 3:517-529. 32. Tertti, R., M. Skurnik, T. Vartio, and P. Kuusela. Submitted for publication. 33. Yother, J., T. W. Chamness, and J. D. Goguen. 1986. A temperature-controlled plasmid regulon associated with the low calcium response in Yersinia pestis. J. Bacteriol. 165:443-447.