Molecular Microbiology (1991) 5{4), 977-986

ADONIS 0950382X91001106

The surface-located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis A. Forsberg,^^ A.-M. Virtanen,^ M. Skurnik^ and H. Wolf-Watz^* 'Department of Celt and Microbiology, National Defence Research Establishment, S'9O1 82 Umei, Sweden. ^Department cf Medical Microbioiogy, University of Turku, SF-20520 Turku, Finiand. ^Department of Cell and Mclecular Biology. University of Umei, S-901 87 Umei. Sweden. Summary The low-calcium response (Icr) is strongly conserved among the pathogenic Yersinia species ar»d is observed when the pathogen is grown at 37°C in Ca^ *-depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence-plasmid-encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature-regulated activator and by Ca^'-regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface-located when Yersmia is grown at 37^0. yopN was found to be part of an operon that is induced during the low-calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild-type phenotype of the yopN mutant. Thus the surface-located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.

Introduction All virulent strains of pathogenic Yersinia species show an unusual requirement for Ca^ * when grown at 37''C (Kupferberg and Higuchi, 1958; Higuchi etaL. 1959). Bacteria, as well as eukaryotic cells, normally maintain a low intracellular concentration of Ca^"^ (Rosen, 1987) and this exclusion system has also been shown to exist in Yersinia pestis

Received 11 July, 1990; revised 11 December, 1990. 'Present address: Imperial Cancer Research Fiind, Untversity of Oxford, Institute of Molecular Medicine. Oxford OX3 OHL, UK. 'For correspondence. Tel. {90) 189230; Fax (90) 186902.

(Perry and Brubaker, 1987), In eukaryotic cells, Ca^^ is an important signal molecule. The generally lew intracellular concentration (less than 0.1 mM) varies between the different compartments of the cell (Haiech ef al.. 1985), while the extracellular calcium concentration is several magnitudes higher (about 1 mM), Virulent Yersinia undergoes growth restriction at 37°C when grown In a Ca^' -depleted medium (Brubaker, 1983). The restricted growth is characterized by a metabolic downshift including a shut-off of stable RNA synthesis, a decrease in adenylate energy charge, and a cessation of DNA and protein synthesis (Zahorchak et ai. 1979). The metabolic downshift observed during low ca\c\um restriction resembles a stringent response in E. coii as observed, for instance, when £ coll is starved for an amino acid (Cashel and Rudd, 1987). However, Charnetzky and Brubaker (1982) showed that the metabolic downshift observed in Yersinia during low calcium restriction is not mediated by the same mechanism as the stringent response of E. coll. Approximately 20 kb (Ca^* region) of the virulence plasmids have been shown to be involved in the expression of the low-calcium response (Icr) (Bolin and WolfWatz, 1984; Cornelis et ai, 1986; Goguen ef ai, 1984; Portnoy and Falkow, 1981; Portnoy ef a/,, 1983). Insertion mutants in this region or strains lacking the plasmid are able to grow at 37°C in the absence of calcium and are usually avirulent. Concomitant with Icr is the induction of a number of virulence-plasmid-encoded genes: those encoding the Yop proteins and the V antigen (Bblin ef a/., 1985). Several Yop proteins have been shown to be important virulence determinants (Bolin and Wolf-Watz, 1988; Forsberg and Wolf-Watz, 1988; Leung ef al., 1990; Mulder ef al.. 1989; Straley and Bowmer, 1986). YopE and YopH are involved in the ability of Yersinia pseudotuberculosis to obstruct the primary host defence in the mouse-infection model (Rosqvist et aL. 1988; 1990). The expression of the Yop proteins is regulated by genes located within or adjacent to the 20kb calcium region, and involves both Ca^*-regulated negative elements (Forsberg and Wolf-Watz, 1988) and at least one temperature-regulated activator (Yother ef at., 1986; Forsberg and Wolf-Watz, 1988; Cornelis ef ai, 1989). Yother and Goguen (1985) were first to describe a mutant with a repressor-negative phenotype (i.e. unable to

978 A. Forsberg, A.-M. Viitanen, M. SkurnikandH. Wotf-Watz

[ M i l l I CI'liPIigD H

BH

krE

icrD tcrGVHyopBD

krX IcrB

pAF68l (pACYCl84) pAf 682 (pACYC 184)

-\

pAF8

(pACYC 184)

pAl-801

{pACYC IS4)

pAF80

kb

form colonies at 37°C). They designated this mutant 'calcium-blind', since it was growth-restricted and expressed high amounts of V antigen irrespective of the Ca^^ concentration of the growth medium. The mutant was obtained via chemical mutagenesis and the mutation was mapped to a 1 kb fragment within the tcrE locus of plasmid pCD1 of Y. pestis (Yother and Goguen, 1985). More recently, several mutants Involving three distinot looi have been isolated or constructed in the different species that are phenotypicaliy repressor-negative (Forsberg and Wolf-Watz, 1988; Mulder et ai. 1989; Prioe and Straley, 1989; Rosqvist ef ai. 1990). These mutants were unable to grow at 37°C and were derepressed for yop transcription at this temperature regardless of the Ca^"^ concentration. We therefore define these mutants as temperature-sensitive (TS). Parts of the tcrE locus in Yersinia enterocotitica 0;3 was recently sequenoed (Viitanen et at., 1990). yopN (in their paper designated IcrE) was shown to be encoded within this multicistronic operon and the calcium blind' mutation {Yother and Goguen, 1985) was found to be part of the same operon (Viitanen et ai, 1990). Herein, we have extended oar analysis of the recently described yopN mutant (Rosqvist ef at., 1990) and present evidence showing that YopN is surface-located and either directly or indirectly involved in sensing the external Ca^"^ concentration. Results DNA sequence The DNA sequence ofthe 2.7kb C/al fragment harbouring

Fig. 1. Restriction endonuclease map of the yopN region of the Y. pseudotuberculosis virulence plasmid, pIBI. The vertical arrows denote the map position ol the insertion mutants in the yopN region used in this study. The horizonlal arrows show the approximate extension and direction of the transcriptional untts identified. Boxes show the positions of the yopt^ gene and 0RF1 and 0RF2 as Identified by Viitanen e( a'. (1990). The hybrid plasmids used in the transcomplementation studies are shown in the lower part of the figure. Abbreviations used to denote restriction enzymes: B. SamHI; Bg, BglW: C, C/al: D, Oral; H, H/ndlil; P. Psfl. These sequence data will appear in the EWBUGenBank/DDBJ Nudeotide Sequence Data Libraries under the accession number X51833.

the yopNgene was recently determined from Y. enterocotitica 0:3 (}J\'\tanen etat.. 1990). Here the region sequenced was about 1.6 kb, between the Cta I site upstream of yopN and 86bp beyond the H/ndlll site downstream of yopW of the corresponding region from virulence plasmid pIBI of y. pseudctubercutosis (Fig. 1). As expected, the two sequences were highly homologous and the three open reading frames identified in the 0:3 strain could also be identified in the sequence from plB1 (Fig. 1). For the open reading frame corresponding to yopN. 19 out of 293 codons were different, revealing an overall homology of 98% at the nucleotide level. Among the 19 codons that differed, 15 led to replacements at the amino acid level (Fig. 2). The YopN protein from pIBI was predicted to have a M, of 32669 Da and a p! value of 4.98, which is very similar to the corresponding protein from the Y. enterocotitica 0:3 strain (Viitanen et ai. 1990). The start codon of yopN was confirmed by sequencing the amino-terminal end of YopN purified from the culture supernatant. However, the amino-terminal methionine was not detected in this analysis. The amino acids obtained were numbers 2-9 in the deduced amino acid sequence of YopN (Fig. 2A). The two short open reading frames (ORFs) downstream of yopN were also found to be highly conserved between the species with only one oodon differing 1or 0RF1 and six lor ORF2. No replacement was evident at the amino acid level for 0RF1, whereas two of the changes led to replacements in 0RF2 (Fig. 2). Neither 0RF1 nor 0RF2 has been shown to be expressed in £ coti or in Yersinia. Therefore, no function has been associated with these putative proteins.

signal transduction in Yersinia

979

A HTTLHNLSYG NTPLHHERPE lASSQIVNQT LGQFRGESVO IVSGTLQSIA DHAEEVTFVF SERKELSLOK RKLSDSOARV SDVEEOVNOY LSKVPELEQK R H K

plBI prV03

QNVSELLSLL SNSPNISLSQ LKAYLEGKSE EPSEQFKMLC GLRDALKGRP ELAHLSHLVE QALVSMAEEQ GETIVLGARI TPEAYRESQS GVNPLQPLRD L V E A

pIBI pYV03

TYRDAVMGYQ GIYAIWSDLQ KRFPNGDIDS VILFLOKALS ADLQSQOSGS GREKLGIVIS DLQKLKEFGS VSDQVKGFUQ FFSEGKTNGV RPF N E E R L 1 L

pIB1 pYV03

B MAYDLSEFMG DIVALVDKRU AGIHDIEHLA NAFSLPTPEI KVRFTQDLKR MFRLFPLGVF SDEEQRONLL QMCQNAIOMA lESEEEELSE LD

plB1 pYV03

C

pIBI pYV03

VSWIEPIISH FCOOLGVPTS SPLSPLIQLE HAOSGTLQLE QHGATLTLUL ARSLAWHRCE DAMVKALTLT AAQKSGALPL RAGULGESQL VLFVSLDERS Q N LTLPLLHQAF EQLLRLQOEV LAP

pIBI pYV03

Fig. 2. The deduced amino acid sequence of yopN (A), orfT (B), and orf2 (C). The deduced sequences trom plasmid pIBI of Y. pseudotuberculosis are shown using the standard one-letter code. Whenever the sequence tor the homologous protein or putative protein from Y. enterocolitica 0;3 (from Viitanen etal.. 1990) differs, this is indicated below the pIBI sequences.

Characterization of mutants The yopN gene (the gene product was previously designated Yop4b) has previously been mapped to SamHI fragment 8 of the virulence plasmid plB1 of Y. pseudotuberculosis {Forsberg etai, 1987). This position has been confirmed in the three pathogenic species (Bolin ef ai., 1988). A number of polar insertion mutants in the region both upstream and downstream of the yopN gene of Y. pseudotubereuiosis were constructed. The position of the inserted kanamycin-resistance gene (GenBlock) relative to the yopN structural gene is shown in Fig. 1, When the mutants were tested for growth characteristics with respect to Ca^^ and temperature, all strains except YPlll/plB82 were found to be Ca^^-independent (Cl) for growth at SZ^'C (Table 1), The Cl mutants were af(

downregulated with respect to yop transcription at 37°C (data not shown), as previously shown for other Ci mutants (Forsberg and Woif-Watz, 1988), including the mutant strain YPIII/pYV19/8 which maps between plB861 and plB84 (Fig. 1). Strain YPIII/plB82 has an internal deletion of about 450 bp within yopNand this mutant has previously been shown to be grov^^h-temperature-sensitive (TS), derepressed for yop transcription, and able to secrete the Yops in the presence of Ca^"^ (Rosqvist etai, 1990). Transeomplementation studies The mutants described above were transcomplemented using different subclones of plB1 (Fig. 1), From the results obtained, the mutants can be grouped into four classes (Table 1). Insertions upstream of yopN could not be

Table 1. Transcomplementation studies of mutants in ttie IcrE region.

Phenotype tor Transcompleted Strains

Strain

No plasmid

pAF681

pAF682

pAF8

pAF80t

pAF80

pAF80 IPTG

YPIII/plB102 YPIII/plB861 YPIII/plB84 YPIII/plB82 YPIII/plB85 YPIII/plB86

CD Cl Cl TS Cl Cl

CD Cl Cl TS NT NT

CD Cl Cl CD NT NT

CD NT Cl CD*

CD NT NT NT CD Cl

CD Cl Cl CD" Cl Cl

CD Cl Cl CD Cl Cl

CD Cl

Abbreviations used tor phenotypes: CD, Ca^' -dependent for growth at 37"C; Cl, Ca^' -independent for growth at 37X1 TS, temperature sensitive for growth at 37°C in^espective ot Ca^*; and NT, not tested. The phenolypes are as defined In ihe Experimental procedures. a. Strain YPIII/plB82, pAF8 is intermediate in plating trequency in the presence ot Ca^* at 37''C and this frequency is about SO times (ower than for wlld-tyf>e strains, but about 200 times higher than for the non-complemented strain, YPIII/plB82. b. Strain YPIII/plB82, pAF80 has a plating frequency about 10 times lower on Ca^ * plates without IPTG induction at 37°C than does the wild-type strain, but the colonies are very small and therefore complementation is only partial.

980 A. Forsberg, A.-M. Viitanen, M. Skurnik and H. Wolf-Watz

i

f

+•

Fig. 3. Northern blotting analysis of -^opB transcription in the yopW mutant. The respective strains were grown as described in the Expenmenfa'pracedures'or analysis of YopN expression. The Northem blotfing analysis of y o p t transcription was performed as previously described (Forsberg and WoH-Watz, 1988). IPTG (linal concentraiion 1 mM) was added at the time of the temperature shift from 26°C to 37°C where indicated. Above each iane.' +' and ' - ' , respectively, denote growth at 37"C in the presence and absence of 2.5 mM Ca^'.

transcomplemented with the hybrid plasmids used. The yopN mutant could be fully transcomplemented only with the plasmids that carried 2,5 kb DNA upstream of yopH. When SamHI fragment 8 (pAF8) was used, only partial complementation was obtained. The two insertions downstream of yopH also fell into two complementation groups. Mutant strain YPlll/plB85 could be fully transcomplemented by hybrid plasmids carrying inserts from BamH\ fragment 8, whereas strain YPIII/plB86, with the insertion within the previously defined \crD locus (Yother and Goguen, 1985), could not be complemented with any of the plasmids used (Table 1). Since yopN is evidently encoded within a multicistronic operon, it was important to show that the phenotype of the polar yopH mutant (plBB2) actually resulted from lack of expression of the YopN protein and not from polar effects on downstreamlocated genes. Therefore the 1.1kb Cla\IDra\ fragment

with the yop/Vstructural gene was inserted downstream of the tac promoter of the vector pMMB66 (Fig. 1). The resulting hybrid plasmid, pAF80. was used to transcomplement the yopA/ mutant. When isopropyl-(3-D-thiogalactoside (IPTG) was added, the mutant was fully restored to wild-type growth characteristics (Table 1). Moreover, the transcomplementation observed in the presence of IPTG also led to a shut down of yop gene transcription in the presence of calcium, as shown for the yof»Egene in Fig. 3. In the absence of IPTG, the Ca^^ phenotype of the yopH mutant was partially complemented, whereas there was no effect on yopEtranscription. Expression of yopA/under the control of the tac promoter did not affect either the Ca^"^ phenotype or the transcription of yop genes in any other strain studied. Neither the wild-type strain YPIII/ plB102 nor the TS mutants YPIII/plB13 {tcrH) and pill/ plB23 {IcrK) were affected (data not shown).

VopA/ expression The pattern of expression of YopN was similar to that of the Yop proteins. As for the yop genes, transcription was induced during low calcium restriction at 37°C (Fig. 4). The overall pattern of yopH transcription in the different regulatory mutants was also similar to that of the yop genes, i.e. derepressed transcription at 37°C in TS mutants and repressed transcription in Cl mutants (Fig. 4). In accordance with the transcomplementation analysis, which indicates that yopH is part of a muiticistronic operon. Northern blottings using yopA/-specific probes revealed two major bands of 1.6 and 2,5-3.0 kb in size (Fig. 4). Using a specific YopN antiserum, the pattern of expression for bacteria-associated YopN was confirmed at protein level (Fig. 5). As expected, the yopN mutant did not express any detectable amount of YopN and upstream insertions were greatly reduced in expression. Downstream insertions were only moderately affected in the bacteria-associated YopN expression (Fig. 5). The

3.0 — Fig. 4. Northem blotting analysis of yopN transctiption. The respective strains were grown as described in tfie Experimental procedures for analysis of YopN expression. Northern btottings were performed as in Fig. 3 using the 0.45kb internal Psf 1 fragment of the yopN gene as a probe. '26' denotes growth at26"C, and ' + ' and ' - ' , respectively, denote growth at 37°C in the presence and absence of Ca^".

signai transduction in Yersinia

Fig. 5. Immunoblotting of whole bacteria using monospecific anti-YopN antiserum. Strains were grown as described in the Experimental procedures. The cultures were harvested and the bacterial pellets were dissolved in SDS sample buffer. The same number of bacteria of each strain, as determined by the OD550 measurements, was run on the SDS-PAGE gels. In each lane, matenal correspondrng to approximate'/ 250111 of culture (OD^M = 0.5) was loaded. The gels were then subjected to immunoblottings using monospecific anti-YopN antiserum. The respective strains are indicated above each lane. Above each lane," + ' and ' ', respectively, denote growth m the presence and absence ot 2.5mM Ca^*. IPTG above the lanes indicates to which cultures 1 mM IPTG was added. The addition of IPTG was carried out at the time of the temperature shift from 26^0 to 37°C.

Mw

69 — 46 — 30 — 215 —

YopN protein was also shown to be expressed after the addition of IPTG in Yersinia (Fig. 5). In the absence of IPTG, low residual calcium-induced YopN expression at 37°C was observed for hybrid plasmid pAF80 expressed in Yersinia. YopN is surface tocated Previous studies on Yersinia grown under calcium-restricted conditions have shown that the majority of the Yop proteins are recovered either in a secreted form or in an outer-membrane-associated form (Heesemann ef at., 1984; Forsberg e(a/., 1987). Western blotting analysis has also confirmed that only low amounts can be recovered from the cytoplasm/periplasm (Forsberg and Wolf-Watz, 1988). The localization of the Yop proteins for cells grown in calcium-containing media is less clear. The overall expression is much lower and the Yops have been shown to be mainly membrane associated, whereas hardly any Yops oan be recovered from the culture medium (Forsberg et ai, 1987). Using the xylene extraction technique employed by Michiels ef at. (1990), the cellular location of YopN was determined in bothOa^^-containing and Ca^^depleted media. The Western blot in Fig. 6 shows that xylene extraction removed the majority of the YopN protein from the surface of the wild-type strain (YPIII/ plB102), irrespective ofthe growth conditions. In contrast, when the IcrK mutant (YPlll/plB23) was analysed, YopN remained cell associated after xylene extraction (Fig. 6). The IcrK mutant is unable to secrete Yop proteins (Rosqvist efa/., 1990). Thus, the results in Fig. 6 indicate that the tcrK mutant does not expose Yop proteins to the surface of the bacteria. In conclusion, the most of the YopN protein is either secreted or surface exposed. Discussion In this paper we present evidence to show that YopN is

981

surface located and either directly or indirectly interacts with calcium to transmit a signal to negatively regulate yop transcription. A model showing the role of YopN in the regulation of yop expression is presented in Fig. 7. This model is supported by the following data, (i) The phenotype of the yopN mutant with respect to growth restriction and yop transcription is as expected for a mutation in a Ca^* sensor, (ii) YopN is either surface exposed or secreted to the medium. Inability to expose YopN on the surface Is therefore expected to result in a TS phenotype, which is also observed for the /crKmutant (Rosqvist efa/., 1990). (iii) The secretion of Yops is also Ca^* regulated and, in contrast to IcrH and tcrK mutants, the yopN mutant is able to secrete Yops in the presence of Ca^^, (Iv) Expression of yopN under the control of the tac promoter complements the phenotype of the yopN mutant, while the wild-type as well as other TS mutants {IcrH and IcrK) are unaffected by the overexpression of YopN. (v) In contrast, overexpression of IcrH results in a Cl phenotype

YPin/plBlO2

YPlll/plB23

4Co^*

no Co^'^

C

C

E

E C

+Co^*

no Co^'*"

E

C

E

Mw

-li 69 — 463021514.3Fig. 6. Immunoblotting analysis of xylene-extracted bacteria. Strains were grown as described in Fig. 5. Parallel samples of xylene-extracted (E) and non-extracted bacteria (O) were subjected to Western blotting analysis using monospecific anti-YopN antiserum.

982 A. Forsberg, A.-M. Viitanen. M. Skurnik andH. Wolf-Watz

*

STIMULUS

RESPONSF. 1 SFNSOR

OM PI RIPIASM

CM

,+ * * TRANSPORI 1

ixrK

I-crH

RF.PRFSSOR

—1 vop-gencs| UrK f

ACTIVATOR

t 37''C

SIIMULUS

Fig. 7. Model showing the role of YopN in Ca^"-regulated yop expression. For simplicity, only yopN, IcrH and IcrK are shown. The arrows indicate where other genes or gene products might interact.

when expressed in the TS insertion mutants that map in lerGVHyopBD{Bergman etal., 1991). This is as expected for a gene acting as a negative element but not involved in the actual sensing of the signal, (vi) Overexpression of the IcrH gene in the yopN mutant restores the growth deficiency of this strain, i.e. a shift of phenotype from TS to Cl (S. H^kansson, unpublished results). Thus, (iii) to (vi) favour the hierarchical order of the negative control elements involved in this regulatory cascade put forward in the model (Fig. 7). yopA/is closer to the Ca^"*" signal than IcrH, and the Ca^' -regulated secretion is also a branch of this regulatory network, with yopN acting close to the signal. One strong argument for the involvement of the surface-located YopN in sensing Ca^^ is the observed high expression of YopN in the /crKmutant. This mutant is TS because of its inability to surface-expose YopN. Most regulatory proteins that respond to Ca^^ show binding affinities in the ^LM range, while the signal sensed by Yersinia is in the mM range (Kupferberg and Higuchi, 1958; Higuchi etai, 1959). Therefore, YopN is unlikely to have a structural motif analogous to those identified for other Ca^"^-binding proteins involved in gene regulation. The conformation as well as the stability of some bacterial proteases are known to be affected by mM concentrations of metal ions such as iron and calcium (McConn et ai, 1964). One group of proteins which depend on Ca^^ for their activity is the toxin family of proteins represented by the E. coli haemoiysin. These toxins have in common a tandennly repeated nine-amino-acid sequence that has been shown to bind Ca^"^ (Boehm et ai., 1990b). In addition, the £ eoli haemoiysin requires about 0,5 mM

Ca^^ for maximal activity (Boehm et ai, 1990a). The hypothesis is that binding of Ca^"*^ induces a conformational change of the haemoiysin and that this change is required for binding of the protein to erythrocytes. In the case of YopN, it is more likely that Ca^+ directly or indirectly affects the conformation rather than the stability of the protein, since there are no signs of degradation of YopN in the absence of Oa^^ (Fig. 5). This change in conformation can be postulated to affect the activity in terms of interacting with another component involved in the cascade of regulatory events that constitutes the lor. Goguen etai (1984) first identified the region between lerA (later divided into /cr5and IcrD) and lerB as a border of divergent transcription. The yopN gene maps within the /crE locus and is contained within a multioistronic operon. Our transcomplementation studies of the yopN mutant show that the major startpoint of transcription for the yopW-containing operon is at least 700 bp upstream of the structural gene itself. This is in agreement with the primer extension analysis performed on the corresponding operon in Y. enteroeolitica 0:3 (Viitanen et ai, 1990). A mutation mapping upstream of yopN shows polarity of YopN expression since the expression is substantially lower than in other Ct mutants mapping downstream of yopN {Fig. 5). Nonetheless, it is likely that there is a weak Ca^^-regulated promoter just upstream of yopN since strain YPIII/plB82, pAF80 shows low but Ca^+-regulated expression of YopN in the absence of IPTG (Fig. 5). Additional support for this promoter activity is the partiai transcomplementation of strain YPIII/plB82 using hybrid plasmids containing SamHI fragment 8 (Fig. 1, Table 1). The two strains carrying insertions upstream of yopN (YPIII/plB861 and YPIII/plB84) could not be transcomplemented using the same plasn:>ids that complemented the yopA/mutant (Table 1). This indicates that these strains are also affectegJ in the divergently transcribed /crSoperon and that this locus extends beyond the Cla\ site in SamHI fragment 6 (Fig. 1). The data presented here indicate that the divergent promoters must have a considerable overlap. Primer extension analysis of Y. enterolitiea 0:3 suggested the presence of a divergent transcript starting within the yopN structural gene, which is close to the Hind\\\ site (Fig. 1), Thus, the overlap in transcription was more than 1 kb (Viitanen et ai, 1990). The yopN mutant could be fully transcomplemented using only the cloned yop/Vgene, making it unlikely that the IcrB promoter maps within the internal Pst\ fragment of the yopN structural gene since the putative IcrB promoter identified in primer extension analysis (Viitanen etai, 1990) has been deleted in the yopA/mutant. The insertion mutants downstream of the yopA/gene fell into two different complementation classes. Strain YPIII/ plB85 could be complemented using hybrid plasmids from SamHI fragment 8, whereas strain YPMI/plB86 could

signal transduction in Yersinia not be complemented with the plasmids used (Fig. 1, Table 1). The insertion in strain YPIIl/plB86 is within the /crD operon and this operon extends into 8amHI fragment 1 {A. Backman, A. Forsberg and H. Wolf-Watz, manuscript in preparation). Therefore, it is not possible to transcomplement the icrD mutant with the hybrid plasmids used in this study. It is also clear from the transcomplementation experiments that there is a locus (denoted IcrX in Fig. 1) between the yopN-containing operon and IcrD. This locus is likely to encode one or more of the open reading frames {ORF2-ORF4) that can be identified from the DNA sequence downstream of the yopN gene (Viitanen etaL. 1990). Signal transduction in bacteria often involves conserved pairs of sensors and regulators (Gross etai, 1989). The list of conserved pairs of sensors and regulators has also been extended to include the regulation of virulence genes, as demonstrated in Salmonella (Miller etaL, 1989) and in Bordetella (Arico e( ai, 1989). No sequence homology with these conserved pairs of sensors and regulators has yet been found for any of the genes so far identified as being involved in regulation of the yop genes. Therefore, it is likely that the Ca^"^-regulated expression of virulence genes in Yersinia is different. Interestingly, in this study we have identified one surface-exposed protein involved in the sensing and transmission into the cell of an environmental signal. This is, to our knowledge, the first time a surface-exposed protein has been implicated in signal transduction. However, it is difficult to understand why an ion sensor should be surface exposed, since Ions easily penetrate the outer membrane. Therefore it is possible that YopN, during the infectious process, interacts with macromolecules and that this interaction mediates a conformational change similar to that postulated to be mediated by Ca^"^ ions in vitro.

Table 2, Yersinia pseudotuberculosis strains used in this study.

983

Experimental procedures Bacterial strains and growth conditions The Yersinia strains used in this study are listed in Table 2. The E. co//strains employed were C600 (Appleyard, 1954),S17-1 {Simon et at., 1983) and MM383 {Monk and Kinross, 1972). In the protein-expression analysis, Yersinia strains were grown in rich medium consisting of 15mM potassium phosphate pH 6.8, lOmM citric acid, lOmM sodium gluconata, lOmM ammonium acetate, 40 mM magnesium sulphate, supplemented with 1 % tryptone, 0.5% yeast extract and 0.2% glucose, Ca^* was depleted by addition of 20mM Na-oxalate. As solid medium. Blood Agar Base {BAB) containing 2.5 mM Ca^' was used. MOX plates {Ca^ ' -free) were BAB with 20 mM Ma-oxalate and 20 mM MgCl2. E. co//strains were grown in Luria broth {LB) or on LB-agar plates.

Nomenclature The gene designated yopN in this paper has been designated previously as IcrE {Viitanen et al., 1990) and the encoded gene product has previously been designated Yop4b (Forsberg etai. 1987) and Yop35 {Cornelis et ai. 1987). Here we have shown that this gene is under the same regulatory control as the yop genes and localization of its encoded gene product is the same as that observed for the other Yop proteins. Therefore, in spite of the fact that this gene is involved in regulation, we suggest that the gene be assigned the name yopN, as proposed by Straley {1988).

Construction of mutants Insertion mutants upstream and downstream of yopN were constructed in the same way as described earlier (Forsberg and Wolf-Watz, 1988; Rosqvist ef a/., 1990). Using this procedure, the virulence-plasmid derivatives plB861, plB84, plB85, and plB86 were constructed. The exact map position of these insertion mutants including the previously described yopN mutant, YPIII/ plB82 {Rosqvist e(a/., 1990), is shown in Fig. 1, Also shown in this

Strain

Comment

Reference

YPIIl/plB102

pIBI derivative with a Tn5 insertion within the yadA gene, CD pIBI derivative with a Tn5-132 insertion within the yadA gene, CD plB103 derivative with a GenBlock insertion in the BamHI site upstream of IcrE. Cl plBiO3 derivative with a GenBlock insertion in the Cla\ site upstream ot icrE, Cl plBiO3 derivative with a GenBlock insertion replacing the internal Pst\ fragment in IcrE, TS plB103 derivative with a GenBlock insertion in the Hind\\\ site downstream of IcrE. Cl pIBI 03 derivative with a GenBlock insertion in the Cla\ site downstream of IcrE. Cl plB103 denvative with a GenBlock insertion in the 63/11 site within the IcrKgene, TS plB103 derivative with a GenBlock insertion in the EcoRI site of the IcrF gene, Cl plB103 denvative with a GenBlock insertion in the EcoRI site of the yopD gene, TS

B6lin and Wolf-Watz (1984)

YPIII/plBI 03 YPIII/plB861 YPIII/plB84 YPIII/plB82 YPIII/plB85 YPIII/plB86 YPlll/plB23 YPIII/plB73 YPni/plB15

Forsberg and Wolf-Watz (1988) This study This study Rosqvist etal. (1990) This study This study Rosqvist e( a/. (1990) A. Forsberg et at. (manuscript in preparation) Bergman etal.. (1991)

984 A. Forsberg, A.-M. Viitanen, M. Skurnik and H. Wolf-Watz figure are the hybrid plasnnlds used in the transcomplementation studies. The vectors used in the clonings were pACYCI 84 (Chang and Cohen, 1978) and pMMB66 (Furste etai, 1986), The mutant strain YPIN/pIB73 is an insertion mutant with the GenBlock inserted into the EcoRI site in the tcrF gene (A. Forsberg, K. Ericksson and H, Wolf-Watz, manuscript in preparation).

Definition of phenotypes The different mutant strains were tested for their phenotypes with respect to temperature and Ca^ * by plating at 26''C and 37°C in the presence and absence of Ca^'. Strains unable to grow at 37°C in the absence of Ca^' are defined as being calcium dependent andplatewithafrequencyofabout 10 ''on plates without Ca^^ at 37°C (compared to plates with 2.5 mM Ca^' at 37°C or plates kept at 26°C). Calcium-independent (Cl) mutants plate equally well on piates at 26X and at 37°C irrespective of the Ca^' concentration. If the strain plates with a frequency of about 10 •* at 37°C compared to 26''C, irrespective of the Ca^' concentration, il is defined as temperature sensitive (TS).

N-fermina/ amfno acid sequence anaiysis Secreted proteins from strain YPIII/plB15 grown in Ca^' -depleted medium at 37°C were precipitated and run on sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Forsberg etai, 1987). Strain YPIII/plB15 has an insertion within the structural gene of yopD (Bergman ef ai, 1991). Therefore YopN is easily separated as a single band by SDS-PAGE. The proteins were then blotted onto Immobilon filters and after Coomassie staining the YopN band was sliced out. The amino-terminal amino acid sequence was then determined using an Applied Biosystems 470A Gas-liquid Phase Sequencer with on-line detection of PTH derivatives. Ten cycles were run and the amino acid residues were identified by comparison with a p-lactoglobulin standard.

Affinity purification of antiserum We have previously described the preparation of a rabbit antiserum directed against both YopD (Yop4a) and YopN (Yop4b) (Forsberg et ai., 1987). Monospecific anti-YopN antiserum was prepared by immunoabsorption of the anti-Yop4a/b antiserum. Released proteins were prepared from strain YPIIl/plB15, which has an insertion in the yopD gene (Bergman et ai, 1991) and therefore expresses a truncated form of YopD. The released proteins were precipitated and subjected to SDS-PAGE as previously described (Forsberg et ai. 1987). The proteins on the get were then blotted onto a nitrocellulose filter and stained with Coomassie Brilliant Blue. The YopN band was cut out and thereafter incubated for one hour at 20°C in a blocking buffer containing 20% fetal calf serum, 50mM Tris-HCl pH 7.0, 0.15M NaCI and 2.5mM ethylenediamine tetraacetic acid. The antiserum was diluted 10 times In the same buffer without serum and then incubated for at least 90 min at 20°C with the nitrocellulose strip. The strip was then washed by mixing in 2 x phosphate-buffered saline (PBS), 2x PBS with 0.1%TritonX-100,3x PBS, and finally in water. The aniibodies were eiuted by mixing the strip in 1 mi 0.1 M glycin-HCI pH 2,5 for 2 min. Finally, the eluate was neutralized by Ihe addition of 100|il o n M Tris-HCl pH 8.0. This affinity-purilied anti-YopN antiserum was used in a 1:10 dilution In Western blotiings.

Anatysis of YopN expression The Yersinia strains were grown at 2 6 ^ to an optical density at 550 nm (0D550) of 0.1. Then the cultures were shifted to 37°C and grown for an additional 2h before harvesting. IPTG to a final concentration of 1 mM was added at the time of the temperature shift to strains with fac-inducible plasmids. The xylene extractions were performed as described by Michieis et ai (1990). Whole bacteria were subjected to SDS-PAGE on 12% acrylamide gels and the subsequent immunoblotting performed as described previously (Forsberg et af., 1987).

DNA methods Preparation of plasmid DNA, restriction enzyme digests, ligation, and transformation of E. coli were performed essentially as described by Maniatis et at. (1982). Transformation of piasmid DNA into / . pseudotubercutosis, extraction of RNA, and Northern blottings were performed as described previously (Forsberg and Wolf-Watz, 1988).

DAM sequencing A region of about 1.6kb, from the Cta\ site extending over the Hind\W site downstream of yopN. was subjected to DNA sequencing (Fig, 1). DNA fragments from this region were subcloned into the M13 sequencing vectors mpl8 and mp19 (Norrander ef ai. 1983). Sequencing was performed using the dideoxy chain termination method (Sanger e( ai., 1977) with modified T7 DNA polymerase (Sequenase version 2.0 supplied by the US Biochemicai Corporation) and (u-^^Sl-dATP (Amersham) as the radioactive label. The DNA sequences were analysed using the Geneus (Harr et af., 1985) and Wisconsin {Devereux ef af., 1984) computer programs.

Acknowledgements We thank Gunnar Bostrom for the artwork. Anna Macellaro and Eleonora Westermark are acknowledged for their excellent technical assistance. We thank the Swedish Medical Research Council (07490-040), the Swedish Natural Science Research Council (BU-4426-301), the Swedish Board for Technical Development (90-00577), the Sigrid Jusetius Foundation, the Emil Aaltonen Foundation, and the Research and Science Foundation of Farmos for support.

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