Download The surface-located YopN protein is involved in calcium signal

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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