analysis of hemolysin operons in actinobacillus pleuropneumoniae
TRANSCRIPT
Gene, 123 (1993) 51-58 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00
GENE 06886
51
Analysis of hemolysin operons in Actinobacillus pleuropneumoniae
(RTX toxins; virulence; serotypes; secretion; deletions)
Joachim Frey, Marianne Beck, Urs Stucki and Jacques Nicolet
Institutefor Veterinary Bacteriology, University of Berne, CH-3012 Berne, Switzerland
Received by M. Bagdasarian: 10 September 1992; Accepted: 20 September 1992; Received at publishers: 16 October 1992
SUMMARY
Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumo-
nia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active
hemolysin II (HlyII) is produced by all serotypes except type 10. In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce
HlyI, the hemolysin (My) operon consists of a structural hlyZA gene, encoding pre-HlyI, an activator gene, hlylC,
necessary for the activation of pre-Hly to active Hly, and two genes, hlyZB and hlylD, involved in Hly secretion. These
genes are clustered in the order, hlylCABD. This is characteristic to RTX toxin (repeats in the structural toxin) operons.
The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, uppA, and its activator
gene, appC. The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter,
hlylB and hlyID, and a small segment of the C terminus of hlylA. This partial HlyI operon might have been formed by
deletion of hlyIC and most of hlylA. In serotype 3, which produces HlyII, but no HlyI, and which releases only minute
amounts of this Hly into the growth medium, none of the hlyl genes and consequently no Hly secretion genes were
found. The above results postulate that HlyII is secreted via the products of hlylB and hlylD, and explain the low
amount of HlyII secreted by serotype 3. Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII
among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related
serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10. The hlyIIA genes, in contrast, seem to be conserved
in all serotypes.
INTRODUCTION
A. pleuropneumoniue, the etiological agent of swine
pleuropneumonia (Shope, 1964), secretes hemolysins Hly
which seem to play an important role in pathogenesis
(Nakai et al., 1984; Frey and Nicolet, 1988b; 1990; Rosen-
da1 et al., 1988). These Hly are strongly immunogenic in
experimentally and naturally infected pigs (Devenish
et al., 1990; Frey and Nicolet, 1991). They were also
shown to induce protective immunity against A. pleuro- pneumoniue infection and are important components for
subunit vaccines (Bhatia et al., 199 1; Van den Bosch et al.,
Correspondence to: Dr. J. Frey, Institute for Veterinary Bacteriology,
Laenggasstrasse 122, CH-3012 Berne, Switzerland. Tel. (41-31) 27 44
84; Fax (41-31) 24 69 22; e-mail: [email protected].
Abbreviations: A., Actinobacillus; aa, amino acid(s); uppA, gene encoding
structural HlyII protein (pre-HlyII); appC, gene encoding activator of
HlyII; bp, base pair(s); CTP, cytosine triphosphate; A, deletion;
dCTP, deoxyCTP; Hly, hemolysis(s); HlyI, hemolysin type I; HlyII,
hemolysin type II; hlylA, gene encoding structural HlyI protein (pre-
HlyI); hlylB and MyID, secretion genes on the operon specifying pre-
HlyI; hlyIC, gene encoding activator of HlyI; kilobase or 1000 bp;
NAD, nicotinamide-adenine dinucleotide; nt, nucleotide(s); oligo, oligo-
deoxyribonucleotide; PCR, polymerase chain reaction; R, resistance/re-
sistant; RTX, repeats in the structural toxin; SDS, sodium dodecyl
sulfate; SSC, 0.15 M NaCl/O.OlS M Na,citrate pH 7.6; Tc, tetracycline;
T,, melting temperature of DNA:DNA duplexes.
52
1990). To date, two different Hly, both proteins with an
apparent molecular weight of 105 kDa, have been iso-
lated (Frey and Nicolet, 1988a; 1988b: 1992). Both the
strongly hemolytic Hlyl which is very similar to the Esch-
erichia coli x-Hly and the weakly hemolytic Hlyll which
resembles more the Pasteurella haemolytica leukotoxin,
belong to the group of RTX toxins as determined from
trarzs-complementation experiments with E. coli x-Hly ac-
tivator and secretion genes and from their DNA derived
aa sequence (Chang et al., 1989; Gygi et al., 1990; Frey
et al., 1992; Smits et al., 199 1). In addition, a third secreted
protein toxin of 120-125 kDa, named pleurotoxin or cy-
tolysin III, was identified among certain serotypes and
was shown to have cytotoxic activity (Rycroft et al., 1991;
Kamp et al., 1991). Among the twelve different serotype
reference strains, the serotypes 1, 5a, 5b, 9, 10 and 1 I,
known generally as virulent strains, have been shown to
secrete Hlyl. All serotypes except serotype-10 secrete
Hlyll (Kamp et al., 1991; Frey et al., 1992), but serotype-
3 seems to secrete substantially lower amounts of Hlyll
than most other strains (Frey and Nicolet, 1991). The
genetic determinant encoding HlyI in A. pleuropneumon- iae serotype-1 is a single operon comprising four contigu-
ous genes, the posttranscriptional activator gene hlylC,
the gene for the structural 105kDa protein hlylA, and
the secretion genes hlyIB and hlylD, arranged in the order
hlylCABD (Gygi et al., 1992). An intragenic Rho-
independent transcription termination signal is located
between hlylA and hlylB (Frey et al., 1991). The genes
encoding the weakly hemolytic Hlyll, also named App,
were postulated to be located on two different operons,
one containing the activator and structural genes appC and appA and a second operon containing appB and
appD (Chang et al., 1991; Smits et al., 1991). Comparison
of the secretion genes appB and appD with the secretion
genes hlylB and hlylD from the hlyl operon shows that
the genes are the same, or at least highly similar and seem
to belong to the hlyl operon (Gygi et al,. 1992). Due to
the fact that certain serotypes of A. pleuropneumoniae
contain two different Hly operons, which were detected
subsequently and by different laboratories, various desig-
nations for these genes and their products have been used,
which are listed in Table I for a better understanding.
In order to clarify the role of the different genes in-
volved in Hlyl and Hlyll production, activation and
secretion in the different serotypes, we have analyzed the
genetic organisation of the hlyl and hlyll determinants
in the serotype reference strains. In addition, the degree
of conservation of the genes encoding the structural pro-
teins of these Hly among the different serotypes was esti-
mated by gene cloning or amplification and subsequent
restriction enzyme fragment analysis.
RESULTS AND DISCUSSION
(a) Analysis of the h&Z operon in serotypes 1-12
In order to get a complete picture of all genes belonging
to the hlyl operon in the different A. pleuropneumoniae serotype reference strains (Table II), Southern blot analy-
sis was performed using total genomic DNA digested
with Pstl and EclXI and probes for the individual hly genes and for a segment upstream from hlylC, containing
the promoter sequences, The location of the different
probes on the hlyl operon is shown in Fig. I. Table II
summarizes the results obtained from the hybridization
experiments. For the serotypes 1, 9, 10 and 11 all probes
for the Hly genes hlylC, hlylA(both N-and C-terminal),
hlylB and hlylD and the segment upstream from hlylC hybridized with a 9.2-kb Pstl-EclXI fragment, indicating
that these genes are closely linked like the genes of the
hlyl operon of serotype 1 (Fig. 2). This genetic arrange-
ment is common to most RTX operons found in many
different pathogenic bacteria (Welch, 1991). In serotypes
5a and 5b, the probes for hlylC, hlylA(N-and C-terminal),
hlylB and hlylD and the probe for the segment upstream
from hlylC hybridized to a 20-kb PstI-EclXI fragment.
When EcoRI + Pstl-digested genomic DNA of serotypes
1, 5a, 5b, 9, 10 and 11 was used: hlylC and hlylA probes
hybridized to a 4.8 kb-fragment in all these serotypes
(Fig. 3 B). Further hybridizations indicated that in sero-
types 5a and 5b the structure of the hlyl operon is the
same as in serotype 1 and that the segment downstream
from the hlyl operon must be different (Fig. 2). The hy-
bridization results with serotypes 2, 4, 6, 7, 8 and 12
showed no signal for h/y16 and no signal for the N-termi-
nal part of the hlylA gene, but a weak reaction with a
6-kb Pstl-EclXI fragment when the C-terminal hlylA probe was used. This 6-kb fragment also hybridized to
the probe for the sequences upstream from hlylC and to
the hlylB and hlylD probes (Table II). When
EcoRI + Pstl-digested genomic DNA was used, a weak
signal of a 1.3-kb fragment, hybridizing to the C-terminal
hlylA probe, could be seen in these serotypes (Fig. 3 B).
The results indicate that the serotypes 2,4, 6, 7, 8 and 12,
which do not produce Hlyl (Frey and Nicolet, 1990; Frey
et al., 1992) contain part of the hlyl operon including the
promoter sequences, the genes hlyIB and hlylD and
aproximately 300-500 bp of the 3’ end of the hlylA gene,
but are lacking hlylC and most of hlyIA (Fig. 2). These
hybridization results and the comparison of the restric-
tion map of the hlyl operon (Gygi et al., 1992) with that
of the genes appB and appD (Chang et al., 1991) show
that the truncated hlyl operon in serotypes 2,4,6,7,8 and
12, which contains a hlylC-hlylA deletion (Fig. 2), is iden-
tical or very similar to the appB and appD gene segment.
Further evidence for our finding that appB and appD
53
TABLE I
Synonyms used for the hemolysin proteins and genes among different Actinobacillus pleuropneumoniae strains
Original designation Other names used
Name Abbreviation Name Abbreviation
toxin protein toxin protein
genes genes
Hemolysin I HlyI”’ Cytolysin I Cly1’5’
hlylC’=’ clylC’6’
hlylA’2J) clylA’6’
hlylB’=’ clylB’“’
hlyZP3’ clyLD’6’
Name
toxin
Abbreviation
protein
genes
.ppBb”’
,pplF)
Name
toxin
Abbreviation
protein
genes
Hemolysin II HlyII’”
hlyZIC”‘4’
hlyllA”‘4’
Cytolysin II ClyI1’5’
clyllC’6’
cly1’1A’~’
Hemolysin App”’ Cytolysin Cyt’9’
appC”’ cyu?
appA’7’ cytA@
The hemolysin of A. pleuropneumoniae were initially named hemolysin I”’ (HlyI), hemolysin II”’ (HlyII). Various groups have later on used different
designations for the proteins and for their genes which are compiled in this table.
a Names for hemolysin II genes in analogy to the protein HlyII.
b Initially believed to belong to the app determinant “) but later found to be hlylB and hlylDc3’ References: (1) Frey and Nicolet, 1988b; (2) Gygi et al., , 1990; (3) Gygi et al., 1992; (4) Frey et al., 1992; (5) Kamp et al., 1991; (6) Jansen et al., 1992; (7) Chang et al., 1989; (8) Chang et al., 1991; (9) Anderson
et al., 1991)
TABLE II
Hybridization of hlyl probes to genomic DNA of Actinobacillus pleuropneumoniae serotypes reference strains digested with PstI and EclXI
Serotype
Probe
1 2 3
Fragment size (kb)”
4 5a 5b 6 7 8 9 10 11 12
Upstream 9.2 6 1 6 20 20 6 6 6 9.2 9.2 9.2 6
hlyIC
hlylC 9.2 20 20 9.2 9.2 9.2 hlyZA N-term 9.2 20 20 - - 9.2 9.2 9.2
hlyIA C-term 9.2 6 6 20 20 6 6 6 9.2 9.2 9.2 6
hlyIB 9.2 6 6 20 20 6 6 6 9.2 9.2 9.2 6 hlylD 9.2 6 - 6 20 20 6 6 6 9.2 9.2 9.2 6
a Numbers indicate the band size in kb. -, indicates no hybridization signal was detected. Probes for Southern blots are described in Fig. 1 and the
conditions for hybridizations are given in Fig. 3 B. The A. pleuropneumoniae serotype reference strains used were: serotype 1, 4074; serotype 2, S1536;
serotype 3, S1421; serotype 4, M62, serotype 5a, K17; serotype 5b, L20; serotype 6, ferna; serotype 7, WF83; serotype 8, 405; serotype 9, CVI 13261;
serotype 10, 13039; serotype 11, 56153; serotype 12, 8329; as described previously (Frey et al., 1992). A. pleuropneumoniae strains were grown in
Columbia broth supplemented with 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, MD. USA) and 10 pg/ml NAD (Sigma Chemicals Co.,
USA) at 37°C with shaking (Frey et al., 1988a). Genomic DNA was isolated by the guanidinium thiocyanate method (Pitcher et al., 1989).
segment is identical to the hlyIB and hlyID genes of the
truncated hlyl operon is provided by the fact that the ‘hly
pseudo-gene’ upstream from appl? and appD (Chang
et al., 1991) corresponds to the 3’ end of the partially
deleted hlyIA gene. In serotype 3 the probe for the se-
quences upstream from hlylC hybridized to a 1-kb PstI-
EclXI fragment indicating that all genes of the hly1 op-
eron are deleted in this serotype (Fig. 2).
The above results indicate that the presence of the
hlyIA gene encoding the structural pre-HlyI protein can
be used as indicator to show whether a strain belongs to
the highly virulent HlyI producing group of A. pleuro-
pneumoniae. PCR amplification with total DNA of sero-
types 1-12 as template and oligo primers HLYIA-L and
HLYIA-R corresponding to the N-terminal and C-termi-
nal coding sequences of the hlyZA gene from serotype-1
strain 4074 (Frey et al., 1991) was used for the detection
of hlyZA. As demonstrated in Fig. 3 A, PCR amplification
produced a 3.1 -kb fragment in serotypes I,9 and I 1 corre-
sponding to the full length DNA of the hlyZA gene. Diges-
tion of this DNA with frequently cutting restriction
enzymes AluI, D&I, Dpd, Hid, HphI, MaeII and RsaI
showed identical restriction fragment profiles indicating
that these genes are highly similar or identical (results not
shown). However, no PCR amplification of a hlylA gene
could be obtained with the above named primers in sero-
types 5a, Sb and 10, even when the annealing temperature
was reduced stepwise from 54°C to 44°C. This was in
I I I I I I I I II I I, II I I III I \II II 1 I I
ml WA II hlylB II hiylD 1
upstream h/y/C
h/y/C
h/y/A
h/y/A N-terminal h/y/A C-terminal
h/v/D
Fig. 1. Location of the different probes for hly genes used for the hybridization experiments. The upper part represents a physical and genetic map
of the h/y1 operon as described (Gygi et al., 1992). The scale is given in kb. The DNA probes for the A. pleuropneumoniae hlyl genes shown in the
lower part were isolated from plasmid pJFF750, containing the full hIyICABD operon from serotype 1 strain 4074 (Gygi et al., 1992). The probe for
the regions upstream from the hlylC gene was a I.l-kb PstI-XhoI fragment. Probe hlylC was a 300-bp SspI-XhoI fragment, hlylB was a 2.1-kb BglII-
BglII fragment and MyID a 800-bp EcoRI-EcoRV fragment. Methods: The hlylA probe was generated by PCR amplification of the 3.1.kb coding
sequence of hlylA with the primers HLYIA-L (5’-TGGCTAACTCTCAGCTCG-3’) and HLYIA-R (5’-ATAGACTAACGGTCCGCC-3’). Cutting
the hlyIA PCR amplified fragment with the restriction enzyme EcoRV gave a 2.3-kb N-terminal hlyIA probe and a 800-bp C-terminal hly1A probe.
Probes for the hly11 genes (not shown in the figure) were produced by PCR amplification. Probe appA was a 2%kb fragment amplified from
chromosomal serotype 1 DNA with the primers APPSA-LT (5’-CCCATATGGATCCGTCAAAAATCACTTTGTCATCATT-3’) and APPSA-RT (5’-
TCCGGAATTCAAGCGGCTCTAGCTAATTGA-3’) corresponding to the beginning and end of the appA sequence (Chang et al., 1989). Probe appC was produced by first amplifying a 3.3-kb appCA fragment with the primers APPSC-LT (5’-CGCGGATCCGTTGCCTTGTTTTCCTTCAC-3’)
corresponding to the beginning of the appC gene (Chang et al., 1989) and oligo APPSA-RT, and subsequent isolation of a 500-bp appC fragment
obtained by digestion with &/I. PCR amplifications were performed in a thermal cycler (Perkin Elmer Cetus, Norwalk, Conn. USA) with the following
parameters: denaturation at 94°C for 1 min, annealing at 54°C for 1 min for the primers HLYIA-L and HLYIA-R or at 52°C for 1 min for the primers
APPSC-LT, APPSA-LT and APPSA-RT, elongation at 74°C for 2.5 min, total of 35 cycles, by using the protocol as described (Innis et al., 1990). The
oligos were produced by Microsynth, Windisch, Switzerland.
contrast to biochemical and serological evidence for the
production of a HlyI protein by serotypes 1, 5a, 5b, 9, 10
and 11 (Kamp et al., 1991; Frey et al., 1992) and to the
results of Southern hybridization using the hlyIA gene as
a probe (Fig. 3 B).
For further analysis of the hly1A genes in serotypes 1,
5a, 5b, 9, 10 and 11 we have cloned the hlyICA genes as
4%kb &I-EcoRI fragments from genomic DNA of these
strains onto vector pBluescriptIISK (Stratagene, La
Jolla, CA, USA). The clones from all these serotypes pro-
duced in E. coli XLl-blue strain (supE44, hsdR17, recA1, endA1, gyrA96, thi, relA1 A[proAB-LX], [F’proAB laclq
lacZAM15, TnZO (TcR)]) (Bullock et al., 1978) a narrow
hemolytic zone without induction of the vector borne lac
promoter. The 105-kDa HlyI protein could be identified
in these clones with monoclonal anti-HlyI antibodies on
immunoblots. These results showed that the recombinant
clones expressed the hlyZCA genes. No HlyI was detected
in culture supernatants of cultures from these clones re-
flecting the absence of the Hly secretory genes hlyZB and
hlyZD. The hlyIA genes from the clones were subcloned
as XhoI-EcoRI fragments onto vector pBluescriptIISK -.
Restriction analysis of the resulting plasmids using the
enzymes AluI, DdeI, DpnI, HinfI, HphI, Mae11 and RsaI
showed two different types of restriction fragment profiles
indicating two classes of genes (Fig. 3 C). The restriction
fragment patterns of the cloned genes from serotypes 1,
9 and 11 were identical (Fig. 3 C) and corresponded to
the DNA sequence as determined (Frey et al., 1991). The
patterns obtained from the clones of serotypes 5a, 5b and
10 were identical among each other but showed clear
differences to hlylA from serotypes 1, 9 and 11 (Fig. 2 C).
In this respect, it is important to note that the strains of
serotypes 1,9 and 11 are serologically closely related
(Mittal, 1990; Nicolet, 1988). The degree of divergence
between the hlylA genes from the group of serotypes 1,
9 and 11 compared to the group of serotypes 5a, 5b and
10 is estimated from the number of different restriction
fragments in the patterns obtained with the frequently
cutting restriction enzymes to be approximately 5%. This
explained the failure of PCR amplification of the hlyZA genes from the serotypes 5a, 5b and 10 with the probes
corresponding to the sequence of hly1A from serotype 1.
The HlyI proteins from both groups of serotypes seem to
be biochemically very similar. So far, no monoclonal anti-
bodies have been found which are able to distinguish
between HlyI from different serotypes. However, the
above described differences in the hlyIA genes might be
useful for taxonomic purposes and as epidemiological
markers.
55
0 1 2 3 4 5 5 7 8 9
I I I I I I I I I I
Serotypes
1
2
3
4
5a
5b
6
7
8
9
10
11
12
h/YlC h/y/A h/y/B h/y/D r-- ._ I :x- . I I I I P
.---._ I E E L ._
.._ .._ !
P ‘.,E‘. E I_ - . . _._- .
_-. L _.. - -.: -__
--G---l P L
7-- ----- h/y/B h/y/D
r I I I P E E I.
h/y/C h,ylA h/y/B hlylD I I I I
II ,
P x E E L
/lryrc h/ylA /IlylE hlylD
I I I I I P x E E I_
i Ii ~-__
h/y/B htyylD I I I P E E L
h/y/B h/y/D
I 3 II I
I
P E E L
1 II h/y/B hlyl0 I I 1 P E E I.
h/y/C My/A hly/B h/y/D
,‘-- 1
I I I x E E L
h/y/C h/y/A hlylt3 hly/D
I I I I I P x E E L
h/y/C h/y/A h/y/B i&ID I I I I I P x E E L
1 II h/y/B h/y/D I I I P E E L
Fig. 2. Genetic organisation of the hlyl operons. Open boxes represent the location of the various hlyl genes. Broken boxes represent the remaining
part of hlyIA in A. pleuropneumoniae serotypes 2, 4, 6, 7, 8 and 12. Broken lines show the possible deletion formations. P, PSI, E, EcoRI, L; EclXI.
The scale on the top is given in kb.
(b) Analysis of the hZyZZ operon tion on Southern blots using genomic DNA from the
The hlyII operon contains the genes appA encoding different serotypes digested with PstI and probes for the
pre-HlyII also named AppA protein (see Table I) and its appA and appC genes confirmed these results and re-
activator gene appC (Chang et al., 1989; Frey et al., 1992). vealed small differences upstream from appC and down-
Downstream of appA only a small segment resembling stream from appA (results not shown). Initially it was
the 5’ beginning of a hlyB-like protein was found (Chang believed that the genes appB and appD were the corre-
et al., 1991; Jansen et al., 1992). PCR amplification using sponding secretion genes belonging to this Hly determi-
total genomic DNA of each serotype as template and the nant (Chang et al., 1991). However, we have shown that
primers APPSC-LT and APPSA-RT, corresponding to appB and appD are indeed hlyIB and hlyID and belong
the beginning of the appC and the end of the appA gene to the HlyI operon as demonstrated in section a. This
(Chang et al., 1989), produced a 3.4-kb fragment in all explains the fact that these secretion genes were initially
serotypes except 10, showing the presence of the linked found to be unlinked to the structural and activator
appIICA genes in these serotypes. DNA:DNA hybridiza- genes. We have found no minor bands on the autoradio-
56
Hlyl Hlyll
M, 1 2 3 4 5a5b 6 7 8 9 1011 12
I ii 1 2 3 4 5a 5b 6 7 8 9 ICI II 12 1 Ii I 2 3 4 5a5b 6 7 8 9 10 Ii 12
M, 1 2 3 4 5a5b 6 7 8 9 1011 12
c
M, i 2 3 4 5a5b 6 7 8 9 1011 12
Fig. 3. Analysis of the structural genes encoding HlyI and HlyII. Left part: hlyI.4 genes encoding the structural protein, HlyI. Right part: app.4 genes encoding the structural protein HlyII. (Panel A) PCR amplified hfq’fA, and upp.4 genes respectively, from serotypes I-12 separated on 0.8% agarose
gels. M, is the molecular size standard, phage 1. DNA digested with Bind111 (23.1, 9,4,6.6,4,4,2.3,2.0,0.56 kb). (Panel B) Southern blot hybridizations
of P~~I~~c~RI-digested total DNA with probes hty1A N-te~inal (left) or appA (right). The open arrowheads give the position of the i. Hind111
markers 4.4 kb, 2.3 kb and 2.0 kb. Black arrowheads indicate the position of the 1.3-kb fragments in serotypes 2,4,6,7,8 and 12. The DNA fragments
were separated on 0.8% agarose gels, depurinated for 10 min in 0.2 M HCL, denatured for 60 min in 0.5 M NaGH/I.S M NaCl, neutralized for 30
min in 1 M TrisHCl pH 7.5/1.5 M NaCI, and transferred to nitrocellulose sheets by passive transfer as described (Ausubel et al., 1990). The DNA
probes were labelled with [a-3ZP]dCTP (3000 Ci/mmol, Amersham plc, UK) using random priming (Feinberg and Vogelstein, 1983). Hybridizations
were carried out for 25 h at 37°C in 5 x SSCjSO% formamide~S% polyethylene glycol 6000~0.5a~ SDS and 100 nglml denatured and sonicated salmon
sperm DNA. Filters were washed in 0.1 x SSC/O.l% SDS at 25°C corresponding to the melting temperature (T,) of DNA:DNA duplexes with
approximately 75% sequence identity. Autoradiography was carried out for 24 h on Fuji RX films using an intensifying screen. (Panel C) left:
Restriction enzyme profile of cloned hl,ylA genes from serotypes 1; 5a, 5b, 9, 10 and 11 digested with D&I. Growth of recombinant E. coli strains,
purificatjon of plasmids and DNA fragments from agarose gels, ligation and selection for recombinant clones are described (Ausubel et al., 1990: Sambrook et al., 1989). (C) right: Restriction enzyme profile of PCR amplified appA genes from the different serotypes digested with Hid. Mb is the molecular size standard, plasmid pBR322 digested with Hi& (1631. 517, 506, 396, 344, 298, 221/220, 154 and 75 bp).
graphs of the Southern hybridizations when hEylB and
I+lD probes were hybridized to chromosomal DNA of
each serotype at low stringency conditions, which pre-
viously allowed the detection of the hEylB gene of A. pleurqneumoniae with an E. coli hlyB probe (Gygi et al.,
1990). Since the genes encoding the HlyB secretion pro-
teins are generally the most conserved genes in the RTX
operons of various bacterial species, we expect that the
strains which we analyzed do not contain secretion genes
specific to the HlyII operon, analogue to h~~~~ and hEyfD.
These results agree with the findings that downstream
from the nppCA genes no genes for Hly secretion were
found (Chang et al., 1989; Smits et al., 1991). Secretion of
the the HlyIf protein seems therefore to occur by ~t”nns-
complementation with the help of the hE_ylB and hlylD gene products from the Hoyt operon. This view is sup-
57
by the fact that in serotypes 2, 4, 6, 7, 8 and 12,
which actively secrete HlyII (Frey and Nicolet, 1988b),
the truncated hlyl operon contains the whole hlyZB and
hlyID genes. Further evidence is given by the serotype 3
strain, which is devoid of hlyIB and hlyZD (Fig. 2), and
which releases only minute amounts of HlyII into the
growth medium (Frey and Nicolet, 1991).
In order to analyze whether the appA genes show heter-
ologies among the various serotypes we have amplified,
by PCR, the uppA genes from chromosomal DNA
(Fig. 3 C right). PCR amplification using oligo APPSA-
LT and APPSA-RT (see legend to Fig. 1) corresponding
to the beginning and end of the appA sequence (Chang
et al., 1989) produced a 2.8-kb fragment in all serotypes
except 10 (Fig. 3 A). Restriction fragment analysis of these
PCR fragments with AM, DdeI, DpnI, Hi&, HphI, Mae11
and RsaI showed no differences indicating that all appA genes in these strains are very similar. In contrast to re-
sults published by Jansen et al. (1992), our experiments
exclude a large insertion in the appA gene (corresponding
to hlyZ1A and to clyllA) in serotype 6. A small difference
among the appA genes was found with EcoRI in serotypes
6, 8, and 12 where appA contained an additional site.
Southern blot hybridization of PstI-EcoRI digested geno-
mic DNA from the different serotypes using the appA gene as a probe showed the internal 2-kb PstI fragment
which contains most of appA in all serotypes except 10
(Fig. 3 B). The second fragment seen on the blot in sero-
types 1, 2, 4, 5a, Sb, 7, 9 and 11 corresponds to a 3.5-kb
PstI fragment containing the N-terminal part of the appA gene and sequences upstream since the same fragment is
obtained with PstI-digested genomic DNA (data not
shown). In serotypes 6, 8 and 12 this fragment is lacking,
but a 500-bp fragment that hybridized with appA is pre-
sent due to the additional EcoRI site which is estimated
to be located about 200 bp downstream from the begin-
ning of uppA. Such EcoRI site could have been created
by a single G + A transition at nt 711 nt in the coding
sequence of appA (Chang et al., 1989) changing the aa
valine to the similar aa isoleucine which is expected not
to change significantly the structure or immunogenic epi-
topes of the HlyII protein. In serotype 3, a 5.5-kb frag-
ment is seen instead of the 3.5-kb fragment, indicating
that sequences outside the appA gene are different
(Fig. 3 B).
(c) Conclusions
(I) All serotypes of A. pleuropneumoniae contain two
different hly operons (hlyl and hly1Z), although frequently
truncated. Our data indicate that the two different Hly
operons did undergo rearrangements in A. pleuropneu- moniue by deletion formation.
(2) During evolution, the hlyll operon has probably
lost its genes encoding the secretion proteins B and D,
possibly due to high similarity to the hlylB and hlylD
genes.
(3) Subsequently, certain serotypes might have un-
dergone further deletions and lost the activator and struc-
tural genes hlyIC and hlyIA from the hlyl operon.
However, they retained the hlyZB and hlyID genes as well
as the segment containing the promoter of this operon
that allows the expression of hlyIB and hlylD. (4) Serotype 3 seems to have lost all genes of the hlyl
operon retaining only some sequences upstream the
hlyIC gene.
(5) One can therefore speculate that ancestors of A. pleuropneumoniae have contained two full hly operons
with all four genes (CABD).
ACKNOWLEDGMENTS
We are grateful to Han van den Bosch and Ruud
Segers, Boxmeer, Netherlands, for valuable help and
stimulating discussions. This work was supported by
grant 31l28401.90 from the Swiss National Science
Foundation.
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