Vol. 60, No. 8

INFECrION AND IMMUNUIY, Aug. 1992, p. 3059-3064 0019-9567/92/083059-06$02.00/0 Copyright C 1992, American Society for Microbiology

Functional Analysis of the Ca2+-Regulated Hemolysin I Operon of Actinobacillus pleuropneumoniae Serotype 1 DANIEL GYGI,"12 JACQUES NICOLET,1 COLIN HUGHES,2 AND JOACHIM FREY`* Institute for Veterinary Bacteriology, University of Berne, Laenggasstrasse 122, CH-3012 Berne, Switzerland and Department ofPathology, Cambridge University, Cambridge CB2 1QP, United Kingdom2 Received 27 January 1992/Accepted 4 May 1992

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlIy; gene designation, hiyl) by ActinobaciUilus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hblyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyL4) to active hemolysin I, while hlyIB and hlyID are necessary for H1yIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyIC4, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher inA. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hdyI determinant.

Actinobacillus pleuropneumoniae, a gram-negative bacterium belonging to the family Pasteurellaceae, is the cause of porcine pleuropneumonia (38), and the strong hemolytic activity of most isolates is believed to be an important factor determining virulence (17, 32). The hemolysin of A. pleuropneumoniae serotype 1 4074T has been purified and shown to be a 105-kDa protein with a pI of 4.3 (16, 17). It was recently demonstrated by gene cloning and by use of monoclonal antibodies that this strain indeed produces two hemolysins with very similar molecular weights, strongly hemolytic HlyI and weakly hemolytic HlyII (19), called also ClyI and ClyII, respectively (23). Both hemolysins were postulated earlier in various A. pleuropneumoniae serotypes by means of biochemical and physiological parameters (18). While HlyII is produced by most serotype reference strains, HlyI seems to be produced only by a few serotypes that seem to be particularly virulent (18, 19, 23). Structural gene hlyIA, encoding inactive 105-kDa prohemolysin I ofA. pleuropneumoniae serotype 1, has been cloned and expressed in Escherichia coli (21). trans-Activation of prohemolysin I (HlyIA) was achieved with the HlyC activator proteins from E. coli and Proteus vulgaris, and HlyIA secretion was effected by the E. coli HlyB and HlyD proteins supplied in trans. The hlyI determinant of A. pleuropneumoniae is therefore assumed to be structurally and functionally related to the E. coli hly operon, despite its low DNA sequence similarity. Sequence analysis of the hlyL4 gene (15) confirmed that A. pleuropneumoniae hemolysin I belongs to the RTX (for repeats in the structural toxin) family of pore-forming hemolysins and shows strong similarities to the E. coli hemolysin. HlyII (19), encoded by the gene appA (6), also belongs to the RTX family but is more related to the Pasteurella haemolytica leukotoxin (15). RTXs are widely spread throughout gram-negative bacterial pathogens of humans and animals, including E. coli, P. haemolytica, P. vulgaris, Morganella morganii, Bordetella pertussis, and Actinobacil*

Corresponding author.

lus actinomycetemcomitans (12, 24, 27, 29, 30; for a review, see reference 42). All the RTXs require Ca2" for their pore-forming activity, but it has been proposed that Ca2+ is also needed for the synthesis of HlyLA in A. pleuropneumoniae, possibly at the transcriptional level (17, 18). We present here the structural and functional characterization of the complete hlyI determinant of A. pleuropneumoniae serotype 1 4074T, confirming that hlyI is analogous to the hemolysin determiniant of E. coli in all respects, except that its expression appears indeed to be specifically regulated by Ca2+.

MATERIALS AND METHOD9 Bacterial strains and plasmids. A. pleuropneumoniae serotype 1 reference strain 4074T (34) was the source of DNA and RNA. The E. coli strains used.were NM 538 (supF hsdR trpR lacY), NM 539 (supF hsdR lacY [P2 coX]) (20), and XL1-Blue {supE44 hsdR17 recAl endAI gyrA96 thi reLAI del(proAB-lac) F' [proAB laclIq lacZdelM15 TnlO (Tcr)]} (4). The A. pleuropneumoniae h*yL4 probe was purified from plasmid pJFF702 (21). The DNA probes for the E. coli hemolysin genes were isolated from plasmids pANN202 (a 600-bp SspI-PstI fragment for hlyC) (33), pEK5OX (a 3,074-bp Tth11lI-PvuII fragment for hlyA) (28), and pLG575 (a 1,918-bp HpaI-AccI fragment for hlyB and a 1,404-bp EcoRI fragment for hlyD) (31). Bacteriophage XEMBL4 (20) and plasmids pBluescriptIIKS+ (Stratagene, La Jolla, Calif.), pUC19 (43), and pACYC184 (5) were used for cloning and subcloning of the hlyI determinant. The Ql fragment was taken from plasmid pHP45-fl (14, 36). All standard recombinant DNA manipulations were performed as described by Ausubel et al. (2) and Sambrook et al. (37). Partially digested A. pleuropneumoniae chromosomal DNA was size fractionated by ultracentrifugation on a 5 to 25% NaCl gradient in Tris-HCl (10 mM)-EDTA (1 mM) (pH 8.0) buffer (2). E. coli strains were grown in Luria-Bertani broth (37), except for expression of the cloned hlyI genes, for which Columbia broth (BBL Microbiology Systems) was used. A. pleuro3059




pneumoniae 4074T was grown in Columbia broth supplemented with 1% IsoVitaleX (BBL) and 10 ,ug of P-NAD (Sigma Chemical Co.) per ml. Media were supplemented with 50 p,g of ampicillin per ml for selection of pBluescript IIKS+, 10 ,ug of tetracycline per ml for propagation of strain XL1-Blue and selection of pACYC184, and 100 ,g of spectinomycin per ml for selection of the Ql fragment. Expression of the cloned genes in pBluescriptIIKS+ was induced in cells at an A650 of 0.5 by the addition of 0.5 mM isopropyl-O-Dthiogalactopyranoside (IPTG) and 1 h of incubation. DNA hybridizations. Plaques of the gene library of A. pleuropneumoniae 4074T were transferred to nitrocellulose (Transblot; Bio-Rad), and lambda DNA was denatured and neutralized by standard methods (37). The hlyL probe was labelled with [32P]dCTP (3,000 Ci/mmol; Amersham) by random priming (11). Hybridization was carried out at 37°C in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0])-50% formamide-5% polyethylene glycol 6000-0.5% sodium dodecyl sulfate (SDS)-100 ,g of denatured and sonicated salmon sperm DNA per ml. Filters were washed in O.lx SSC-0.1% SDS at 55°C. For Southern hybridization, DNA fragments were separated in an 0.8% agarose gel and transferred to nitrocellulose (Transblot) by use of the LKB 2016 Vacugene blotting system. DNA probes for the E. coli hemolysin genes were labelled as described above. Hybridization was carried out at 37°C in 5 x SSC-5% polyethylene glycol 6000-0.5% SDS100 ,ug of denatured and sonicated salmon sperm DNA per ml. Filters were washed in O.lx SSC-0.1% SDS at 25°C, corresponding to the melting temperature of DNA-DNA duplexes with approximately 75% sequence identity. Autoradiography was carried out for 24 h at -80°C on Fuji RX film with an intensifying screen. Northern blot and dot blot hybridizations. A. pleuropneumoniae 4074T was grown to anA650 of 0.7 in Columbia broth supplemented with 1% IsoVitaleX and 10 ,g of P-NAD per ml. The addition of 5 mM CaCl2 resulted in a medium with a concentration of free Ca2+ of 1.5 mM, as measured with a Ca2' electrode, and the addition of 1 mM ethylene glycolbis(3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in a medium with

Functional analysis of the Ca(2+)-regulated hemolysin I operon of Actinobacillus pleuropneumoniae serotype 1.

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype...
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