An Aeromonas salmonicida gene required for the establishment of infection in rainbow trout (Oncorhynchus mykiss)

GENE AN I N T E R N A T I O N A L , J O U R N A L O N GENES AND GENOHES

ELSEVIER

Gene 175 (1996) 127-131

An Aeromonas salmonicida gene required for the establishment of infection in rainbow trout (Oncorhynchus mykiss) Brian Noonan, Trevor J. T r u s t * Department of Biochemistry and Microbiology, and Canadian Bacterial Diseases Network, University of Victoria, Victoria, B.C. V8W3P6, Canada Received 5 October 1995; revised 2 January 1996; accepted 19 January 1996

Abstract

The asoB gene of Aeromonas salmonicida is located approximately 9 kb downstream of the structural gene (vapA) for the surface layer (A-layer). The nucleotide sequence of asoB was determined and found to encode a putative polytopic cytoplasmic membrane protein which exhibited homology to a number of bacterial transport proteins. Allele exchange mutagenesis of asoB resulted in a mutant (A449-D) which was avirulent when administered by bath immersion. However, when administered by intraperitoneal injection, A449-D is as lethal as wild type. Characterization of the phenotype of A449-D showed that there were pleiotropic effects on VapA secretion, haemolysis and outer membrane protein composition. Mobilization of cloned asoB on a broad-host-range plasmid into A449-D resulted in the complementation of VapA translocation, haemolytic activity and virulence. Keywords: Secretion; Virulence; Complementation; A-layer; VapA translocation; A449-D, A. salmonicida asoB mutant

1. Introduction

The paracrystalline surface protein layer (A-layer) of the fish pathogen, Aeromonas salmonicida, is a tetragonal array of protein subunits of Mr 50 800, which contributes to the ability of the bacteria to colonise fish and cause disease (Trust, 1993). The A-layer appears to be multifunctional, contributing to serum resistance (Munn et al., 1982), protecting against the action of proteases (Chu et al., 1991) and phagocytic cells (Trust et al., 1983; Gardufio and Kay, 1992). The A-layer also binds certain porphyrins, immunoglobulins, as well as a range of extracellular matrix proteins (Trust, 1993). Mutants of A. salmonicida which are unable to secrete VapA through the outer m e m b r a n e accumulate large quantities of VapA

in the periplasm and are avirulent (Belland and Trust, 1985; N o o n a n and Trust, 1995a). The asoA gene of A. salmonicida, which is located approximately 7 kb downstream of vapA, has been characterized ( N o o n a n and Trust, 1995b) and shown to be required for the organization of the outer surface of the cell. Mutagenesis of the asoA gene of A. salmonicida results in strands of membrane and A-layer protruding from the cell. These mutants display increased systemic virulence, emphasizing the important role the A. salmonicida surface plays in pathogenesis ( N o o n a n and Trust, 1995b). This study deals with the molecular characterization of an open reading frame (asoB) 3' of asoA, required for the maximal secretion of VapA through the cytoplasmic membrane. Allele exchange mutagenesis of asoB resulted in mutants with reduced ability to colonize fish and cause disease.

* Corresponding author. Tel. + 1 604 7217079; Fax + 1 604 7216134; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ap, ampicillin; As, Aeromonas salmonicida; asoB, gene encoding AsoB; bp, base pair(s); Cm, chloramphenicol; ip, intraperitoneal; kb, kilobase(s)or 1000 bp; Kin, kanamycin; LD, lethal dose; LPS, lipopolysaccharide;mAb, monoclonal antibody(ies); nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamidegel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; Tc, tetracycline; TSA, tryptic soy agar; TSB, tryptic soy broth; vapA, gene encoding VapA; wt, wild type. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0378-1119(96)00137-0

2. Results and discussion

2.1. Cloning and nucleotide sequence of an O R F downstream of asoA The asoA gene of A. salmonicida was previously cloned on a 6-kb fragment in pUC18 (pB42)(Noonan and Trust, 1995b) (Fig. 1). The region of D N A downstream

128

B. Noonan, T.J. Trust/Gene 175 (1996) 127-131

A vapA

abcA

'~ iii~iiiii!i!!i!!!!!iiiiiiiil-lliii!ii!i!i!]iii!~

asoA

o 3

asoB B

1 kb

B asoA

pB42 (pUC 18)

I H

I B

I C

K

asoB

SK

H

B

Km

asoA

I C

p P M C B K m (pPM2101)

K

asoB

SK

H

B

aso B

p M M B S B (pMMB67EH)

S

H

B

Fig. 1. A. Chromosomal organization of aso genes. B. Principal clones used in this study. Vectors used indicated in brackets. Direction of transcription of all genes shown is from left to right. B = BamHI, C = ClaI, H = HindlII, K = KpnI, S =SalI, K m = kanamycin resistance gene. The E. coli strains used were DH5ct (Hanahan, 1983) and S17.1 (Simon et al., 1983). E. coli strains were grown in TSB or on TSA plates at 37°C. Aeromonas strains were grown on the same media but at 20°C. For E. coli, Ap and Km were used at a final concentration of 50 #g/mE For A. salmonicida, Ap and Km were used at a final concentration of 50/~g/ml and Tc and Cm were used at a final concentration of 100/~g/ml. The plasmids used in this study for cloning were pUC18/19 (Yanisch-Perron et al., 1985) and pMMB67EH/HE (FOrste et al., 1986). Small scale plasmid preparations were performed using the Wizard Minipreps DNA purification system (Promega) and all enzymes were utilized as recommended by the manufacturers.

of asoA was subcloned in pUC18 and the nucleotide sequence was determined in both strands using universal and custom oligonucleotide primers (Fig. 2). Analysis of the nucleotide sequence revealed an ORF, 1278 bp in length, which begins 145 bp downstream of the stop •

•

.

•

GCCTGATAAC~ACCGGTTAACACTGTTTGAATGAAGAJL%C A t a~oA aeoB ATGTCTGCTTT~GGATC M S A L K

K

D

L

TC GGGC TCTGGCAGAGAAT~C G L W Q R M

TC GC TC TGG~CTGGGTGTT~TCATCGTGCTGGTC, L V L P I A F T F A R C CTGC GGC CCTTACTATC P A A L T I

GC C~ A A

ATTGCCTTGGCCATATT~TGACC I A L A I L O GAAGCTTTCGC E A F A

L

GGCAAGCTGC C CACCTC G K L P T S

.

L

TC~TCACAACC L T T

GC TGGCTCGTC TCTCGGCC L A R L S A

GGC CTC TTCTC CCTGTG~CGCC G L F S L W R

R

G

D

Y

TCACTGCTGGGTACC S L L G T

.

.

W

R

C C TGTGGGC L W A

.

.

L

P

~ G A C ~ A T A

~ C ~ G ~ C ~ C A F G N G

CATC CAGC TGCTGAC I Q L L T

CCTGC TCGGGGT L L G V

GCGAC GCTGC C CAGC CACTGC CGACGGCAGCTGAGTGGC D A A Q P L P T A A E W

CC GC TGGC CC TGC TC GGCGGTGTCTT~ P L A L L G G V L

GC CGCTCAATGCTCTGTGCTGGGTGC P L N A L C W V

GC TATCTGC GC CGCAC C CGGGC CAC GGAGAATATCTGC Y L R R T R A T E N I C

CTG~GTTA~ L G Y

L

TC ~C A

A~ I

~ L

L

.

.

CTTAACAAGCAAGGGTTTGCGGCCTAACATGGC

G G T A T C ~ G ~ C G I F V V

GGAGCGC CGCACTTGATTGGTC G A P H L I G

CCTAC C GCCAAGTGGCTGGTGGCAATC P T A K W L V A I

CGGGGGGC R G A

.

13TTTGAGCAAGGGATAGTC

GCTGTTTGATC TGGGGGAGTGGAC L F D L G E W T

CTGCTCATCTGGTTCAAGGGGGATCTCAGCTGGC L L I W F K G D L S

CCTOCTCTCGCT~TGTTCGGC L L S L L F

.

G~

C TGC CCATC GC CTTCACCTTC~CC G R R Y P H A ~

C GGTTTC TGGCATGC G F W H A

T

G

C CACATGGOGGAGGAGTTCAAGCGACCTGAGCGGGACTAC H M G E E F K R P E R

~-%C G C C A C C O C C A T T C C G G C N A T A I P A

G CAGGTTAC A G Y

L

.

CCC CTTCCAAAAAGGC

codon of asoA. The G + C content of the ORF was 64%, which is higher than the reported G + C content of 55% for the A. salmonicida genome and the G + C content of the asoA gene (57%) immediately 5' of this ORF. 3' of the ORF, there is an inverted repeat followed by a

A

G

T S

~C A

GAGC ~ E R

P

CC ~ A

T C ~ C T G ~ A G C A ~ C A ~ S C P G S I N

GC CATC AAUTAACCAGC A I K •

~ CA~AC S I T

C~ATCC L I

CCGACAAAGAGC

~ T ~ A G

C G

C S

A

I

L

CAT~AC M G ~ S

V

150 50

~ G ~ G V G L

300 100

~ C ~ C S G N

C ~ ~ C ~ L Q L L

450 150

L

~ T

~ T ~ T G ~ T G C V M F W C

~ C U ~ C ~ P V G L

600 200

~ A ~

A

L

A

~ W

~ L

~ R

~C S

V

L

L

P

CCCA~C~ L I

~ T C ~ T A ~ T ~ A ~ C ~ C G A ~ C ~ V V L K Y G L Y G D E

T A T C ~ A ~ ~ C Y L Q G F A TCT~ W

~ G ~ A L W A

~ C ~ C ~ C C T G ~ A C ~ C F S A F L F L T V

ATTC C T G C T G G ~ G C ~ F L L G L R G

~ C A ~ T A ~ G T C C A C ~ G L I Y W V H

~ C ~ T C C L V

C L

A

. CC GATGTGTGGTTATTAATC~AC~T

~ C R

L

CCC GCAGTGCGGGGTTCTTTGT

P

R

L

A

~ A T ~ G C A T G ~ G A G I W S M A R

AC ~A~AGC~ATC L D E L

I

C GCTACGC R Y A

C~C N

K

750 250

E

900 300

~C G

1050 350

1320 4.26

Fig. 2. Nucleotide sequence of the asoB gene of A. salmonicida (accession No. L47259). A putative transcriptional terminator sequence is underlined. DNA sequencing reactions were performed using the Taq DyeDeoxy Terminator Cycle sequencing kit (Applied Biosystems Inc) using M13 forward and reverse primers as well as custom primers. Custom oligonucleotide primers were synthesised on an ABI PCR-MATE Oligosynthesiser using 40 nM columns using the protocols described by the manufacturer. Cycle sequencing reactions were performed using a Perkin Elmer DNA Thermal Cycler (Model 480).

B. Noonan, T.J. Trust/Gene 175 (1996) 127-131

T-rich region which may act as a transcriptional terminator (Fig. 2). The deduced aa sequence of asoB was determined and was found to encode a putative polytopic cytoplasmic membrane protein of predicted molecular mass 46 113 Da. The predicted protein consists of 60.1% hydrophobic aa and the Kyte-Doolittle hydropathic index, using an interval of 9 aa, gives an average hydrophobicity of 0.86 (Kyte and Doolittle, 1982). The deduced protein also contained 27% polar, 8.7% basic and 4.2% acidic aa and has a predicted pl of 9.87. A search of the protein sequence data bases showed that the deduced protein exhibited sequence homology to a number of polytopic cytoplasmic membrane transport proteins, sharing 25% identity with PotE (Kashiwagi et al., 1991) and 21% identity with CadB (Meng and Bennett, 1992).

2.2. Allele exchange mutagenesis of DNA downstream of asoA In order to examine the function of the ORF located 3' of asoA, a ClaI/BamHI fragment from pB42 (Fig. 1), containing this region, was cloned into the mobilizable vector, pPM2101 (Sharma et al., 1989). A 1.3-kb Km R cassette (Barany, 1988) was cloned into the HindlII site approximately 1 kb downstream of asoA (Fig. 1). This construct, p P M C B K m was mobilized into A. salmonicida A449 by conjugation. Exconjugants were initially screened for Km R and Ap s. Allele exchange was confirmed by Southern blotting of total DNA from putative mutants digested with BamHI using a KpnI/HindlII fragment containing the 3' region of the ORF, labeled with Digoxygenin, as a probe. In the allele exchange mutants the probe hybridized to a band 1.3 kb larger than in the wild type, which confirms the insertion of the Km R cassette (Fig. 3). One such mutant, A449-D, was taken for further study. A 2.5-kb SalI/BamHI fragment from pB42 (Fig. 1) containing the DNA downstream of asoA was cloned into pMMB67EH (pMMBSB) and conjugated into A449-D for use in complementation studies.

129

A

B

23.1-

9°4 m

6.6-

i

iiiii!ii!i!!!!?

4.4-

2.3Fig. 3. Southern blot confirming allele exchange mutagenesis in A. salmonicida A449-D.Total DNA from A449 (lane A) and A449-D (lane B) digested with BamHI. Figures on left are DNA molecular weight markers. Hybridization conditions were as described previously (Noonan and Trust, 1995b). The probes used in the hybridization experiments were isolated from an agarose gel using the QIAEX DNA purification system (QIAGEN) and labeled with Digoxygenin11-dUTP as described by the manufacturer (Boehringer Mannheim). The probe was used at a final concentration of 50ng/ml of hybridisation buffer. Table 1 Virulence of an A. salmonicida asoB mutant with rainbow trout (0. mykiss) ~ Strain

A449 A449-D A449-D (pMMBSB)

LDso Ip injection

Bath immersion

1.0 x 104 1.3 × 104 2.5 × 103

3.2 > 1.3 1.6

× × x

105 108 106

aExperimental fish (weight 8-10 g) were maintained at 13°C (+ I°C) in a continuous flow of dechlorinated city water. 40 fish were used for each LDs0 determination. Bacterial cultures were grown overnight at 20°C in TSB and diluted to the required concentrations with PBS (8 g/l NaC1, 0.2 g/1 KC1, 1.44 g/l NazHPO4, 0.24 g/1 KH2PO 4, pH 7.4). The bacterial cells were administed in volumes of 0.1 ml by ip injection or by immersion for 15 min with aeration. LDs0 determinations were carried out as described previously (Reed and Muench, 1938).

with pMMBSB resulted in the restoration of virulence almost to wild type levels (Table 1).

2.3. Virulence of A. salmonicida A449-D The asoA gene is required for the biogenesis of a normal outer surface and asoA mutants display increased systemic virulence. To determine if the mutation introduced downstream of asoA affected virulence, rainbow trout (Oncorhynchus mykiss) were challenged with A449, A449-D and A449-D (pMMBSB) by ip injection and by bath immersion (Table 1). No significant differences in LDso values were observed when the challenge was by ip injection, which indicates that the insertion mutation does not affect systemic virulence. However, when the mode of challenge used was bath immersion, A449-D was found to be avirulent. Complementation of A449-D

2.4. Pleiotropic effects of the insertion mutation in A449-D The A-layer of A. salmonicida is a principal component in virulence. To determine if the mutation in A449-D affected the synthesis or secretion of the A-layer subunits, cell fractionation studies were performed to localize VapA in the cell. SDS-PAGE analysis of cytoplasmic and outer membrane fractions of A449-D determined that the majority of VapA was localized in the outer membrane fraction as in wild type (data not shown). However, VapA was also readily detected associated with the cytoplasmic membrane fraction of A449-D.

130

B. Noonan, T.J. Trust/Gene 175 (1996) 127-131

W e s t e r n blot analysis of c y t o p l a s m i c m e m b r a n e p r e p a r a tions from A449 a n d A 4 4 9 - D with a m o n o c l o n a l antib o d y specific to A - p r o t e i n ( m A b A A 6 ) ( D o i g et al., 1993) c o n f i r m e d t h a t this b a n d was indeed V a p A (Fig. 4). T h e L P S of A 4 4 9 - D was a n a l y s e d b y S D S - P A G E of p r o t e i n a s e K digested whole cells, followed b y Silver stain or western b l o t t i n g with p o l y c l o n a l antisera. This s h o w e d t h a t the L P S of A 4 4 9 - D was i n d i s t i n g u i s h a b l e from t h a t of the wild type, A449 ( d a t a n o t shown). H o w e v e r , S D S - P A G E analysis of the p r o t e i n profiles of m e m b r a n e fractions of A 4 4 9 - D a n d A449 d e m o n s t r a t e d m a r k e d differences. T h e o u t e r m e m b r a n e of A 4 4 9 - D c o n t a i n e d r e d u c e d levels of a n u m b e r of proteins, b u t in p a r t i c u l a r , a c o n s i d e r a b l e r e d u c t i o n in the m a j o r p o r i n b a n d with an a p p a r e n t m o l e c u l a r m a s s of 41 k D a . O t h e r o u t e r m e m b r a n e a s s o c i a t e d p r o t e i n s that were present in r e d u c e d a m o u n t s were b a n d s with a p p a r e n t m o l e c u l a r masses of 47, 36, 27 a n d 24 k D a ( d a t a n o t shown). T h e h e m o l y t i c activity of A 4 4 9 - D was also affected b y the m u t a t i o n . It was o b s e r v e d t h a t the zone of h a e m o l y s i s a r o u n d A 4 4 9 - D g r o w n on b l o o d a g a r (5% horse b l o o d in T S A ) plates was significantly larger ( > 3-fold) t h a n t h a t of the p a r e n t strain. T h e O R F was n a m e d asoB (Aeromonas surface o r g a n i z a t i o n ) b e c a u s e of its role in cell surface biogenesis a n d its p r o x i m i t y to asoA. C o m p l e m e n t a t i o n with p M M B S B was f o u n d to be p a r t i a l l y effective. V a p A was no longer a s s o c i a t e d with the cytoplasmic membrane fraction in A 4 4 9 - D ( p M M B S B ) (Fig. 4). T h e presence of p M M B S B also resulted in a 53% r e d u c t i o n in the size of the zone of h e m o l y s i s ( d a t a n o t shown). H o w e v e r , r e s t o r a t i o n of n o r m a l o u t e r m e m b r a n e p r o t e i n levels was n o t o b s e r v e d in A 4 4 9 - D ( p M M B S B ) . To d e t e r m i n e if the p l e i o t r o p i c effects o b s e r v e d with A 4 4 9 - D were typical of all m u t a n t s g e n e r a t e d with p P M C B K m , the allele exchange experim e n t was repeated. M u t a n t s isolated from a further two

1

2

3

6645-

31Fig. 4. Western blot of cytoplasmic membrane fractions from A449 (lane 1), A449-D (lane 2) and A449-D-pMMBSB (lane 3) with a 1/5000 dilution of monoclonal antibody mAbAA6, specific to VapA. Cells were fractionated by 3 passages through a French pressure cell and the cytoplasmic and outer membrane fractions were prepared as described previously (Belland and Trust, 1985). SDS-PAGE was performed using the method of Laemmli (Laemmli, 1970). Protein samples were stacked in 4.5% acrylamide at 10mA followed by separation in 12.5% acrylamide at 20 mA. Proteins were stained with Coomassie Blue or else transferred to nitrocellulose or nylon membranes. Western blotting was carried out as described previously (Towbin et al., 1979).

m u t a g e n e s i s e x p e r i m e n t s were all s h o w n to possess the same p h e n o t y p e as A449-D.

3. Conclusions T h e c o m p l e t e nt sequence of a 1278-bp O R F 3' of the A. salmonicida asoA gene was d e t e r m i n e d a n d s h o w n to e n c o d e a p u t a t i v e p o l y t o p i c c y t o p l a s m i c m e m b r a n e protein. T h e asoB gene begins 145 b p d o w n s t r e a m of the s t o p c o d o n of asoA a n d is in the same orientation. H o m o l o g y between AsoB a n d o t h e r b a c t e r i a l transp o r t e r s suggests t h a t A s o B is involved in t r a n s l o c a t i o n across the c y t o p l a s m i c m e m b r a n e . Allele exchange m u t a genesis of asoB resulted in m u t a n t s which exhibited p l e i o t r o p i c effects on V a p A t r a n s l o c a t i o n across the c y t o p l a s m i c m e m b r a n e , h e m o l y t i c activity a n d o u t e r membrane protein composition. W h e n a d m i n i s t e r e d to fish by ip injection an asoB m u t a n t , A449-D, was as virulent as wild type. H o w e v e r when the m o d e of delivery was b y b a t h immersion, A 4 4 9 - D was shown to be avirulent. The increase in the h e m o l y t i c activity of A 4 4 9 - D d i d n o t c o u n t e r a c t the overall effect of the asoB m u t a t i o n . It has been well established t h a t the A - l a y e r plays a significant role in the virulence of A. salmonicida (reviewed b y Trust, 1993). T h e r e d u c e d t r a n s l o c a t i o n of V a p A across the cytop l a s m i c m e m b r a n e in A 4 4 9 - D m a y affect the synthesis a n d a s s e m b l y of the A-layer, resulting in increased susceptibility to host defense mechanisms.

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cholerae: molecular cloning of genes involved in pilus biosynthesis and evaluation of TCP as a protective antigen in the infant mouse model. Microb. Pathogen., pp. 437 448. Simon, R., Priefer, U. and Puhler, A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1,784-791. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Trust, T.J. (1993) Molecular, structural and functional properties of Aeromonas S-layers. In: Beveridge, T.J. and Koval, S.F. (Eds.), Advances in Bacterial Paracrystalline Surface Layers. Plenum Publishing Corporation, New York, pp. 159-171. Trust, T.J., Kay, W.W. and Ishiguro, E.E. (1983) Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida. Curr. Microbiol. 9, 315-318. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119.