Riboflavin mutants as vaccines against Actinobacillus pleuropneumoniae

Described is a vaccine against Actinobacillus pleuropneumoniae (APP) comprising genetically defined biochemically attenuated mutant of APP that requires riboflavin and is attenuated in vivo.

TECHNICAL FIELD 
The invention relates to vaccines and in particular, live vaccines against 
Actinobacillus pleuropneumoniae (APP) and related bacterial pathogens. The 
invention is also concerned with recombinant techniques for preparing such 
a vaccine. 
BACKGROUND OF THE INVENTION 
An organism known as Actinobacillus pleuropneumoniae (APP) is a gram 
negative coccobacillus organism that is found in the pig and causes 
pneumonia in the pig. 
This disease is characteristically an acute necrotizing hemorrhagic 
bronchopneumonia, with accompanying fibrinous pleuritis (Fenwick, B. and 
S. Henry. 1994. Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 
204:1334-1340)(Sebunya, T.N.K. and J.R. Saunders. 1983. Haemophilus 
pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 
182:1331-1337). Porcine pleuropneumonia is an economically devastating, 
severe and often fatal disease with clinical courses ranging from 
hyperacute to chronic infection (Fenwick, B. and S. Henry. 1994. Porcine 
pleuropneumonia. J. Am. Vet. Med. Assoc. 204:1334-1340)(Hunneman, W. A. 
1986. Incidence, economic effects, and control of Haemophilus 
pleuropneumoniae infections in pigs. Vet. Quarterly 8:83-87). The 
existence of at least twelve antigenically distinct capsular serotypes 
(Perry, M. B., E. Altman, J. -R. Brisson, L. M. Beynon, and J. C. 
Richards. 1990. Structural characteristics of the antigenic capsular 
polysaccharides and lipopolysaccharides involved in the serological 
classification of Actinobacillus pleuropneumoniae strains. Serodiag. 
Immunother. Infect. Dis. 4:299-308) has made development of a 
cross-protective vaccine difficult. Killed whole cell bacterins provide at 
best serotype-specific protection (Nielsen, R. 1984. Haemophilus 
pleuropneumoniae serotypes--Cross protection experiments. Nord. Vet. Med. 
36:221-234)(Nielsen, R. 1976. Pleuropneumonia of swine caused by 
Haemophilus pleuropneumoniae. Studies on the protection obtained by 
vaccination. Nord. Vet. Med. 28:337-338) (Rosendal, S., D. S. Carpenter, 
W. R. Mitchell, and M. R. Wilson. 1981. Vaccination against 
pleuropneumonia in pigs caused by Haemophilus pleuropneumoniae. Can. Vet. 
J. 22:34-35)(Thacker, B. J., and M. H. Mulks. 1988. Evaluation of 
commercial Haemophilus pleuropneumoniae vaccines. Proc. Int. Pig Vet. Soc. 
10:87). In contrast, natural or experimental infection with a highly 
virulent serotype of A. pleuropneumoniae elicits protection against 
reinfection with any serotype (Nielsen, R. 1979. Haemophilus 
parahaemolyticus serotypes: pathogenicity and cross immunity. Nord. Vet. 
Med. 31:407-413)(Nielsen, R. 1984. Haemophilus pleuropneumoniae 
serotypes--Cross protection experiments. Nord. Vet. Med. 
36:221-234)(Nielsen, R. 1974. Serological and immunological studies of 
pleuropneumonia of swine caused by Haemophilus parahaemolyticus. Acta Vet. 
Scand. 15:80-89). In several recent studies, attenuated strains of A. 
pleuropneumoniae produced by chemical mutagenesis, serial passage, or 
other undefined spontaneous mutation have been tested as live vaccines, 
with promising results (Inzana, T. J., J. Todd, and H. P. Veit. 1993. 
Safety, stability and efficacy of nonencapsulated mutants of 
Actinobacillus pleuropneumoniae for use in live vaccines. Infect. Immun. 
61:1682-1686) (Paltineanu, D., R. Pambucol, E. Tirziu, and I. Scobercea. 
1992. Swine infectious pleuropneumonia: Aerosol vaccination with a live 
attenuated vaccine. Proc. Int. Pig. Vet. Soc. 12:214) (Utrera, V., C. 
Pijoan, and T. Molitor. 1992. Evaluation of the immunity induced in pigs 
after infection with a low virulence strain of A. pleuropneumoniae 
serotype 1. Proc. Int. Pig Vet. Soc. 12:213). However, the use of live 
vaccines in the field is problematic, particularly when the attenuating 
lesion in the vaccine strain has not been genetically defined. A 
well-defined mutation that prevents reversion to wild-type would be 
extremely desirable for the development of a live attenuated vaccine 
against Actinobacillus pleuropneumoniae. 
A variety of mutations in biosynthetic pathways are known to be attenuating 
in other organisms. Lesions in aro(Hoiseth S. K. and B. A. D. Stocker. 
1981. Aromatic-dependent Salmonella typhimurium are non-virulent and 
effective as live vaccines. Nature (london). 291: 238-239)(Homchampa, P., 
R. A. Strugnell and B. Adler. 1992. Molecular analysis of the aroA gene of 
Pasteurella multocida and vaccine potential of a constructed aroA mutant. 
Mol. Microbiol. 6: 3585-3593)(Homchampa, P., R. A. Strugnell and B. Adler. 
1994. Construction and vaccine potential of an aroA mutant of Pasteurella 
haemolytica. Vet. Microbiol. 42:35-44) (Karnell, A., P. D. Cam, N. Verma 
and A. A. Lindberg. 1993. AroD deleteion attenuates Shigella flexneri 
strain 2457T and makes it a safe and efficacious oral vaccine in monkeys. 
Vaccine 8:830-836.) (Lindberg, A. A., A. Karnell, B. A. D. Stocker, S. 
Katakura, H. Sweiha and F. P. Reinholt. 1988. Development of an 
auxotrophic oral live Shigella flexneri vaccine. Vaccine 
6:146-150)(O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. 
Dougan. 1988. Characterization of aromatic and purine dependent Salmonella 
typhimurium: attenuation, persistence and ability to induce protective 
immunity in BALB/c mice. Infect. Immun. 56:419-423)(Vaughan, L. M., P. R. 
Smith, and T. J. Foster. 1993. An aromatic-dependent mutant of the fish 
pathogen Aeromonas salmonicida is attenuated in fish and is effective as a 
live vaccine against the Salmonid disease furunculosis. Infect. Immun. 
61:2172-2181), pur (O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. 
Easmon and G. Dougan. 1988. Characterization of aromatic and purine 
dependent Salmonella typhimurium: attenuation, persistence and ability to 
induce protective immunity in BALB/c mice. Infect. Immun. 
56:419-423)(Sigwart, D. F., B. A. D. Stocker, and J. D. Clements. 1989. 
Effect of a purA mutation on the efficacy of Salmonella live vaccine 
vectors. Infect. Immun. 57:1858-1861), and thy (Ahmed, Z. U., M. R. 
Sarker, and D. A. Sack. 1990. Protection of adult rabbits and monkeys from 
lethal shigellosis by oral immunization with a thymine-requiring and 
temperature-sensitive mutant of Shigella flexneri Y. Vaccine. 8:153-158) 
loci, which affect the biosynthesis of aromatic amino acids, purines, and 
thymine, respectively, are attenuating because they eliminate the ability 
of the bacterium to synthesize critical compounds that are not readily 
available within mammalian hosts. For example, aro mutants of Salmonella 
and Shigella species have been shown to be attenuated in their natural 
hosts (Hoiseth S. K. and B. A. D. Stocker. 1981. Aromatic-dependent 
Salmonella typhimurium are non-virulent and effective as live vaccines. 
Nature (london). 291: 238-239)(Homchampa, P., R. A. Strugnell and B. 
Adler. 1992. Molecular analysis of the aroA gene of Pasteurella multocida 
and vaccine potential of a constructed aroA mutant. Mol. Microbiol. 6: 
3585-3593)(Homchampa, P., R. A. Strugnell and B. Adler. 1994. Construction 
and vaccine potential of an aroA mutant of Pasteurella haemolytica. Vet. 
Microbiol. 42:35-44)(Karnell, A., P. D. Cam, N. Verma and A. A. Lindberg. 
1993. AroD deletion attenuates Shigella flexneri strain 2457T and makes it 
a safe and efficacious oral vaccine in monkeys. Vaccine 
8:830-836)(Lindberg, A. A., A. Karnell, B. A. D. Stocker, S. Katakura, H. 
Sweiha and F. P. Reinholt. 1988. Development of an auxotrophic oral live 
Shigella flexneri vaccine. Vaccine 6:146-150) (O'Callaghan, D. D. Maskell, 
F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of 
aromatic and purine dependent Salmonella typhimurium: attenuation, 
persistence and ability to induce protective immunity in BALB/c mice. 
Infect. Immun. 56:419-423). Lesions that affect the biosynthesis of LPS 
(Collins, L. V., S. Attridge, and J. Hackett. 1991. Mutations at rfc or 
pmi attenuate Salmonella typhimurium virulence for mice. Infect. Immun. 
59:1079-1085)(Nnalue, N. A., and B. A. D. Stocker. 1987. Tests of the 
virulence and live-vaccine efficacy of auxotrophic and galE derivatives of 
Salmonella cholerasuis. Infect. Immun. 55:955-962) and of cyclic AMP 
(Kelly, S. M., B. A. Bosecker and R. Curtiss III. 1992. Characterization 
and protective properties of attenuated mutants of Salmonella cholerasuis. 
Infect. Immun. 60:4881-4890) (Tacket, C. I., D. M. Hone, R. Curtiss III, 
S. M. Kelly, G. Losonsky, L. Guers. A. M. Harris, R. Edelman. M. M. 
Levine. 1992. Comparison of the safety and immunogenicity of .DELTA.aroC 
.DELTA.aroD and .DELTA.cya.DELTA.crp Salmonella typhi strains in adult 
volunteers. Infect. Immun. 60:536-541) have also been shown to be 
attenuating in Salmonella species. It is important to note that not all 
attenuating mutations are good vaccine candidates in different organisms 
because some attenuating mutations result in poor persistence and 
immunogenicity (O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon 
and G. Dougan. 1988. Characterization of aromatic and purine dependent 
Salmonella typhimurium: attenuation, persistence and ability to induce 
protective immunity in BALB/c mice. Infect. Immun. 56:419-423)(Sigwart, D. 
F., B. A. D. Stocker, and J. D. Clements. 1989. Effect of a purA mutation 
on the efficacy of Salmonella live vaccine vectors. Infect. Immun. 
57:1858-1861). 
Riboflavin (vitamin B2), a precursor of the coenzymes flavin adenine 
dinucleotide (FAD) and flavin mononucleotide (FMN), is essential for basic 
metabolism. It is synthesized by plants and by most microorganisms but not 
by higher animals (Bacher, A. 1991. Biosynthesis of flavins. p. 215-59. In 
F. Muller (ed.), Chemistry and Biochemistry of Flavins, Vol. 1. Chemical 
Rubber Company, Boca Raton, Fla.). Many pathogenic bacteria are apparently 
unable to utilize flavins from their environment and are entirely 
dependent on endogenous production of riboflavin (Schott, K., J. 
Kellerman, F. Lottspeich and A. Bacher. 1990. Riboflavin synthases of 
Bacillus subtilis: purification and amino acid sequence of the 
.alpha.-subunit. J. Biol.Chem. 265:4204-4209). Even with the ability to 
utilize exogenous riboflavin, there may not be enough of the vitamin 
present in mammalian host tissues to permit growth, particularly not in 
sites devoid of normal bacterial flora. 
Vaccines are preparations used to prevent specific diseases in animals by 
inducing immunity. This is accomplished by exposing a patient to an 
antigen from an agent capable of causing a particular disease which, in 
turn, causes the immune system of the patient to produce large quantities 
of antibody. The presence of the antibody in the patient's blood protects 
the patient from a later attack by the disease-causing agent. Vaccines may 
either be composed of subunits of the agent, or the live or killed agent 
itself. If a live vaccine is to be used, its virulence must be attenuated 
in some way; otherwise, the vaccine will cause the disease it is intended 
to protect against. See U.S. Pat. No. 5,429,818, Col. 1. 
Most current vaccines against APP are killed whole cell bacterins, that is, 
whole bacterial cells killed by heat treatment or formalinization, 
suspended in an adjuvant solution. Some alternative ways of attempting to 
develop vaccines against APP are the use of subunit vaccines and the use 
of non-encapsulated mutants. 
The use of a protease lysate of the outer membrane of A. pleuropneumoniae 
cells as a vaccine against APP infection is described in U.S Pat. No. 
5,332,572. 
The use of extracellular proteins and/or hemolysins from APP as vaccines 
against APP infection is described in U.S. Pat. Nos. 5,254,340, WO Patent 
No. 9409821, EP No. 595,188, CA 2045950, and EP No. 453,024. 
The use of non-encapsulated mutants of APP is described in U.S. Pat. No. 
5,429,818. It disclosed that the capsule of such bacteria is required for 
virulence. Therefore, the preparation of a mutant of APP that was a 
non-capsulated mutant was described as a vaccine. 
A method of administering vaccines to pigs by a transthoracic 
intrapulmonary immunization is described in U.S. Pat. No. 5,456,914. 
A vaccine for the immunization of an individual against Salmonella 
choleraesuis utilizing derivatives that are incapable of producing 
functional adenylate cyclase and/or cyclic AMP receptor protein is 
described in U.S. Pat. No. 5,468,485. The avirulent S. choleraesuis was 
made avirulent by an inactivating mutation in a cya gene and an 
inactivating mutation in a crp gene. Similar techniques are described in 
other bacteria in U.S. Pat. Nos. 5,424,065; 5,389,386; 5,387,744 and 
4,888,170. 
To protect animals from lung disease, it is needed to achieve a 
sufficiently high level of antibodies, particularly IgA antibodies, in the 
lungs to prevent adherence of invading microorganisms to mucosal surfaces 
and neutralize potentially damaging virulence factors. Antibodies in the 
patient's serum or at the mucosal surfaces can be important to protection. 
One of the reasons for using a live vaccine instead of a killed whole cell 
bacterin is that a live vaccine, given intranasally or orally, can induce 
specific local secretory antibody in the secretions that cover mucosal 
surfaces. This local antibody is often quite helpful for protection 
against diseases that infect at or through mucosal surfaces. 
None of the patents pertain to a recombinant technique for a relatively 
convenient method for obtaining genetically defined mutants for use in a 
vaccine against APP. 
It is believed that a mutation in a critical biosynthetic pathway which 
limits growth in vivo but does not otherwise alter expression of important 
antigens such as capsular polysaccharide, lipopolysaccharide and 
extracellular toxins, could produce an attenuated vaccine strain capable 
of inducing cross-protective immunity against A. pleuropneumoniae. 
It is believed that riboflavin biosynthesis would be essential for survival 
of A. pleuropneumoniae in vivo, and that mutations in the riboflavin 
biosynthetic pathway would be attenuating due to the scarcity of 
riboflavin present on the mucosal surfaces of the respiratory tract. 
It is an object of the present invention to describe the use of mutations 
in the riboflavin biosynthetic pathway to construct attenuated strains of 
pathogenic bacteria for use as live vaccines, with a riboflavin-requiring 
mutant of APP used as a specific example. 
It is an object of the present invention to describe a live vaccine against 
APP utilizing a riboflavin mutation in the APP genome. 
SUMMARY OF THE INVENTION 
Described is a live vaccine against bacterial pathogens comprising a 
recombinant riboflavin-requiring mutant having a mutation that inactivates 
riboflavin biosynthesis therein. In particular, this includes bacterial 
pathogens in the family Pasteurellaceae, which include animal pathogens as 
Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus 
parasuis, Pasteurella haemolytica and Pasteurella multocida, as well as 
human pathogens Haemophilus influenzae and Haemophilus ducreyi. 
Also described is a live vaccine against Actinobacillus pleuropneumoniae 
(APP) comprising a recombinant APP having an attenuating inactivating 
mutation therein.

DESCRIPTION OF PREFERRED EMBODIMENTS 
The present application pertains to the development of attenuated mutants 
of the pathogenic bacterium A. pleuropneumoniae which contain mutations in 
the genome, specifically in the genes encoding the enzymes involved in the 
biosynthesis of riboflavin. By "mutation" is meant not just a random 
selection of variations of the genome of APP but utilization of well known 
recombinant techniques for specifically modifying the genome of APP. 
Accordingly, therefore, it is desirable to ascertain the riboflavin 
biosynthesis genes of APP. 
By "attenuated" is meant a reduction in the severity, virulence or vitality 
of the disease causing agent. 
After determining the sequence and organization of the riboflavin genes, 
one is then able to modify APP by removing some or all of such genes, 
thereby attenuating the pathogen, i.e., making the pathogen avirulent. 
After a strain of avirulent APP is obtained, it could then be utilized as a 
live vaccine. Described below are the detailed steps broadly outlined 
above. 
IDENTIFYING, CLONING, AND SEQUENCING OF THE RIBOFLAVIN BIOSYNTHESIS GENES 
FROM APP 
Cloning of riboflavin genes from APP is described in the paper entitled 
"Characterization of APP Riboflavin Biosynthesis Genes", Journal of 
Bacteriology, December, 1995, pages 7265-7270 by Fuller and Mulks. This is 
incorporated herein by reference. 
Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine 
pleuropneumonia (9,23,39). The disease is characteristically an acute 
necrotizing hemorrhagic bronchopneumonia, with accompanying fibrinous 
pleuritis (9,39). Pleuropneumonia is an economically devastating, severe 
and often fatal disease with clinical courses ranging from peracute to 
chronic infection (9,14). The existence of at least twelve antigenically 
distinct capsular serotypes (31) has made development of a 
cross-protective vaccine difficult. Killed whole cell bacterins provide at 
best serotype-specific protection (25,26,35,43). In contrast, natural or 
experimental infection with virulent APP frequently elicits protection 
against reinfection with any serotype (24,25,27). Avirulent strains of APP 
have been tested as live vaccines and have elicited cross-protective 
immunity against subsequent challenge (15,28,44). However, the use of live 
vaccines in the field is problematic, particularly when the attenuating 
lesions in the vaccine strain have not been genetically defined. 
Development of attenuated strains with defined biochemical mutations that 
limit growth in vivo and prevent reversion to wild type is a promising 
approach to improved vaccines against APP infection. 
Riboflavin (vitamin B2), a precursor of the coenzymes flavin adenine 
dinucleotide (FAD) and flavin mononucleotide (FMN), is essential for basic 
metabolism. It is synthesized by plants and by most microorganisms but not 
by higher animals (1). Many pathogenic bacteria are apparently unable to 
utilize flavins from their environment and are entirely dependent on 
endogenous production of riboflavin (38). Therefore, riboflavin 
biosynthesis may be essential for survival of pathogens in vivo, and 
mutations in the riboflavin biosynthetic pathway may be attenuating. 
The proposed metabolic pathway for bacterial riboflavin synthesis shown in 
FIG. 1 begins with guanosine triphosphate (GTP ) as the precursor (for a 
review see reference 1). The most extensively studied system for bacterial 
riboflavin synthesis is Bacillus subtilis (for a review see reference 29). 
The B. subtilis riboflavin synthesis genes are located and coregulated in 
an operon structure (12) that consists of five open reading frames 
designated as ribG, rib B, rib A, ribH and ribT (19,29) . The ribGBAHT 
genes encode, respectively, a rib-specific deaminase; the .alpha.-subunit 
of riboflavin synthase (lumazine synthase); a bifunctional enzyme 
containing GTP cyclohydrase and 3,4-dihydroxy 2-butanone 4-phosphate 
synthase (DHBP) activities; the .beta.-subunit of riboflavin synthase; and 
a rib-specific reductase (29). The complete sequence of the B. subtilis 
riboflavin operon has been determined in two individual laboratories 
(19,30). The B. subtilis structural ribGBAHT genes code for predicted 
proteins of 361 (MW 39,700), 215 (MW 23,600), 398 (MW 43,800), 154 (MW 
16,900), and 124 (MW 13,600) amino acids in length (19, 29). Two 
functional promoters have been identified in the B. subtilis rib operon. 
The main promoter, P1, for which a transcriptional start site has been 
determined 294 base pairs (bps) upstream of ribG (12,30), is responsible 
for transcription of all five structural genes (12). Another promoter, P2, 
produces a secondary transcript encoding the last three genes of the 
operon, ribAHT (12). A possible third promoter has been postulated that 
would express ribH (7). In addition, the operon has been shown to be 
transcriptionally coregulated (30) by a transacting repressor, RibC (3,6), 
which acts at a regulatory site, ribO (3,20), upstream of ribG, apparently 
by a transcription terminationantitermination mechanism (29). The RibC 
repressor appears to respond to FMN and FAD, as well as to riboflavin and 
several of its biosynthetic intermediates, and regulates expression from 
both ribPl and ribP2 (4,20,29). 
E. coli is the second most chemically characterized system for riboflavin 
synthesis. In contrast to B. subtilis, the rib genes of E. coli are 
scattered around the chromosome and are expressed constituitively (2,46). 
Rather than having a bifunctional ribA, E. coli has two separate genes, 
ribB and ribA, that encode the functions of 3,4-DHBP synthase (34) and GTP 
cyclohydrase II (33), respectively. ribB is homologous to the 5' end of B. 
subtilis ribA while ribA is homologous to the 3' end (33,34). E. coli 
genes with sequence homology to the B. subtilis ribG (42), ribH (42), and 
ribB genes have also been identified. 
Identified herein is a fragment of APP serotype 5 chromosomal DNA that 
triggers overproduction of riboflavin when cloned in E. coli. Nucleotide 
sequence analysis demonstrated four open reading frames with significant 
identity and a similar operon arrangement to the ribGBAH genes from 
Bacillus subtilis. 
MATERIALS AND METHODS 
Bacterial strains and media. A. pleuropneumoniae ISU178, a serotype 5 
strain, was cultured at 37.degree. C. in brain heart infusion broth or 
agar (Difco Laboratories, Detroit, Mich.) containing 10 .mu.g/ml 
nicotinamide adenine dinucleotide (NAD) (Sigma Chemical Company, St. 
Louis, Mo.). E. coli DH5-.alpha. (supE44), .DELTA.lacU169, (.o 
slashed.80lacZ.DELTA.M15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was 
used for construction of the APP genomic library. E. coli strain DS410 
(azi-8, tonA2, minA1, minB2, rpsL135, xyl-7, mtl-2, thi-1, .lambda.-) was 
used for minicell isolation and protein labeling experiments. E. coli 
ribA:Tn5 (BSV18), ribB:Tn5 (BSV11) and ribC:Tn5 (BSV13) mutants used for 
complementation studies were described by Bandrin et al (2) and are 
available from Barbara Bachmann (E. coli Genetic Stock Center, Yale 
University). E. coli strains were cultured in Luria-Bertani medium or in 
M9 (36) supplemented with 15 g/L NZ (amine (Sigma) and with riboflavin at 
200 .mu.g/mL when necessary. Ampicillin was added to 100 .mu.g/ml for 
plasmid selection. 
DNA manipulations. DNA modifying enzymes were supplied by 
Boehringer-Mannheim Biochemicals (Indianapolis, Ind.) and used according 
to manufacturer's specifications. Genomic and plasmid DNA preparations, 
gel electrophoresis, and E. coli transformation were all performed by 
conventional methods (36). 
Cloning and sequencing. APP serotype 5 genomic DNA was digested with 
HindIII and fragments ranging in size from 4 to 7 kb were ligated into the 
HindIII site in the polylinker of the plasmid vector pUC19 (45). A 
recombinant plasmid, designated PTF10, which overproduced riboflavin was 
isolated from this library. Unidirectional nested deletions were 
constructed with exonuclease III and S1 nuclease digestion, using the 
Erase-a base system (Promega Corp., Madison, Wis.). Nucleotide sequencing 
was performed on alkali-denatured double-stranded DNA by the dideoxy 
chain-termination method of Sanger et al. (37) using the Sequenase 2.0 kit 
(U.S. Biochemical, Cleveland, Ohio) and .sup.35 S!dATP (adenosine 
triphosphate) (Amersham Corp., Arlington Heights, Ill.). Sequencing 
primers used included universal forward and reverse primers for pUC 
sequencing (U.S. Biochemicals), as well as several oligonucleotide primers 
designed from previously obtained sequence data. These internal primers 
were synthesized by the Michigan State University Macromolecular Structure 
Facility and included MM4 (5'-AAT-CCG-GCA-AAA-ATT-GAA-GGC-3') (Sequence ID 
No: 1), MM5 (5'-GCA-CCG-TGA-CGC-ACT-AAC-G-3') (Sequence ID No: 2), MM6 
(5'-GCG-CCA-ATA-CTT-GCT-CAC-CG-3') (Sequence ID No: 3), MM9 
(5'-GGT-TTC-TTT-ATT-CGT-ATG-CGG-3') (Sequence ID No: 4), MM10 
(5'-TGA-AGA-AGT-CGG-CAA-ATT-GCT-C-3') (Sequence ID No: 5), MM11 
(5'-CGG-ATT-GGG-ATT-CGT-CCA-GCC-3') (Sequence ID No: 6), MM13 
(5'-GGC-GAC-ACG-ATT-GCG-GTG-3') (Sequence ID No: 7), MM14 
(5'-GCC-AGT-TAG-TGC-AGA-CAG-CG-3') (Sequence ID No: 8), and MM38 
(5'-CTC-ACC-GGT-TCC-TGC-CAA-ACC-3') (Sequence ID No: 9). 
DNA sequences were analyzed using the GCG sequence analysis programs (11). 
Mass spectroscopy. Positive and Negative Ion Fast Atom Bombardment (FAB) 
mass spectroscopy was performed at the Michigan State University Mass 
Spectroscopy Facility. 
Quantification of riboflavin. Bacterial cells were pelleted in a 
microcentrifuge, and the absorbance at 445 nm of the culture supernatant 
was measured using a Beckman DU-7 spectrophotometer (Beckman Instruments, 
Fullerton, Calif.). The absorbance at 445 nm was multiplied by a factor of 
31.3 to yield the riboflavin concentration in mg/liter (10). 
Minicell Analysis. The minicell-producing E. coli strain DS410 (32) was 
transformed by calcium chloride/heat shock treatment with pUC19 or pTF rib 
clones. Transformant colonies which produced a large number of minicells 
were selected by microscopy. Cultures were grown overnight at 37.degree. 
C. in 500 mL LB broth, and minicells were isolated by differential 
centrifugation followed by glass fiber filtration as described by Christen 
et al (8). Minicells were resuspended to an OD.sub.590 of 0.5-1.0 in 200 
.mu.l labeling mix (22.0 ml M9 media, 20.0 ml 50 mM HEPES 
(N-2-hydroxyethyl! piperazine)-N'-2 ethanesulfonic acid) pH 7.5, 2.5 ml 
of 20% glucose, 0.05 ml of 10 mg/ml adenine, 0.05 ml of 10 mg/ml 
pyridoxine, 5.0 ml of NEDA amino acid stock (21) lacking methionine and 
cysteine, and 0.2 ml of 10 mg/ml cycloserine-D) and incubated at 
37.degree. C. for 30 minutes. Trans-label (.sup.35 S!methionine plus 
.sup.35 S!cysteine, ICN Biomedicals, Irvine, Calif.) was added to a final 
concentration of 22 .mu.Ci per reaction and cells were incubated at 
37.degree. C. for 1 hour. Total and TCA (trichloroacetic acid) 
precipitable counts were measured by liquid scintillation counting to 
determine amount of incorporation. Cells were pelleted in a 
microcentrifuge and washed with cold HEPES (50 mM,pH7.5) plus 10 mM 
methionine plus 10 mM cysteine. Labeled proteins (50,000 cpm/lane) were 
separated by discontinuous SDS-PAGE on a 12% polyacrylamide gel and were 
visualized by autoradiography on Kodak XAR-5 film. 
Nucleotide sequence accession number. The nucleotide sequence of the A. 
pleuropneumoniae ribGBAH genes has been submitted to GenBank and assigned 
an accession number of: U27202. 
RESULTS 
Identification of a riboflavin producing clone. A genomic library of A. 
pleuropneumoniae serotype 5 DNA was constructed in pUC19 and transformed 
into E. coli DH5-.alpha.. A single clone, designated pTF10 (FIG. 2), 
containing a 5.2 kbp insert, was identified that produced a bright yellow 
extracellular, water-soluble compound that fluoresced under ultraviolet 
light. The compound was crudely purified by filtration through a 3000 Da 
cut off membrane filter (Amicon Corporation, Bedford, Mass.). Absorbance 
spectra of this compound in aqueous solution under neutral conditions 
showed absorbance peaks at 373 and 443 nm, which coalesced to a single 
peak at 388 nm under acidic conditions; these results compared well to a 
riboflavin standard (FIG. 3). Positive and negative ion fast atom 
bombardment mass spectroscopy indicated that the compound was a flavin 
(data not shown). Culture of E. coli DH5-.alpha./pTF10 in M9 medium plus 
NZ amine plus 0.6% glucose yielded 10 mg riboflavin per liter in 24 hours. 
Sequence of APP rib genes. The nucleotide sequence and corresponding 
predicted amino acid sequence of a 4312 bp region of the insert in pTF10 
is shown in FIG. 4. Four open reading frames of 1232, 647, 1205, and 461 
bp were observed that encoded proteins with predicted molecular masses of 
45,438 Da, 23,403 Da, 44,739 Da and 16,042 Da, respectively. Based on 
homology with the riboflavin biosynthetic genes of B. subtilis (see 
below), these ORFs were designated ribG, ribB, ribA, and ribH, 
respectively. All four ORFs were preceded by potential ribosome binding 
sites (RBS), although the RBS upstream of ribG is not as strong as the 
other three. Production of riboflavin by this clone is not dependent on 
its orientation in pUC19 or on induction by IPTG, indicating that it is 
produced under the control of a native promoter included in the cloned DNA 
fragment. A consensus promoter sequence of the -35/-10 type (12) was 
identified within the sequenced region 224 bp upstream from the ribG start 
codon. A second potential consensus promoter was identified between the 
genes ribA and ribH. However, no consensus promoter was identified between 
ribB and ribA, as is found in B. subtilis. The ORF encoding ribH is 
followed by an inverted repeat stem-loop structure with a .DELTA.G=-56.0, 
that may function as a rho-dependent transcriptional terminator (13) 
Homology of APP rib genes. Predicted amino acid sequences of the APP 
RibGBAH proteins were compared with B. subtilis RibGBAH (19); E. coli 
RibA, RibB, RibC, RibG, and RibH (33,34,42); Photobacterium leiognathi 
RibI-III (17), Photobacterium phosphoreum RibI-IV (16), and Vibrio harveyi 
LuxH (41) proteins, using the GCG Gap program (Table 1). APP RibG showed 
62-63% similarity to the RibG proteins from B. subtilis and E. coli. APP 
RibB showed 58-69% and APP RibH showed 69-83% similarity to homologous 
genes from B. subtilis, E. coli, and Photobacterium species. APP RibA 
showed 73% similarity to the entire RibA protein of B. subtilis and 61% to 
the RibII protein of P. leiognathi, both of which encode a bifunctional 
enzyme catalyzing two distinct steps in the riboflavin pathway. In 
addition, the carboxy terminal half of APP RibA, encompassing .about.200 
amino acids, shows 59-63% similarity to E. coli RibB, and V. harveyi LuxH, 
which encode 3,4-DHBP synthase. The N-terminal region of APP RibA, 
encompassing the remaining .about.200 amino acids, shows 63-73% similarity 
to E. coli RibA and P. phosphoreum RibIV, which encode GTP cyclohydrase 
II. 
TABLE 1 
__________________________________________________________________________ 
Percent similarity of amino acid sequences of riboflavin synthesis 
proteins.sup.a 
% Similarity with A. pleuropneumoniae 
RibG RibB RibA RibH 
Compared 
Compared 
Compared 
Compared 
Bacterium 
With % With % With % With % 
__________________________________________________________________________ 
B. subtilis 
RibG 63 RibB 69 RibA 73 RibH 83 
E. coli.sup.b 
RibG 62 RibC 58 RibB 63 RibH 74 
RibA 73 
H. influenzae.sup.c 
RibG 58 RibC 60 RibB 65 RibE 75 
RibA 71 
P. leiognathi 
NA RibI 64 RibII 
61 RibIII 
69 
P. phosphoreum.sup.d 
NA RibI 63 RibII 
59 RibIII 
73 
RibIV 
63 
V. harveyi NA NA LuxH 59 NA 
__________________________________________________________________________ 
.sup.a Identity is expressed in percent similarity as calculated by the 
Genetics Computer Group NeedlemanWunsch algorithm (22). Proteins with 
partial identify were compared with the entire appropriate A. 
pleuropneumoniae Rib protein. 
.sup.b E. coli RibB is homologous to the 5' end of A. pleuropneumoniae 
RibA. E. coli RibA is homologous to the 3' end of A. pleuropneumoniae 
RibA. 
.sup.c H. influenzae RibB is homologous to the 5' end of A. 
pleuropneumoniae RibA H. influenzae RibA is homologous to the 3' end of A 
pleuropneumoniae RibA. 
.sup.d P. phosphoreum RibIV is homologous to the 3' end of A. 
pleuropneumoniae RibA. 
Complementation of E. coli mutants. The original pTF10 clone and several 
deletion derivatives were tested for their abilities to complement ribA 
(GTP cyclohydrase II), ribB (3,4-DHBP synthase), and ribC (.beta.-subunit 
of riboflavin synthase) mutations in E. coli (2) (FIG. 5) Complementation 
of the E. coli mutation was determined by restoration of the ability to 
grow on M9 minimal medium in the absence of riboflavin. Plasmids 
containing a complete copy of the APP ribB gene complemented the E. coli 
ribC mutation. Plasmids containing the 5' end of APP ribA complemented the 
E. coli ribB mutation. Plasmids containing a complete copy of APP ribA 
complemented both E. coli ribB and ribA mutations. 
Minicell analysis. Plasmid pTF10 and its deletion derivatives were 
transformed into the minicell-producing E. coli strain DS410, and the 
proteins encoded by these plasmids were radioactively labeled, separated 
by SDS-PAGE, and visualized by autoradiography. Compared with the pUC19 
vector, plasmid pTF10 shows four unique proteins with apparent molecular 
masses of 45 kDa, 27.7 kDa, 43.7 kDa, and 14.8 kDa (FIG. 6), which 
correspond well with the sizes predicted for the RibG, RibB, RibA, and 
RibH proteins by amino acid sequence data. The RibG protein did not appear 
to be as strongly expressed as RibB, RibA, and RibH. Analysis of proteins 
encoded by plasmid pTF19 (FIG. 5), which contains no ribH and a slightly 
truncated ribA gene, eliminates the 14.8 kDa protein (RibH) and truncates 
the 43.7 kDa protein (RibA) to 42.5 kDa (FIG. 6). Plasmid pTF12 (FIG. 5), 
which does not contain ribb, ribA, or ribH genes, does not express the 
27.7, 43.7, or 14.8 kDa proteins (data not shown). 
Described above is the identification, cloning and complete nucleotide 
sequence of four genes from Actinobacillus pleuropneumoniae that are 
involved in riboflavin biosynthesis. The cloned genes can specify 
production of large amounts of riboflavin in E. coli, can complement 
several defined genetic mutations in riboflavin biosynthesis in E. coli, 
and are homologous to riboflavin biosynthetic genes from both E. coli and 
Bacillus subtilis. The genes have been designated APP ribGBAH due to their 
similarity in both sequence and arrangement to the B. subtilis ribGBAH 
operon. 
The DNA sequence data, complementation, and minicell analysis strongly 
suggest that the four rib genes are transcribed from a single APP promoter 
upstream of the ribG gene. This promoter, among the first described for 
housekeeping genes in APP, is a good match for an E. coli consensus 35/-10 
promoter. There is a 4 of 6 bp match at the -35 region, a 17 bp interval, 
a 4 of 6 bp match at the -10 region, an 8 bp interval, and a CAT box at 
the -1/+1 site. There is also a second potential promoter located between 
ribA and ribH, although it is not clear whether this promoter is 
functional. 
Biosynthesis of riboflavin by APP appears to be more similar to that in the 
gram-positive bacterium B. subtilis than in the gram-negative bacterium E. 
coli. First, APP rib genes are arranged in an operon similar to that seen 
in B. subtilis, rather than scattered throughout the chromosome as is 
found in E. coli. However, the B. subtilis rib operon contains a fifth 
gene, ribT, that is proposed to mediate the third step in riboflavin 
biosynthesis; it is unlikely that a ribT homologue is present as part of 
the operon in APP because of the presence of a strong inverted repeat 
following ribH and the lack of a likely reading frame downstream. Second, 
APP contains a ribA gene that encodes a bifunctional enzyme with both GTP 
cyclohydrase II and DHPB synthase activities, as is found in B. subtilis; 
E. coli has two genes, ribA and ribB, that encode these two enzymes 
separately. Finally, the APP riboflavin biosynthetic enzymes are more 
similar at the amino acid level to the enzymes of B. subtilis than to 
those of E. coli, although alignment of the proteins from all three 
sources shows highly conserved sequences (data not shown). 
It should be noted that in three bioluminescent species from the family 
Vibrionaceae, Vibrio harveyi, Photobacterium leiognathi, and P. 
phosphoreum, riboflavin biosynthesis genes have been shown to be closely 
linked to the lux operon (10, 11, 41). FMNH.sub.2 is the substrate for the 
light-emitting reaction, and therefore an increase in bioluminescence 
requires an increased supply of the cofactor. Since riboflavin is the 
precursor for FMN, linkage and possibly coordinate regulation of lux and 
rib genes may facilitate the expression of bioluminescence in these 
bacteria. 
The recombinant E. coli DH5-.alpha. containing plasmid pTF10 showed a 
marked increase in extracellular riboflavin production, most likely due to 
the lack of regulation (40) and high copy number of the cloned synthetic 
genes (45). Although the APP rib operon is similar in structure to that of 
B. subtilis, it is not yet known whether the genes are tightly regulated 
in APP by a repressor similar to B. subtilis RibC, or whether they are 
constituitively expressed as appears to be true in E. coli (33). It is 
believed APP must synthesize riboflavin to meet its own metabolic demands 
during infection, since riboflavin is not synthesized by mammals and 
therefore is not likely to be freely available to APP within its porcine 
host. 
ATTENUATION OF RIB- MUTANTS OF A. PLEUROPNEUMONIAE 
Applicants have constructed deletion-disruption riboflavin-requiring 
mutants of A. pleuropneumoniae serotypes 1 and 5. 
Applicants have conducted experiments to confirm that the Rib- APP mutants 
constructed are attenuated in swine. 
In a preliminary experiment, seven 8-to-10 week old pigs were used. Three 
pigs were infected endobronchially with Nal.sup.R (resistant to the 
antibiotic malidixic acid) derivatives of wild type virulent APP-1 or 
APP-5; three were infected with APP-5 Rib- mutants; and one was used as an 
uninfected control. The APP strains, dosages used for infection, and 
results are summarized below in Table 2. Animals were euthanized when 
clinical signs became severe or at 12 hours post infection. The animals 
were necropsied and the lungs examined for gross pathology and 
histopathology, and lungs were cultured to recover APP. 
TABLE 2 
______________________________________ 
Summary of clinical signs, gross pathology, and histopathology 
seen in pigs challenged with either wild type APP serotype 1 or 
5 or Rib- mutants of App Serotype 5 
APP Strain and 
Description Dosage Results 
______________________________________ 
AP225: APP-1, Nal.sup.R 
2 .times. 10.sup.8 cfu 
Died, &lt;4 hrs; 4+ peracute 
hemorrhagic pneumonia lesions 
AP227: APP-5, Nal.sup.R 
1 .times. 10.sup.9 cfu 
Died, &lt;4 hrs: 4+ peracute 
hemorrhagic pneumonia lesions 
AP228: APP-5, Nal.sup.R 
1 .times. 10.sup.9 cfu 
Died, &lt;4 hrs: 4+ peracute 
hemorrhagic pneumonia lesions 
AP229: APP-5, Nal.sup.R,, 
1 .times. 10.sup.9 cfu 
Mild clinical signs; 1+ mild 
Km.sup.R, Rib- pneumonia lesions 
AP230: APP-5, Nal.sup.R,, 
1 .times. 10.sup.9 cfu 
Mild clinical signs; 1+ mild 
Km.sup.R, Rib- pneumonia lesions 
AP231: APP-5, Nal.sup.R,, 
1 .times. 10.sup.9 cfu 
Mild clinical signs; 1+ mild 
Km.sup.R, Rib- pneumonia lesions 
None -- 1+ mild 
pneumonia lesions (mycoplasma?) 
______________________________________ 
Note that the dosage used in all of these animals was about 200 times the 
LD.sub.50 (50% lethal dose, or the dose that will kill 50% of the animals 
exposed) for the wt (wild type) APP parent strains. The Nal.sup.R 
derivatives of the wild type) parent strains retained virulence, 
triggering severe fibrinosuppurative hemorrhagic pneumonia and death 
within 4 hours. The Rib- mutants caused minimal clinical signs (increased 
respiration rate and slight fever) and at most mild signs of pneumonia, 
including some consolidation but no hemorrhagic necrosis, as compared to 
the uninfected control. These were not SPF (specific pathogen free) pigs, 
and there were histologic lesions suggestive of mild mycoplasma infection, 
in all of the pigs, including the uninfected control (Table 2). 
Described below is the construction of a deletion-disruption riboflavin 
mutant of A. pleuropneumoniae serotype 1 (APP-1) and detailed analysis of 
the attenuation of this APP-1 Rib- mutant in vivo in swine. 
MATERIALS AND METHODS 
Bacterial strains and media. The bacterial strains and plasmids used in 
this study are listed in Table 1. A. pleuropneumoniae strains were 
cultured at 37.degree. C. in either brain heart infusion (BHI), heart 
infusion (HI), or tryptic soy agar (TSA) (Difco Laboratories, Detroit, 
Mich.) containing 10 .mu.g/ml NAD (V factor) (Sigma Chemical Company, St. 
Louis, Mo.). Riboflavin (Sigma) was added to a final concentration of 200 
.mu.g/ml when needed. E. coli strains were cultured in Luria-Bertani 
medium. Ampicillin was added to 100 .mu.g/ml and kanamycin to 50 .mu.g/ml 
for plasmid selection in E. coli strains. For A. pleuropneumoniae strains, 
50 .mu.g/ml kanamycin sulfate and 25 .mu.g/ml nalidixic acid were added as 
required, except for selection after matings which were performed with 100 
.mu.g/ml kanamycin sulfate and 50 .mu.g/ml nalidixic acid. 
DNA manipulations. DNA modifying enzymes were supplied by 
Boehringer-Mannheim Biochemicals (Indianapolis, Ind.) and used according 
to the manufacturer's specifications. Genomic DNA was prepared according 
to the lysis/proteinase K method of the Gene Fusion Manual (Silhavy, T. J. 
1984. DNA extraction from bacterial cells. p. 137-139. In Experiments with 
Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.34). 
Plasmid DNA preparations, agarose gel electrophoresis, and E. coli 
transformation were all performed by conventional methods (Sambrook, J., 
E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory 
Manual, 2nd. ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). 
Filter mating targeted mutagenesis. Filter mating between E. coli S17-1 
(.lambda.pir) /pTF67A and AP225 was performed according to the protocol of 
Mulks and Buysse (Mulks, M. H. and J. M. Buysse. 1995. A targeted 
mutagenesis system for Actinobacillus pleuropneumoniae. Gene 165:61-66). 
Briefly, bacterial cultures were grown overnight at 37.degree. C. Equal 
cell numbers of donor and recipient cultures, as determined by optical 
density at 520 nm, were added to 5 ml 10 mM MgSO.sub.4 and the bacteria 
pelleted by centrifugation. The pellet containing the cell mating mixture, 
resuspended in 100 .mu.l of 10 mM MgSO.sub.4, was plated on a sterile 
filter on BHIV+riboflavin agar and incubated for 3 h at 37.degree. C. 
Cells were washed from the filter in sterile phosphate buffered saline (pH 
7.4), centrifuged, resuspended in 400 .mu.l BHIV broth and plated in 100 
.mu.l aliquots on BHIV containing riboflavin, kanamycin, and nalidixic 
acid. Kanamycin and nalidixic acid resistant colonies were selected from 
filter mating plates and screened for riboflavin auxotrophy by replica 
plating onto TSAV, observing for inability to grow in the absence of added 
riboflavin. 
Southern Analysis of Transconjugants. Chromosomal DNA and plasmid controls 
were digested with the appropriate restriction enzymes and the DNA 
fragments were separated on an 0.7% ultrapure agarose gel in TAE buffer. 
Southern blots were performed as described by Sambrook et al (Sambrook, 
J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory 
Manual, 2nd. ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). DNA 
probes were labeled with digoxygenin by random priming using the Genius V. 
3.0 kit from Boehringer Mannheim. Probes included the 5.2 Kb insert from 
pTF10 containing the intact riboflavin operon from AP106 (Rib), the 1.4 Kb 
ClaI/NdeI fragment deleted from the riboflavin operon in the construction 
of pTF67a (R.Del.), the 1.2 Kb kanamycin cassette from pUC4K (Km) and the 
intact plasmid pGP704 (pGP704). Hybridization was carried out in 50% 
formamide at 42.degree. C. for 16 h. Blots were washed twice in 
2.times.SSC/0.1% SDS for 15 min at room temperature, then twice in 
0.1.times.SSC/0.1% SDS for 30 min at 65.degree. C. Blots were developed 
with alkaline phosphatase-conjugated anti-digoxygenin and calorimetric 
substrate (Boehringer Mannheim) according to the manufacturer's 
instructions. 
Phenotypic analysis of mutant strains. Whole cell lysates and supernatants 
of AP100, AP225 (Nal.sup.R), and AP233 (Km.sup.R, Nal.sup.R, Rib-) were 
prepared from overnight cultures grown in HIV+5 mM CaCl.sub.2 +appropriate 
antibiotics. Cells were separated by microcentrifugation and resuspended 
in SDS-PAGE sample buffer (Laemmli, U. K. 1970. Cleavage of structural 
proteins during the assembly of the head of bacteriophage T4. Nature 
227:680-685). The culture supernatant was precipitated with an equal 
volume of 20% trichloroacetic acid (TCA) and resuspended in SDS-PAGE 
(sodium dodecyl sulfate--polyacrylamide gel electrophoresis) sample 
buffer. Cellular polysaccharides, including lipopolysaccharide (LPS) and 
capsular polysaccharide, were prepared according to the cell 
lysis/proteinase K method of Kimura et al (Kimura, A. and E. J. Hansen. 
1986. Antigenic and phenotypic variations of Haemophilus influenzae type B 
lipopolysaccharide and their relationship to virulence. Infect. Immun. 
51:69-79). All samples were analyzed on a 0.125% SDS-12% acrylamide gel 
using a discontinuous buffer system (Laemmli, U. K. 1970. Cleavage of 
structural proteins during the assembly of the head of bacteriophage T4. 
Nature 227:680-685). Samples were transferred to nitrocellulose according 
to standard protocols (Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. 
Molecular Cloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor Press, 
Cold Spring Harbor, N.Y.) and probed with convalescent serum from a pig 
infected with A. pleuropneumoniae serotype 1. Antigen-antibody complexes 
were detected with horseradish peroxidase-conjugated protein A (Boehringer 
Mannheim) and the colorimetric substrate 4-chloro-naphthol (BioRad, 
Hercules, Calif.). 
Production of serotype-specific capsular polysaccharide was measured by 
coagglutination assay using hyperimmune rabbit anti-sera complexed to 
Staphylococcus aureus whole cells (Jolie, R. A. V., M. H. Mulks, and B. J. 
Thacker. 1994. Antigenic differences within Actinobacillus 
pleuropneumoniae serotype 1. Vet. Microbiol. 38:329-349). 
Electroporation of A. pleuropneumoniae. AP233 was grown in 100 ml BHIV with 
riboflavin at 37.degree. C., with shaking at 150 RPM, to an OD.sub.520 of 
0.7. Cells were chilled on ice and centrifuged at 5,000.times.g at 
4.degree. C. for 10 min. Cells were washed twice in ice cold sterile 15% 
glycerol. Cells were resuspended in 2 ml 15% glycerol and frozen in 50 
.mu.l aliquots using a dry ice-ethanol bath. Plasmid DNA was added to an 
aliquot of competent cells thawed on ice and then transferred to a 0.1 cm 
gap electroporation cuvette (BioRad). Cells were electroporated using a 
Gene Pulser II (BioRad) with the following settings: voltage, 1.8 kV; 
resistance, 200 .OMEGA.; capacitance, 25 .mu.Fd. 
Experimental infections. Eight-week-old, specific-pathogen-free, castrated, 
male pigs (Whiteshire Hamroc, Inc., Albion, Ind.) were allotted to six 
challenge groups by a stratified random sampling procedure, balancing each 
group for body weight. Each challenge group was housed in a separate BSL-2 
(biosafety level) isolation room at the Michigan State University Research 
Containment Facility. All experimental protocols for animal experiments 
were reviewed by the Michigan State University All University Committee on 
Animal Use and Care, and all procedures conformed to university and USDA 
regulations and guidelines. 
For preparation of challenge inocula, bacteria were grown in 30 ml HIV+5 mM 
CaCl.sub.2 +riboflavin and antibiotics as needed, in 300 ml baffled 
side-arm flasks, at 37.degree. C. with shaking at 160 RPM, to an 
OD.sub.520 (optical density) of 0.8. Ten ml of each culture was harvested 
by centrifugation at room temperature and washed once with sterile 0.9% 
saline. The cell pellet was resuspended in 10 mL of saline and diluted in 
saline to obtain the desired cfu/ml. The actual inoculating doses were 
retrospectively calculated by viable cell counts on agar plates. 
For the challenge procedure, pigs were anesthetized by intravenous 
injection with ketamine (4.4 mg/kg) and xylazine (1.65 mg/kg) and 
inoculated by percutaneous intratracheal injection with the appropriate 
dose of bacteria suspended in 10 mL saline. Clinical signs of 
pleuropneumonia, including increased respiration rate, fever, dyspnea, 
decreased appetite and activity/attitude (depression), were monitored and 
scored as previously described (Jolie, R. A. V., M. H. Mulks, and B. J. 
Thacker. 1995. Cross-protection experiments in pigs vaccinated with 
Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 
45:383-391). Seriously ill animals, as determined by severe dyspnea and/or 
depression, were euthanized immediately. Survivors were euthanized three 
days post-challenge. All animals were necropsied, and lungs were examined 
macroscopically for A. pleuropneumoniae lesions, including edema, 
congestion, hemorrhage, necrosis, abscessation, fibrosis, and pleuritis. 
The percentage of lung tissue and pleural surface area affected was 
estimated for each of the seven lung lobes, and the total % pneumonia and 
% pleuritis calculated using a formula that weights the contribution of 
each lung lobe to the total lung volume (Jolie, R. A. V., M. H. Mulks, and 
B. J. Thacker. 1995. Cross-protection experiments in pigs vaccinated with 
Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 
45:383-391). Representative lung samples were collected for histopathology 
and for bacterial culture. 
RESULTS 
Construction of A. pleuropneumoniae rib mutants. To construct 
riboflavin-requiring auxotrophic mutants of A. pleuropneumoniae, a suicide 
plasmid with part of the riboflavin operon deleted and replaced with a 
kanamycin-resistance (Km.sup.R) cassette was designed (FIG. 7). A 2.9 kb 
EcoRI fragment from pTF10 (Fuller, T. E. and M. H. Mulks. 1995. 
Characterization of Actinobacillus pleuropneumoniae riboflavin 
biosynthesis genes. J. Bacteriol. 177:7265-7270) containing the A. 
pleuropneumoniae ribBAH genes was cloned into the EcoRI site of the 
conjugative suicide vector pGP704 (18) to create plasmid pTF66. pTF66 was 
digested with ClaI and NdeI to excise the 3' end of ribB and the entire 
ribA gene. After Klenow treatment of the DNA, the 1.2 kb Km.sup.R 
cassette, excised with EcoRI from pUC4K , was blunt-end ligated into the 
rib deletion site to create pTF67a. 
pTF67a was transformed into E. coli S17-1 (.lambda.pir) and mobilized into 
AP225 (Nal.sup.R) to produce &gt;100 transconjugant colonies demonstrating 
resistance to both nalidixic acid and kanamycin. Transconjugants were 
replica plated onto TSAV and TSAV+riboflavin to assess the requirement for 
riboflavin and the stability of the riboflavin auxotrophy. Two classes of 
transconjugants were found. The majority of the transconjugants, e.g. 
AP234, were unstable and produced revertants capable of growth without 
supplemental riboflavin in the absence of kanamycin selection. One 
transconjugant, AP233, was very stable, maintaining kanamycin resistance 
as well as the inability to grow without exogenous riboflavin. All 
transconjugants were confirmed as A. pleuropneumoniae by gram stain, 
colonial morphology, and requirement for V factor (.beta.-NAD). 
Southern blot analysis of transconjugants. Two transconjugants were 
selected for further analysis based on their phenotypes as potential 
single (AP234) and double cross-over mutants (AP233). Southern blot 
analysis of transconjugant genomic DNA from the two mutants indicated that 
AP233 and AP234 were indeed double and single cross-over insertion mutants 
respectively (FIG. 8A). Predicted band sizes for single and double 
cross-over events are shown in FIG. 8A. Genomic DNA from AP233 contained a 
2.2 Kb HindIII fragment that hybridized with the riboflavin operon (Rib) 
probe, as well as 1.7 and 1.3 Kb fragments that hybridized with both the 
Rib and Km probes; however, there was no reaction with either pGP704 nor 
the deleted portion of the riboflavin operon (FIG. 8B). This is the 
pattern of hybridization predicted in transconjugants that replaced the 
wild type riboflavin operon with the mutated rib: :Km.sup.R locus by a 
double-crossover event (FIG. 8A). In contrast, genomic DNA from AP234 
shows the presence of DNA homologous to the fragment deleted from the 
riboflavin operon (R. del), pGP704, and the kanamycin cassette (FIG. 8B). 
This is the pattern of hybridization predicted in transconjugants that 
inserted the entire pTF67a plasmid into the wild type rib operon by a 
single crossover event (FIG. 8A). 
Phenotypic analysis of the A. pleuropneumoniae rib mutant. Whole cell 
lysates, TCA-precipitated culture supernatants, and polysaccharide 
preparations were analyzed on silver stained SDS-PAGE and on immunoblots 
developed with convalescent swine sera. No differences in protein, LPS, 
extracellular toxin, or capsular polysaccharide profiles were detected 
between wild type AP100, its Nal.sup.R derivative AP225, and the 
riboflavin mutant AP233 (data not shown). There was no difference in 
reactivity with serotype-specific antisera as determined by 
coagglutination assay (data not shown). 
Complementation of the rib mutation with a cloned wild type rib operon. The 
5.2 Kb insert from pTF10, containing the wild-type A. pleuropneumoniae 
riboflavin operon, was cloned into pGZRSl9, an E. coli-A. pleuropneumoniae 
shuttle vector (West, S. E. H., M. J. M. Romero, L. B. Regassa, N. A. 
Zielinski, and R. A. Welch. 1995. Construction of Actinobacillus 
pleuropneumoniae-Escherichia coli shuttle vectors: expression of 
antibiotic resistance genes. Gene 160: 81-86), to form pTF76. pTF76 was 
transformed into AP233 by electroporation, restoring the ability of AP233 
to grow in the absence of exogenous riboflavin and restoring the virulence 
of the mutant (see below). 
Attenuation of virulence of the rib mutant in swine. Six groups of three 
pigs each were infected with: group 1, 1 LD.sub.50 (5.times.10.sup.6 cfu) 
of AP225; groups 2-5, AP233 at doses equivalent to 4, 20, 100, and 500 
times the wild-type LD.sub.50 ; and group 6, 1 wild-type LD.sub.50 of 
AP233/pTF76. Mortality, lung score, and clinical score data, shown in 
Tables 3, 4 and 5, all indicate that the riboflavin auxotroph is avirulent 
in pigs at doses as high as 500 times the wild-type LD.sub.50. The pigs 
infected with the rib mutant AP233 displayed no dyspnea, elevated 
respiration rate, depression, or loss of appetite, and had no typical 
pleuropneumonic pathology at necropsy, at even the highest dose tested. In 
contrast, 1 of 3 pigs infected with the wild-type AP225 strain died, and 
all three exhibited significant clinical signs of APP disease, including 
elevated respiration rates, dyspnea, depression, loss of appetite, and 
fever, and severe pneumonia and pleuritis was evident at necropsy. Pigs 
infected with AP233 containing the riboflavin genes in trans (pTF76) also 
exhibited obvious clinical signs and significant pneumonia and pleuritis, 
although somewhat less severe than the wild-type strain. These results 
indicate that restoration of the ability to synthesize riboflavin does 
restore virulence. 
Bacteria were readily reisolated at necropsy from the lungs of pigs 
receiving AP225 and AP233/pTF76. All reisolated organisms were 
characterized by gram stain, colonial morphology, requirement for V factor 
(.beta.-NAD), antibiotic sensitivity, and serotyping by coagglutination. 
Reisolated organisms showed no differences from the initial inocula, 
including the presence of plasmid pTF76 in bacteria reisolated from pigs 
infected with AP233/pTF76. In contrast, we were unable to recover 
organisms from the lungs of animals infected with AP233 and euthanized 48 
hours post infection. 
TABLE 3 
______________________________________ 
Characteristics of bacterial strains and plasmids 
Strain/ Source/ 
Plasmid 
Characteristics Reference 
______________________________________ 
Strain 
E. coli 
supE44, .DELTA.lacU169, (.phi.80lacZ.DELTA.M15), 
BRL (USA) 
DH5-.alpha. 
hsdR17, recA1, endA1, gyrA96, thi-1, 
relA1 
E. coli 
.lambda.pir, supE44, .DELTA.lacU169, 
Mulks & Buysse 
DH5-.alpha. 
(.phi.80lacZ.DELTA.M15), hsdR17, recA1, endA1, 
(.lambda.pir) 
gyrA96, thi-1, relA1 
E. coli 
.lambda.pir, recA, thi, pro, hsd, (r-m+), 
Simon et al. 
S17-1 RP4-2, (Tc::Mu), (Km::Tn7), TmpR!, 
(.lambda.pir) 
SmR! 
AP100 A. pleuropneumoniae ATCC 27088, 
ATCC 
serotype 1, passaged through pigs 
AP106 A. pleuropneumoniae ISU178, a 
Iowa State 
serotype 5 field isolate, passaged 
University 
through pigs 
AP225 A spontaneous nalidixic acid 
This work 
resistant mutant of AP100 
AP233 A double cross-over riboflavin 
This work 
auxotroph of AP225 
AP234 A single cross-over riboflavin 
This work 
auxotroph of AP225 
Plasmid 
pUC19 Ap.sup.R cloning vector 
Vieira & 
Messing 
pUC4K Ap.sup.R, Km.sup.R vector, source of the kan 
Pharmacia 
cassette (USA) 
pGP704 Ap.sup.R broad host range suicide vector 
Miller & 
Mekalanos 
pGZRS19 
Ap.sup.R APP-E. coli shuttle vector 
West et al. 
pTF10 AP106 ribGBAH genes cloned into 
Fuller & Mulks 
pUC19 
pTF66 A 2.9 kb fragment containing AP106 
This work 
ribBAH in pGP704 
pTF67a pTF66 with all of ribA and part of 
This work 
ribB deleted and replaced with the 
kan cassette from pUC4K 
pTF76 5.2 Kb insert from pTF10 cloned into 
This work 
pGZRS19 
______________________________________ 
TABLE 4 
______________________________________ 
Mortality and Lung Lesion Data 
Dose Mor- % % 
Group Strain (LD.sub.50).sup.a 
tality 
Pneumonia.sup.b 
Pleuritis.sup.c 
______________________________________ 
1 AP225 (WT) 1 1/3 66.7 71.7 
2 AP233 (Rib-) 
4 0/3 0 0 
3 AP233 (Rib-) 
20 0/3 0 0 
4 AP233 (Rib-) 
100 0/3 0 0 
5 AP233 (Rib-) 
500 0/3 0 0 
6 AP233 + pTF76 
1 0/3 27.6 20.2 
______________________________________ 
.sup.a Doses are multiples of the established wildtype APP 225serotype 1 
LD.sub.50 of 5.0 .times. 10.sup.6 cfu (12) 
.sup.b Percentage of lung tissue exhibiting A. pleuropneumoniae lesions 
.sup.c Percentage of pleural surface area exhibiting pleuritis 
TABLE 5 
__________________________________________________________________________ 
Clinical Score Data 
Group 
Strain Dose (LD.sub.50).sup.a 
RR Max.sup.b 
Temp Max.sup.c 
Dyspnea.sup.d 
Depression.sup.e 
Appetite.sup.f 
__________________________________________________________________________ 
1 AP225 1 20 105.7 5.5 6.7 4.2 
2 AP233 5 8 102.5 0 0 0 
3 AP233 20 8 103.3 0 0 0 
4 AP233 100 8 103.5 0 0 0 
5 AP233 500 8 102.8 0 0 0 
6 AP233 + pTF76 
1 19.3 105.4 4.5 4.7 3.7 
Normal 8.0 &lt;103.0 0 0 0 
Maximum 25 15 15 5 
__________________________________________________________________________ 
.sup.a Doses are multiples of the established wildtype APP225serotype 1 
LD.sub.50 of 5.0 .times. 10.sup.6 cfu (12) 
.sup.b Maximum respiratory rate observed after challenge. Respiratory rat 
recorded as number of breaths per 15 sec observation period. 
.sup.c Maximum rectal temperature after challenge, in degrees Fahrenheit. 
.sup.d Dyspnea score measures degree of respiratory distress and labored 
breathing. Scored as 0 = normal; 1 = slight; 2 = moderate; 3 = severe. 
Total score = sum of scores taken at 12 hour intervals after challenge. 
.sup.e Depression score evaluates attitude and activity. Scored as 0 = 
normal; 1 = slight inactivity; 2 = moderate; 3 = severe. Total score = su 
of scores taken at 12 hour intervals after challenge. 
.sup.f Appetite was scored as 0 = did eat; 1 = did not eat. Total score = 
number of 12 hour periods not eating over 60 hour observation period. 
DISCUSSION 
Above is shown the construction of a serotype 1 Actinobacillus 
pleuropneumoniae deletion-disruption riboflavin mutant that is attenuated 
in vivo. The A. pleuropneumoniae ribGBAH operon was disrupted by deleting 
an internal segment of the operon (ribBA) and replacing it with a Km.sup.R 
cassette using a targeted mutagenesis technique (Mulks, M. H. and J. M. 
Buysse. 1995. A targeted mutagenesis system for Actinobacillus 
pleuropneumoniae. Gene 165:61-66). A stable riboflavin-requiring, Km.sup.R 
mutant, AP233, was phenotypically identical to its wild-type parent based 
on analysis of proteins, extracellular toxin, LPS, and capsular 
polysaccharide by SDS-PAGE, immunoblot, and coagglutination. 
A riboflavin mutant of A. pleuropneumoniae serotype 5 was also constructed 
and was also found to be attenuated in a preliminary animal challenge 
experiment. However, further studies were conducted in serotype 1 because 
serotype 5 seemed to be very resistant to transformation by standard heat 
shock or electroporation procedures. In order to complement the rib 
mutation in trans, and for ease of future genetic manipulations, it was 
desirable to use a serotype 1 strain for these studies. 
Experimental infection of pigs, the only natural host for A. 
pleuropneumoniae, demonstrated that the riboflavin-requiring mutant was 
unable to cause disease at dosages as high as 500 times the LD.sub.50 for 
the wild-type parent. In the four groups of pigs infected with AP233 by 
intratracheal inoculation, there was no mortality, no significant clinical 
signs were observed, and no typical pleuropneumonic lesions were observed 
at necropsy. Complementation of AP233 in trans with the wild-type A. 
pleuropneumoniae riboflavin operon restored both the ability to grow 
without exogenous riboflavin and virulence, demonstrating that the 
riboflavin mutation itself is responsible for the attenuation in vivo. 
It is important to note that the riboflavin-requiring mutant used in these 
studies is a deletion mutant, with .about.1.4 Kb of the riboflavin operon 
removed from the chromosome and replaced with an antibiotic resistance 
marker. Neither reversion to prototrophy nor loss of kanamycin resistance 
in this mutant in the laboratory was observed. In the preliminary 
experiment with a serotype 5 riboflavin mutant, it was possible to 
reisolate the mutant from the lungs at 16 hours post-infection. All 
colonies isolated in this experiment were kanamycin-resistant, nalidixic 
acid-resistant, and riboflavin requiring, suggesting that reversion to 
prototrophy and thus virulence will not occur in vivo. 
In the dosage trial experiment, AP233 was not recovered from the lungs of 
infected swine at 48 hours post-infection. These results may indicate poor 
persistence of the organism in vivo. If necessary, sufficient exogenous 
riboflavin could be added to the vaccine to allow the organism to 
replicate minimally and therefore persist long enough to induce a 
protective immune response. The above represents a new addition to the 
group of biosynthetic mutations that can be used to construct attenuated 
strains of bacteria. It also shows a genetically modified attenuated 
mutant of APP that is capable of production of all of the major virulence 
factors of this organism, including extracellular toxins and capsular 
polysaccharide. 
EVALUATION OF A RIBOFLAVIN-REQUIRING AUXOTROPHIC MUTANT OF ACTINOBACILLUS 
PLEUROPNEUMONIAE AS A GENETICALLY DEFINED LIVE ATTENUATED VACCINE AGAINST 
PORCINE PLEUROPENUMONIA 
The applicants have evaluated a genetically defined riboflavin-requiring 
attenuated mutant of Actinobacillus pleuropneumoniae as a live avirulent 
vaccine that provides immunity against experimental challenge with a 
virulent strain of A. pleuropneumoniae. 
The specific aims of this study were: 1) to evaluate whether respiratory 
exposure to a live attenuated vaccine APP strain elicits protection 
against subsequent experimental challenge with virulent A. 
pleuropneumoniae; and 2) to determine whether addition of exogenous 
riboflavin to the vaccine dosage improves persistence, and therefore 
immunogenicity and protection; and 3) to compare the protection afforded 
by respiratory exposure to that elicited by intramuscular (IM) 
immunization with the live vaccine, which is a more commercially feasible 
vaccination route. 
MATERIALS AND METHODS 
Animals. In this study, 6-to-8 week old crossbred (Yorkshire/Landrace) 
barrows from a herd known to be free of A. pleuropneumoniae and related 
respiratory pathogens were used. Pigs were housed in the Michigan State 
University Research Containment Facility and fed a standard 
antibiotic-free diet provided by the MSU Swine Research and Teaching 
Center. 
Preparation of Vaccines. 
1. Live vaccine: The bacterial strain used to prepare the live attenuated 
vaccine was AP233, a derivative of the species type strain, ATCC27088 
(here designated APP-1A) that is resistant to nalidixic acid (Nal.sup.R), 
resistant to kanamycin (Kan.sup.R), and that requires riboflavin (Rib-) 
because it contains a riboflavin biosynthetic operon that has been mutated 
by deletion-disruption with a kanamycin resistance cassette. Bacteria for 
the live vaccine were grown in heart infusion broth containing 10 .mu.g/ml 
NAD (nicotine adenine dinucleotide)+5 mM CaCl.sub.2 +200 .mu.g/ml 
riboflavin, at 37 C., to an optical density at 520 nm of 0.8. Bacteria 
were harvested, washed once in phosphate buffered saline (PBS), pH 7.0, 
diluted in phosphate buffered saline (PBS) or PBS containing 5 .mu.g/ml 
riboflavin to the appropriate cell density, and used immediately as 
vaccine. 
2. Bacterin: Virulent APP-1A bacteria were grown in heart infusion broth 
containing 10 .mu.g/ml NAD (nicotine adenine dinucleotide)+5 mM CaCl.sub.2 
at 37 C., shaking at 160 rpm, to an optical density at 520 nm of 0.8. 
Bacteria were harvested by centrifugation and washed once with 
Tris-acetate-EDTA-DTT buffer. Bacteria were resuspended in buffer 
containing 0.2% formalin to a concentration of 5.times.10.sup.9 cfu/ml, 
and kept at room temperature for 1 hour, then stored at 4.degree. C. Each 
vaccine dose contained 1 ml formalinized cells, 0.5 ml saline, and 0.5 ml 
Emulsigen adjuvant (MVP Laboratories, Ralston, Nebr.). 
Vaccine groups. There were six treatment groups (six pigs/group) in this 
study. Pigs were blocked by starting weight and randomly assigned to 
treatment groups. The animals were vaccinated twice at a 3 week interval, 
and challenged with virulent APP serotype 1A (APP-1) two weeks after the 
second vaccination. Group 1 received 5.times.10.sup.8 cfu (100.times. the 
50% lethal dose previously established for the wild type parent strain WT 
LD.sub.50 !) of live AP233, our APP-1 riboflavin-requiring mutant, in 10 
ml of sterile PBS, by percutaneous transtracheal inoculation, as in our 
challenge model (described below). Group 2 received the same treatment as 
Group 1, except the bacteria were suspended in 10 ml of PBS containing 5 
.mu.g/ml riboflavin, a concentration of exogenous riboflavin sufficient to 
permit 2-3 generations of growth. Group 3 received 5.times.10.sup.8 cfu of 
live AP233, intramuscularly in 2 ml PBS. Group 4 received the same 
treatment as Group 3, except the bacteria were suspended in PBS plus 5 
.mu.g/ml riboflavin. Group 5 received a formalinized whole cell bacterin 
prepared from APP-1, which contained the equivalent of 5.times.10.sup.9 
cfu per dose, in 2 ml of 25% Emulsigen adjuvant (MVP Laboratories, 
Ralston, Nebr.). Group 6 were unvaccinated controls. 
Experimental challenge. Two weeks after the second vaccination, all groups 
of pigs were challenged with virulent wild type APP-1A, using an 
experimental challenge model (Jolie, R. A. V., M. H. Mulks, and B. J. 
Thacker. 1995. Cross-protection experiments in pigs vaccinated with 
Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 45: 
383-391; Thacker, B. J., M. H. Mulks, B. Yamini, & J. Krehbiel. 1988. 
Clinical, immunological, hematological, microbiological, and pathological 
evaluation of a percutaneous intratracheal injection Haemophilus 
pleuropneumoniae challenge model. Proc. Int. Pig Vet. Soc. 10: 69). For 
the challenge inoculum, bacteria were grown to late exponential phase in 
heart infusion broth containing 10 .mu.g/ml NAD+5 mM CaCl.sub.2, washed 
once in sterile saline, and diluted in saline to the appropriate cell 
density. Pigs were anesthetized by intravenous injection with a mixture of 
ketamine (6.6 mg/kg) and xylazine (1.65 mg/kg) and inoculated 
transtracheally with 1 LD.sub.50 (5.times.10.sup.6 cfu) of APP-1 suspended 
in 10 ml saline. Clinical signs, including increased rectal temperature, 
increased respiration rate, dyspnea, decreased appetite, and depression, 
were monitored at 4 hour intervals for the first 24 hours post infection, 
and at 12 hour intervals thereafter. Severely ill animals, as determined 
by the severity of clinical signs, were euthanized by overdose with a 
pentobarbital solution (Beuthanasia) delivered intravenously and 
necropsied immediately. Three days post-infection, all surviving pigs were 
euthanized and necropsied, and gross pathology of the lungs examined and 
compared. Lungs were examined macroscopically for APP lesions, including 
edema, congestion, hemorrhage, infarction, necrosis, abscess, fibrosis, 
and pleuritis. The percentage of lung tissue and surface area affected was 
estimated for each of the seven lung lobes, and the data inserted into a 
formula that weights the contribution of each lung lobe to give a total 
percentage of lung involvement and affected pleural surface (Thacker, B. 
J., M. H. Mulks, B. Yamini, & J. Krehbiel. 1988. Clinical, immunological, 
hematological, microbiological, and pathological evaluation of a 
percutaneous intratracheal injection Haemophilus pleuropneumoniae 
challenge model. Proc. Int. Pig Vet. Soc. 10: 69). Tissue samples were 
collected and processed for histopathology, and for culture of APP to 
confirm infection. Protection of pigs against challenge was measured as a 
reduction in mortality, in the severity of lung lesions, and in the 
severity and duration of clinical signs as compared to the unvaccinated 
control animals. Statistical analysis of the data was conducted using the 
Statistix microcomputer program (Analytical Software, Tallahassee, Fla.) 
for analysis of variance (ANOVA) and Epistat (T. L. Gustafson, Round Rock, 
Tex.) for nonparametric analyses. 
RESULTS 
Safety. Pigs were monitored post-vaccination for any clinical signs of APP 
disease, such as fever, dyspnea, and increased respiratory rate, and for 
injection site reactions in Group 3, 4, and 5 animals. The bacterin 
vaccinated animals (Group 5) showed mild fever, depression, and decrease 
in appetite for 8-16 hours post-vaccination, which is a common reaction to 
bacterin vaccines. Several of the Group 5 animals had granulomatous 
reactions at the injection site in the neck muscle, which were detected at 
necropsy. The Group 1 and 2 animals, which received intratracheal 
immunizations, showed increased respiratory rates, fever, decreased 
appetite, and mild depression for 8-16 hours post-immunization. The Group 
3 and 4 animals, which received the intramuscular vaccine, showed only 
slight depression and decreased appetite for &lt;8 hours, and no significant 
fever or increase in respiratory rate. No injection site reactions were 
detected in the Group 3 or 4 animals at necropsy. These results 
demonstrate that the live intramuscular vaccine is at least as safe as, if 
not safer than, a formalinized bacterin of the type routinely used 
commercially at this time. 
Immunogenicity. The immune responses of the pigs to vaccination were 
evaluated by ELISA against APP outer membranes (Jolie, R. A. V., M. H. 
Mulks, and B. J. Thacker. 1995. Cross-protection experiments in pigs 
vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. 
Microbiol. 45: 383-391); by hemolysin neutralization titer (Montaraz, J. 
A., B. Fenwick, H. Hill, and M. Rider. 1996. Evaluating antibody 
isotype-specific ELISA, complement fixation, and ApxI hemolysin 
neutralization tests to detect serum antibodies in pigs infected with 
Actinobacillus pleuropneumoniae serotype 1. Swine Health and Production 4: 
79-83); and by complement fixation (CF) (Hoffman, L. J. 1989. 
Actinobacillus (Haemophilus) pleuropneumoniae: Use of coagglutination and 
complement fixation to determine the relationship between presence of 
organisms and antibody titer in slaughterhouse pigs. J. Vet. Diagn. 
Invest. 1:12-15)(Table 1). 
At challenge, the bacterin-vaccinated animals showed significant ELISA and 
complement fixation titers, but low or negative hemolysin neutralization 
titers. The four groups receiving live vaccines showed low or negative 
ELISA and CF titers. However, the Group 3 and 4 animals did show 
significant hemolysin neutralization titers. 
TABLE 6 
______________________________________ 
Serologic analysis of serum samples collected at challenge 
Group # 
Vaccine HN.sup.1 ELISA-APP1.sup.2 
CF.sup.3 
______________________________________ 
1 Live, IT, 3129 .+-. 1478.sup.b 
227 .+-. 90.sup.b 
1.7 .+-. 2.8.sup.b 
PBS 
2 Live, IT, 2520 .+-. 741.sup.b 
164 .+-. 73.sup.b,c 
1.6 .+-. 3.1.sup.b 
PBS + 
riboflavin 
3 Live, IM, 10760 .+-. 6245.sup.a 
120 .+-. 32.sup.b,c 
0.0 .+-. 0.0.sup.b 
PBS 
4 Live, IM, 6293 .+-. 2662.sup.a,b 
236 .+-. 173.sup.b 
2.0 .+-. 4.0.sup.b 
PBS + 
riboflavin 
5 APP-1A 3035 .+-. 285.sup.b 
1119 .+-. 170.sup.a 
24.3 .+-. 7.4.sup.a 
bacterin 
6 Unvac- 2240 .+-. 243.sup.b 
67 .+-. 21.sup.c 
0.0 .+-. 0.0.sup.b 
cinated 
control 
______________________________________ 
.sup.1 Hemolysin neutralization titer; &lt;3000 = negative; 3000-6000 = 
suspect; &gt;6000 = positive. Assays performed in the laboratory of Dr. Brad 
Fenwick, Kansas State University. 
.sup.2 ELISA vs APP1 outer membranes; &lt;200 = negative, 200-300 = suspect, 
&gt;300 = positive. Assays performed in the laboratory of Dr. Martha H. 
Mulks, Michigan State University. 
.sup.3 Complement fixation test; reported as geometric mean titer 0 = 
negative; &gt;0 = positive. Assays performed at the Veterinary Diagnostic 
Laboratory, Iowa State University. 
Addition of riboflavin to the inoculum. In preliminary studies, it was 
found that riboflavin-requiring strains of APP failed to persist in the 
porcine respiratory tract for more than 16-24 hours. Poor persistence of 
live vaccine strains in vivo can lead to a failure to elicit a protective 
immune response. A. pleuropneumoniae and other related pathogens can 
produce infection-associated antigens when grown in an appropriate host. 
These are antigens that are only produced by the bacterium when it is 
grown within a host animal, presumably due to specific environmental 
stimuli such as temperature, lack of available iron, pH, or osmotic 
conditions (Mekalanos, J. J. 1992. Environmental signals controlling 
expression of virulence determinants in bacteria. Infect. Immun. 
174:1-7.). Such infection associated antigens are not produced when the 
bacterium is grown in vitro in standard laboratory media. In order to 
assure that such infection-associated antigens would be expressed by the 
live attenuated vaccine strain of bacteria after immunization of pigs, it 
was necessary to ensure that the bacteria had sufficient available 
riboflavin to permit 2-3 generations of growth. It was determined that 
addition of 5-10 .mu.g of riboflavin per ml of the vaccine inoculum was 
sufficient to permit this amount of growth. Therefore, as part of this 
vaccine trial, intratracheal (IT) and intramuscular (IM) administration of 
the live attenuated vaccine, with and without the addition of 5 .mu.g/ml 
exogenous riboflavin, were compared. 
Riboflavin may be added to permit two generations of growth such that the 
amount may vary from about 1 to about 10 .mu.g/ml. 
Protection against challenge. In this experiment, the live attenuated 
vaccine prepared with exogenous riboflavin and delivered intramuscularly 
(Group 4) provided complete protection against mortality (0/5 animals 
died) and a significant reduction in lung damage and in some clinical 
signs of pleuropneumonia (Tables 2 and 3). In contrast, 6/6 unvaccinated 
control animals died from overwhelming pleuropneumonia as a result of this 
experimental challenge. Other live vaccine formulations, as well as the 
formalinized bacterin, afforded less protection than the intramuscular 
immunization containing riboflavin. It is concluded that 1) intramuscular 
immunization with this live vaccine does elicit significant protection 
against APP infection; 2) that intratracheal immunization does not elicit 
the same degree of protection; and 3) that the addition of exogenous 
riboflavin improves the efficacy of the live vaccine. 
Lung cultures. APP was cultured from the lungs of all the challenged pigs 
except for 1 animal in Group 4. All cultures were confirmed as APP-1A by 
gram stain, requirement for NAD, and coagglutination. 
TABLE 7 
______________________________________ 
Mortality and Lung Score Data 
Group Vaccine.sup.1 
Mortality 
% Pneumonia.sup.2 
% Pleuritis.sup.3 
______________________________________ 
1 Live, IT, PBS 
3/5 58.6 .+-. 23.5.sup.a,b 
73.3 .+-. 39.3.sup.a 
2 Live, IT, PBS + 
6/6 63.2 .+-. 8.2.sup.a,b 
66.7 .+-. 51.6.sup.a 
riboflavin 
3 Live, IM, PBS 
4/6 57.7 .+-. 23.2.sup.b 
73.3 .+-. 42.5.sup.a 
4 Live, IM, PBS + 
0/5 24.5 .+-. 15.0.sup.c 
21.5 .+-. 20.7.sup.a 
riboflavin 
5 APP-1A bacterin 
3/6 54.1 .+-. 24.8.sup.b 
73.9 .+-. 41.2.sup.a 
6 Unvaccinated 6/6 80.9 .+-. 13.2.sup.a 
83.3 .+-. 40.8.sup.a 
control 
______________________________________ 
.sup.1 IT: live vaccine administered by intratracheal inoculation; IM: 
live vaccine administered by intramuscular injection. 
.sup.2 Percentage of lung tissue exhibiting A. pleuropneumoniae lesions; 
results presented as mean .+-. standard deviation. 
.sup.3 Percentage of pleural surface area exhibiting pleuritis; results 
presented as mean .+-. standard deviation. 
.sup.a . . . c Values with different superscripts among the six vaccine 
groups were significantly different (p &lt; 0.05) by Least Significant 
Difference (LSD) analysis. 
TABLE 8 
__________________________________________________________________________ 
Clinical Score Data 
Group 
Vaccine.sup.1 
RR Max.sup.2 
Temp Max.sup.3 
Dyspnea.sup.4 
Depression.sup.5 
Appetite.sup.6 
__________________________________________________________________________ 
1 Live, IT, PBS 
22.0 .+-. 5.2.sup.a 
104.7.sup.a 
1.80 .+-. .45.sup.a,b 
1.40 .+-. .55.sup.a 
2.00 .+-. .71.sup.a 
2 Live, IT, PBS + 
19.7 .+-. 5.7.sup.a 
104.0.sup.a 
2.17 .+-. .41.sup.a 
1.67 .+-. .82.sup.a 
0.75 .+-. .96.sup.b,c 
riboflavin 
3 Live, IM, PBS 
19.2 .+-. 1.2.sup.a 
104.7.sup.a 
1.83 .+-. 41.sup.a,b 
1.27 .+-. .75.sup.a 
2.33 .+-. 1.21.sup.a 
4 Live, IM, PBS + 
18.2 .+-. 3.4.sup.a 
104.2.sup.a 
1.20 .+-. .75.sup.b 
0.40 .+-. .89.sup.a 
0.20 .+-. .45.sup.c 
riboflavin 
5 APP-1A bacterin 
23.3 .+-. 1.6.sup.a 
104.7.sup.a 
1.83 .+-. 52.sup.a,b 
1.83 .+-. .75.sup.a 
1.67 .+-. 1.03.sup.a,b 
6 Unvaccinated control 
23.0 .+-. 5.8.sup.a 
104.9.sup.a 
2.33 .+-. .52.sup.a 
1.83 .+-. .75.sup.a 
1.83 .+-. .90.sup.a,b 
Normal 8.0 &lt;103.0 0 0 0 
Maximum 25 3 3 3 
__________________________________________________________________________ 
.sup.1 IT: live vaccine administered by intratracheal inoculation; IM: 
live vaccine administered by intramuscular injection. 
.sup.2 Maximum respiratory rate observed after challenge. Respiratory rat 
recorded as number of breaths per 15 second observation period. 
.sup.3 Maximum rectal temperature after challenge, in degrees Fahrenheit. 
.sup.4 Maximum dyspnea score observed after challenge. Dyspnea score 
measures degree of respiratory distress and labored breathing. Scored as 
= normal; 1 = slight; 2 = moderate; 3 = severe. 
.sup.5 Maximum depression score observed after challenge. Depression scor 
evaluates attitude and activity. Scored as 0 = normal; 1 = slight 
inactivity; 2 = moderate; 3 = severe. 
.sup.6 Appetite was scored as 0 = did eat; 1 = did not eat. Total score = 
number of 12 hour periods not eating over 36 hour observation period. 
.sup.a . . . c Values with different superscripts among the six vaccine 
groups were significantly different (p &lt; 0.05) by Least Significant 
Difference (LSD) analysis. 
It is concluded that intramuscular vaccination with the live attenuated 
riboflavin-requiring A. pleuropneumoniae mutant, with the addition of a 
limited amount of exogenous riboflavin, led to complete protection against 
mortality and to significant reduction in lung damage and clinical signs 
of pleuropneumonia. 
The applicants have determined that other related species of pathogenic 
bacteria in the Family Pasteurellaceae contain homologous genes encoding 
riboflavin biosynthetic enzymes. Southern blot analysis of genomic DNA 
from Actinobacillus suis and Pasteurella haemolytica demonstrated that 
these species contain DNA fragments that are highly homologous to the rib 
genes from A. pleuropneumoniae. Genomic DNA from these species was 
digested with the restriction endonucleases EcoRI and HindIII, fragments 
separated on an agarose gel, and the fragments transferred to 
nitrocellulose. The nitrocellulose blot was probed with a 
digoxigenin-labelled probe prepared from the ribGBAH operon from APP 
serotype 5, at 42.degree. C., in a hybridization cocktail that included 
50% formamide, 5.times.SSC (20.times.SSC contains 3 M NaCl and 0.3 M 
sodium citrate, pH 7.0), 0.1% N-lauroylsarcosine, and 0.02% SDS. The blot 
was washed under high stringency conditions, including two 15 minute 
washes at room temperature in 2.times.SSC, 0.1% SDS, followed by two 30 
minute washes at 68.degree. C. in 0.1.times.SSC, 0.1% SDS. P. haemolytica 
contains an .about.12 kb DNA HindIII fragment that hybridized with the rib 
probe, while A. suis contains three EcoRI fragments of .about.4.4, 2.5, 
and 1.0 kb that are highly homologous to the APP-5 rib probe. These data 
suggest that these species of bacteria contain riboflavin operons that are 
similar to that analyzed from APP serotype 5. 
A live avirulent vaccine against APP is desirable. There are a variety of 
different kinds of vaccines produced to elicit protection against 
bacterial diseases. Some of the most effective are purified toxins 
converted to toxoids. These toxoid vaccines are often very safe, and can 
be very effective against diseases where a toxin is the major virulence 
factor. Examples would be current vaccines against tetanus and diphtheria. 
These vaccines do not prevent acquisition and carriage of the causative 
organism, e.g, Clostridium tetani, the causative agent of tetanus, or 
Corynebacterium diphtheriae, the agent of diphtheria. Rather, they prevent 
the deleterious effects of the toxin by eliciting antibodies that 
neutralize the toxin. In other cases where a key virulence factor has been 
identified, purified protein or polysaccharide vaccines have been 
produced. Examples here would be the E. coli pilin vaccine against porcine 
colibacillosis and the capsular polysaccharide vaccines now available 
against Haemophilus influenzae B, Streptococcus pneumoniae, and some 
serotypes of Neisseria meningitidis. These vaccines either prevent initial 
adherence of the pathogen, as in the case of the pilin vaccine, or enhance 
phagocytosis and clearance of the pathogen, as in the case of the capsular 
polysaccharide vaccines. In the veterinary field, it is also common to use 
bacterin vaccines, that is, killed whole cell vaccines. Because these 
bacterins can induce a wide range of side effects, they are not commonly 
used for human vaccines. A problem with all of these types of vaccines is 
that they generally induce systemic humoral immunity, i.e., serum 
antibodies. It is difficult to induce local secretory immunity with these 
types of vaccines. Live avirulent vaccines, where the recipient of the 
vaccine receives a dose of infectious but not virulent bacteria, can be an 
improvement over purified subunit or killed whole cell vaccines, for 
several reasons. First, the vaccine dose can often be administered to the 
same region of the body that is normally infected by the pathogen, e.g., 
orally for a gastrointestinal pathogen or as a nasal spray for a 
respiratory pathogen, This can elicit local secretory immunity as well as 
systemic humoral immunity. Second, live avirulent vaccines can often be 
administered as a single dose rather than multiple doses, because the 
organism can continue to grow and replicate within the host, providing a 
longer term exposure to important antigens that a single dose of killed 
vaccine. Finally, live avirulent vaccines may provide exposure to 
important bacterial antigens not contained in killed vaccines grown in the 
laboratory. For example, if a bacterium produces important antigens or 
virulence factors whose expression is induced by in vivo environmental 
signals, these antigens may not be contained in a vaccine prepared from 
bacteria grown in vitro in laboratory media. It is desirable for a vaccine 
to elicit cross-protective immunity against the different serotypes of 
APP. It is known that vaccination with a killed whole cell vaccine 
prepared from a single serotype of APP will usually not elicit 
cross-protective immunity against other serotypes. However, infection with 
a virulent strain of APP will generally elicit at least some degree of 
cross-protection against other serotypes. One explanation for this 
phenomenon is that antigens may be expressed by APP during growth in vivo 
that elicit a cross-protective immune response, and that these antigens 
are not contained in most bacteria vaccines. 
Specifically, it has been shown that extracellular toxins, referred to as 
hemolysins/cytolysins, are produced by APP in vivo but are not produced 
under the culture conditions typically used for producing killed whole 
cell vaccines. 
The applicants have shown that riboflavin-requiring mutants of APP can be 
effective as a live avirulent vaccine. There are two basic methods for 
producing live avirulent vaccine strains. One is to knock out a critical 
virulence factor necessary for survival in vivo and perhaps also for 
disease/damage to the host. An example would be Inzana's non-capsulated 
APP mutants. These mutants are unable to synthesize capsular 
polysaccharide, which acts in vivo to protect the bacterium from 
phagocytosis and clearance by alveolar macrophages. Non-capsulated mutants 
simply can not survive long enough in vivo to cause disease. They do, 
however, presumably express all the other important virulence factors and 
therefore should elicit an immune response against antigens other than 
capsular polysaccharide. 
A second method to produce live avirulent vaccines is to knock out genes in 
biosynthetic pathways known to be critical for survival in vivo. For 
example, the availability of compounds such as purines and aromatic amino 
acids is limited in mammalian hosts. Bacterial pathogens must be able to 
synthesize these compound themselves, or scavenge them from host tissues. 
Mutations in the biosynthetic pathways for purines and aromatic amino 
acids have been used to construct bacterial mutants that can not survive 
long in vivo, and thus have potential for use as attenuated vaccines. Much 
of the current research on genetically engineered live avirulent vaccines 
has been done with members of the genus Salmonella. These studies show 
that purA mutants are avirulent but poorly immunogenic (O'Callaghan et al, 
1988), while mutations in the chorismate pathway, including aroA, aroc, 
and aroD, are attenuated and can be effective as live oral vaccines 
(Doggett & Curtiss, 1992; Tacket et al, 1992). In addition, Salmonella 
strains carrying cya and crp mutations, which produce mutants that lack 
the enzyme adenylate cyclase and the cyclic AMP receptor protein, which 
are required for the expression of numerous critical genes in bacteria, 
have been shown to be both avirulent and immunogenic (Doggett & Curtiss, 
1992; Tacket et al, 1992; Kelly et al, 1992). 
Riboflavin is an essential vitamin and biosynthetic precursor for the 
coenzymes FMN and FAD. It is synthesized by most bacteria, but not by 
mammals. Therefore, it is expected that riboflavin would be in limited 
supply in a mammalian host and that a bacterium incapable of synthesizing 
its own riboflavin would be attenuated. This has been shown above. It has 
also been shown above that Rib- mutants can survive long enough in the 
host to be immunogenic and effective as a live avirulent vaccine. 
The rib-APP mutant may be combined with a sterile, buffered, isotonic, 
pharmaceutically-acceptable and compatible aqueous carrier such as saline, 
or saline derivative such as citrate-buffered saline, tris-buffered 
saline, Ringer's Solution or tissue culture medium, and the like, 
preferably having a physiologic pH. An antigen composition may also 
include a suitable compatible adjuvant such as aluminum hydroxide, 
paraffin-based oils, averdine, muramyl dipeptide, and the like, to 
stabilize the antigen in solution, and/or an immunomodulator such as a 
recombinant cytokine or interleukin such as IL-1, IL-5, IL-6, TGF-beta, or 
gamma interferon, and the like, to enhance the IgA antibody response. 
However, the adjuvant chosen should not contain any preservative, such as 
formalin, that would be deleterious to a live vaccine. In the experiments 
described above, no adjuvant was used. 
The vaccine composition may be formulated for administration as a single 
injection of about 0.5 to 10 ml. The composition may also be in the form 
for administration in a series of biweekly or monthly injections of about 
0.5 to 10 ml each, until the desired level of immunity is achieved. 
Preferably,the composition is formulated for a single administration to 
the animal. 
The vaccine composition as described herein may be formulated with 
conventional pharmaceutically acceptable vehicles for administration by 
transthoracic intrapulmonary injection, intratracheal innoculation, 
subcutaneous, intraperitoneal or intramuscular injection. The vaccine may 
also be supplied orally or intranasally. These vehicles comprise 
substances that are essentially nontoxic and nontherapeutic such as saline 
and derivatives of saline such as citrate-buffered saline, tris-buffered 
saline and Ringer's Solution, dextrose solution, Hank's Solution, tissue 
culture medium, and the like. The antigen composition may also include 
minor but effective amounts of pharmaceutically-accepted adjuvants, 
buffers and preservatives to maintain isotonicity, physiological pH, and 
stability. Adjuvants useful in the composition include, but are not 
limited to, for example, paraffin based oils, averdine, muramyl dipeptide, 
and oil-in-water-based adjuvants, andthe like. Examples of suitable 
buffers include but not limited to, phosphate buffers, citrate buffers, 
carbonate buffers, TRIS buffers, and the like. It is also envisioned that 
the antigen may be combined with a biocompatible, and optimally 
synergistic, immunomodulator that cooperatively stimulates IgA antibody 
production, as for example, but not limited to, recombinant cytokines such 
as TGF-beta, interferons, activating factors, chemoattractants, 
interleukins such as IL-1, IL-2, IL-4, IL-5, IL-6 and the like, and other 
like substances. 
While the forms of the invention herein disclosed constitute presently 
preferred embodiments, many others are possible. It is not intended here 
to mention all the possible equivalent forms or ramifications of the 
invention. It is understood that the terms used herein are merely 
descriptive, and that various changes may be made without departing from 
the spirit or scope of the invention. 
FIGURE LEGENDS 
FIG. 1. Proposed bacterial riboflavin biosynthesis pathway. Proposed gene 
functions are as indicated although the functions of ribG and ribT have 
not been determined conclusively. Structures correspond to the following: 
I, GTP; II, 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone 5'-phosphate; 
III, 5-amino-6-(ribosylamino)-2,4(1H,3H)-pyrimidinedione 5'-phosphate; IV, 
5-amino-6-(ribitylamino)-2,4(1H,3H)-pyrimidinedione 5'-phosphate; V, 
5-amino-6-(ribitylamino)-2,4(1H,3H) -pyrimidinedione; VI, ribulose 
5'-phosphate; VII, 3,4-dihydroxy-2-butanone 4-phosphate; VIII, 
6,7-dimethyl-8-ribityllumazine; IX, riboflavin. Structures are adapted 
from Bacher (1). 
FIG. 2. Physical map of the construct, pTF10, which contains the APP 
riboflavin synthesis genes. 
FIG. 3. Absorbance spectra of aqueous solutions at neutral pH (Panel A) and 
acidified aqueous solutions (Panel B) of the product excreted into the 
growth medium by E. coli DH5.alpha./pTF10 (solid line) and of a standard 
riboflavin preparation (dotted line). 
FIG. 4. Complete nucleotide sequence of APP ribGBAH genes and flanking 
regions. The amino acid translations are shown for ribG, ribB, ribA, and 
ribH and correspond to base pairs 330-1560, 1685-2330, 2393-3596 and 
3709-4168. Putative ribosome binding sites are underlined. Potential 
promoters for the operon and for ribH are double-underlined. An inverted 
repeat that may function as a transcription terminator is indicated with 
arrows. 
FIG. 5. Complementation of E. coli mutants by cloned APP rib genes. A 
physical map for the APP ribGBAH genes is shown as well as several 
deletions that were made from the 3' end of the APP rib clone. The E. coli 
gene designations are indicated above their APP homologues. A "+" 
indicates complementation of the indicated E. coli mutation by the 
recombinant plasmid. nd=not done. 
FIG. 6. Minicell analysis of pTF10 and deletions. Minicells contained: Lane 
1, pUC19; Lane 2, pTF10; Lane 3, pTF19. Molecular weight standards are 
indicated on the left. Proteins encoded by the APP genes are indicated by 
the arrows on the right. Apparent molecular weights for the APP Rib 
enzymes are: RibG, 45 kDa; RibA, 43.7 kDa; RibB, 27.7 kDa, and RibH, 14.8 
kDa. 
FIG. 7. Construction of pTF67A. The entire riboflavin operon, containing 
the ribGBAH genes from AP106 was cloned into pUC19 to make pTF10 (5). A 
2.9 kb fragment containing the ribBAHportion of the riboflavin operon was 
excised from pTF10 with EcoRI and ligated into the EcoRI site of the 
conjugative suicide vector pGP704 to form pTF66. A 1.4 kb ClaI/NdeI 
fragment, which contains all of ribA and part of ribb was deleted and 
replaced with the Km.sup.R cassette from pUC4K to create pTF67a. 
FIG. 8. Southern blot analysis of chromosomal DNA from AP100 rib mutants. 
(A) Schematic structure of the rib locus of parent and mutant strains in 
double and single cross-over events. The predicted sizes of HindIII 
genomic fragments are shown for two possible single cross-over events and 
for a double cross-over event. The results show that for AP233 the 
chromosomal rib operon has been replaced with the cloned riboflavin operon 
containing the Km.sup.R cassette by a double cross-over event, while AP234 
is the result of a single cross-over event either upstream or downstream 
of the kanamycin cassette. Restriction enzymes used: E=EcoRI; H=HindIII. 
(B) Southern blots of HindIII or EcoRI digested DNA from mutants and 
controls. Blots were prepared in quadruplicate and hybridized at high 
stringency with one of four probes: Rib, the entire ribGBAH operon from 
pTF10; R. Del., the deleted portion (ClaI/NdeI fragments) of the ribGBAH 
operon; pGP704, the entire plasmid; Km, the kanamycin cassette from pUC4K. 
Lanes: 1, pTF10 digested with HindIII; 2, AP106+HindIII; 3, AP100+HindIII; 
4, pTF67a+EcoRI; 5, AP233+HindIII; and 6, AP234+HindIII. 
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# SEQUENCE LISTING 
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# 50ATTTTC AAAAGTAGAA GATGCGATCG AAGCGATTCG 
# 100TTTTAG TGACTGACGA TGAAGATCGC GAAAACGAAG 
# 150GCGGCG GAATTTGCCA CACCGGAAAA TATCAATTTT 
# 200CAAAGG TTTGATTTGT ACGCCGATTT CAACCGAAAT 
# 250ATTTCC ATCCGATGGT TGCGGTCAAT CAAGATAATC 
# 300ACCGTA TCGGTGGATC ATATTGATAC GGGAACGGGT 
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# 850GCGGTC AGCAATTTGC CGCAGCAATG ACCCAAATTG 
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- Thr Gly Thr Gly Ile Ser Ala Phe Glu Arg Se - #r Ile Thr Ala Met Lys 
# 110 
- Ile Val Asp Asp Asn Ala Lys Ala Thr Asp Ph - #e Arg Arg Pro Gly His 
# 125 
- Met Phe Pro Leu Ile Ala Lys Glu Gly Gly Va - #l Leu Val Arg Asn Gly 
# 140 
- His Thr Glu Ala Thr Val Asp Leu Ala Arg Le - #u Ala Gly Leu Lys His 
145 1 - #50 1 - #55 1 - 
#60 
- Ala Gly Leu Cys Cys Glu Ile Met Ala Asp As - #p Gly Thr Met Met Thr 
# 175 
- Met Pro Asp Leu Gln Lys Phe Ala Val Glu Hi - #s Asn Met Pro Phe Ile 
# 190 
- Thr Ile Gln Gln Leu Gln Glu Tyr Arg Arg Ly - #s His Asp Ser Leu Val 
# 205 
- Lys Gln Ile Ser Val Val Lys Met Pro Thr Ly - #s Tyr Gly Glu Phe Met 
# 220 
- Ala His Ser Phe Val Glu Val Ile Ser Gly Ly - #s Glu His Val Ala Leu 
225 2 - #30 2 - #35 2 - 
#40 
- Val Lys Gly Asp Leu Thr Asp Gly Glu Gln Va - #l Leu Ala Arg Ile His 
# 255 
- Ser Glu Cys Leu Thr Gly Asp Ala Phe Gly Se - #r Gln Arg Cys Asp Cys 
# 270 
- Gly Gln Gln Phe Ala Ala Ala Met Thr Gln Il - #e Glu Gln Glu Gly Arg 
# 285 
- Gly Val Ile Leu Tyr Leu Arg Gln Glu Gly Ar - #g Gly Ile Gly Leu Ile 
# 300 
- Asn Lys Leu Arg Ala Tyr Glu Leu Gln Asp Ly - #s Gly Met Asp Thr Val 
305 3 - #10 3 - #15 3 - 
#20 
- Glu Ala Asn Val Ala Leu Gly Phe Lys Glu As - #p Glu Arg Glu Tyr Tyr 
# 335 
- Ile Gly Ala Gln Met Phe Gln Gln Leu Gly Va - #l Lys Ser Ile Arg Leu 
# 350 
- Leu Thr Asn Asn Pro Ala Lys Ile Glu Gly Le - #u Lys Glu Gln Gly Leu 
# 365 
- Asn Ile Val Ala Arg Glu Pro Ile Ile Val Gl - #u Pro Asn Lys Asn Asp 
# 380 
- Ile Asp Tyr Leu Lys Val Lys Gln Ile Lys Me - #t Gly His Met Phe Asn 
385 3 - #90 3 - #95 4 - 
#00 
- Phe 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 645 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS:single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
# 50TATTGA AGAAGTCGGC AAAATTGCTC AAATTCATAA 
# 100CGGTAG TCACAATTAA TGCGACCAAA GTATTACAAG 
# 150GACACG ATTGCGGTGA ACGGCGTATG TTTAACCGTA 
# 200TAATCA GTTTACCGCC GATGTAATGT CGGAAACGTT 
# 250TAGGCG AATTAAAGTC GAATAGTCCG GTTAATTTAG 
# 300GCAAAC GGACGTTTCG GCGGACACAT CGTTTCGGGG 
# 350CGGCGA AATTGCGGAA ATCACACCGG CACATAATTC 
# 400TTAAAA CCTCTCCAAA ATTAATGCGT TATATTATTG 
# 450ACCATT GACGGTATTA GCCTGACCGT AGTCGATACC 
# 500CCGTGT ATCGATTATT CCGCATACGA TTAAAGAAAC 
# 550AAAAAA TCGGCAGTAT TGTCAATTTA GAAAATGATA 
# 600ATCGAA CAGTTTTTAC TGAAAAAGCC GGCGGATGAG 
# 645TT AGACTTTTTA AAGCAGGCGG GATTT 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 215 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- Met Phe Thr Gly Ile Ile Glu Glu Val Gly Ly - #s Ile Ala Gln Ile His 
# 15 
- Lys Gln Gly Glu Phe Ala Val Val Thr Ile As - #n Ala Thr Lys Val Leu 
# 30 
- Gln Asp Val His Leu Gly Asp Thr Ile Ala Va - #l Asn Gly Val Cys Leu 
# 45 
- Thr Val Thr Ser Phe Ser Ser Asn Gln Phe Th - #r Ala Asp Val Met Ser 
# 60 
- Glu Thr Leu Lys Arg Thr Ser Leu Gly Glu Le - #u Lys Ser Asn Ser Pro 
# 80 
- Val Asn Leu Glu Arg Ala Met Ala Ala Asn Gl - #y Arg Phe Gly Gly His 
# 95 
- Ile Val Ser Gly His Ile Asp Gly Thr Gly Gl - #u Ile Ala Glu Ile Thr 
# 110 
- Pro Ala His Asn Ser Thr Trp Tyr Arg Ile Ly - #s Thr Ser Pro Lys Leu 
# 125 
- Met Arg Tyr Ile Ile Glu Lys Gly Ser Ile Th - #r Ile Asp Gly Ile Ser 
# 140 
- Leu Thr Val Val Asp Thr Asp Asp Glu Ser Ph - #e Arg Val Ser Ile Ile 
145 1 - #50 1 - #55 1 - 
#60 
- Pro His Thr Ile Lys Glu Thr Asn Leu Gly Se - #r Lys Lys Ile Gly Ser 
# 175 
- Ile Val Asn Leu Glu Asn Asp Ile Val Gly Ly - #s Tyr Ile Glu Gln Phe 
# 190 
- Leu Leu Lys Lys Pro Ala Asp Glu Pro Lys Se - #r Asn Leu Ser Leu Asp 
# 205 
- Phe Leu Lys Gln Ala Gly Phe 
# 215 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH:1230 bases 
(B) TYPE:nucleic acid 
(C) STRANDEDNESS:single 
(D) TOPOLOGY:linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
# 50TAAGCG GTGGTTTTTC CTATCTTTTT TACAAGCCTT 
# 100TCAAGG CTTTTTTCAT CATTAGGGTA AACATGCCTG 
# 150CTGCCC TCAAATAGTT TCAAAACAAT GACGGATTTA 
# 200TGCCAT TGCACTGGCA AAACAAGGTT TAGGCTGGAC 
# 250TTGTCG GTTGTGTAAT TGTCAAAAAC GGTGAAATCG 
# 300CATGAA AAGATTGGTG GATGGCATGC GGAACGTAAT 
# 350TAAGGA AGATCTTTCC GGGGCGACTG CTTATGTAAC 
# 400GTCATC ACGGCCGCAC GCCGCCTTGT TCGGATTTAT 
# 450ATTAAA AAAGTATTTA TCGGTTCGAG CGATCCGAAT 
# 500GCGGGG AGCAAATCAG CTACGCCAAG CCGGCGTGGA 
# 550TACTCA AAGAAGAATG TGATGCGTTA AACCCGATTT 
# 600CAAACT AAACGTCCGT ATGTGCTAAT GAAATATGCC 
# 650CAAAAT TGCAACCGGT AGCGGCGAAT CCAAATGGAT 
# 700CAAGAG CAAGAGTGCA GCAAACACGT CATCAATATA 
# 750GGTGTA GATACGGTAC TTGCCGATAA CCCGATGTTA 
# 800GAATGC GAAACAACCG GTCCGGATTG TCTGCGATAG 
# 850CGTTAG ATTGCCAGTT AGTGCAGACA GCGAAAGAAT 
# 900GCAACC GTTAGTGACG ATTTGCAAAA AATTGAACAA 
# 950CGTAGA TGTATTAGTG TGTAAAGCAC GAAACAAGCG 
# 1000ATCTTT TGCAAAAGCT CGGTGAAATG CAGATCGACA 
# 1050GGCGGT TCAAGTTTGA ATTTCAGTGC GTTAGAAAGC 
# 1100AGTACA TTGTTATATT GCGCCTAAAT TAGTCGGTGG 
# 1150CCCCAA TCGGCGGTGA GGGAATTCAA CAAATCGACC 
# 1200AAATTG AAATCGACCG AACTCATCGG CGAAGATATT 
# 1230 TCAT CTCCCCTCTT 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 410 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- Met Lys Leu Pro Cys Lys Arg Trp Phe Phe Le - #u Ser Phe Leu Gln Ala 
# 15 
- Leu Arg Ser Lys Asp Phe Lys Ala Phe Phe Il - #e Ile Arg Val Asn Met 
# 30 
- Pro Val Met Cys Phe Pro Leu Pro Ser Asn Se - #r Phe Lys Thr Met Thr 
# 45 
- Asp Leu Asp Tyr Met Arg Arg Ala Ile Ala Le - #u Ala Lys Gln Gly Leu 
# 60 
- Gly Trp Thr Asn Pro Asn Pro Leu Val Gly Cy - #s Val Ile Val Lys Asn 
# 80 
- Gly Glu Ile Val Ala Glu Gly Tyr His Glu Ly - #s Ile Gly Gly Trp His 
# 95 
- Ala Glu Arg Asn Ala Val Leu His Cys Lys Gl - #u Asp Leu Ser Gly Ala 
# 110 
- Thr Ala Tyr Val Thr Leu Glu Pro Cys Cys Hi - #s His Gly Arg Thr Pro 
# 125 
- Pro Cys Ser Asp Leu Leu Ile Glu Arg Gly Il - #e Lys Lys Val Phe Ile 
# 140 
- Gly Ser Ser Asp Pro Asn Pro Leu Val Ala Gl - #y Arg Gly Ala Asn Gln 
145 1 - #50 1 - #55 1 - 
#60 
- Leu Arg Gln Ala Gly Val Glu Val Val Glu Gl - #y Leu Leu Lys Glu Glu 
# 175 
- Cys Asp Ala Leu Asn Pro Ile Phe Phe His Ty - #r Ile Gln Thr Lys Arg 
# 190 
- Pro Tyr Val Leu Met Lys Tyr Ala Met Thr Al - #a Asp Gly Lys Ile Ala 
# 205 
- Thr Gly Ser Gly Glu Ser Lys Trp Ile Thr Gl - #y Glu Ser Ala Arg Ala 
# 220 
- Arg Val Gln Gln Thr Arg His Gln Tyr Ser Al - #a Ile Met Val Gly Val 
225 2 - #30 2 - #35 2 - 
#40 
- Asp Thr Val Leu Ala Asp Asn Pro Met Leu As - #n Ser Arg Met Pro Asn 
# 255 
- Ala Lys Gln Pro Val Arg Ile Val Cys Asp Se - #r Gln Leu Arg Thr Pro 
# 270 
- Leu Asp Cys Gln Leu Val Gln Thr Ala Lys Gl - #u Tyr Arg Thr Val Ile 
# 285 
- Ala Thr Val Ser Asp Asp Leu Gln Lys Ile Gl - #u Gln Phe Arg Pro Leu 
# 300 
- Gly Val Asp Val Leu Val Cys Lys Ala Arg As - #n Lys Arg Val Asp Leu 
305 3 - #10 3 - #15 3 - 
#20 
- Gln Asp Leu Leu Gln Lys Leu Gly Glu Met Gl - #n Ile Asp Ser Leu Leu 
# 335 
- Leu Glu Gly Gly Ser Ser Leu Asn Phe Ser Al - #a Leu Glu Ser Gly Ile 
# 350 
- Val Asn Arg Val His Cys Tyr Ile Ala Pro Ly - #s Leu Val Gly Gly Lys 
# 365 
- Gln Ala Lys Thr Pro Ile Gly Gly Glu Gly Il - #e Gln Gln Ile Asp Gln 
# 380 
- Ala Val Lys Leu Lys Leu Lys Ser Thr Glu Le - #u Ile Gly Glu Asp Ile 
385 3 - #90 3 - #95 4 - 
#00 
- Leu Leu Asp Tyr Val Val Ile Ser Pro Leu 
# 410 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH:459 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
# 50AGGTAA CTTAGTTGCG ACAGGTTTAA AATTCGGTAT 
# 100TCAACG ATTTTATCAA CGATAAATTA TTAAGCGGTG 
# 150GTGCGT CACGGTGCGT ATGAAAACGA TATTGATACG 
# 200TGCATT TGAGATTCCA TTAGTTGCGA AAAAAATGGC 
# 250ATGATG CGGTAATCTG TTTAGGTACG GTAATTCGCG 
# 300TATGAT TACGTATGTA ATGAAGCGGC AAAAGGTATC 
# 350AGAAAC CGGCGTACCG GTAATTTTCG GTGTATTAAC 
# 400AACAGG CGATTGAACG CGCGGGTACT AAAGCAGGTA 
# 450TGTGCA TTAGGCGCAA TCGAAATAGT AAACGTATTA 
# 459 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 153 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- Met Ala Lys Ile Thr Gly Asn Leu Val Ala Th - #r Gly Leu Lys Phe Gly 
# 15 
- Ile Val Thr Ala Arg Phe Asn Asp Phe Ile As - #n Asp Lys Leu Leu Ser 
# 30 
- Gly Ala Ile Asp Thr Leu Val Arg His Gly Al - #a Tyr Glu Asn Asp Ile 
# 45 
- Asp Thr Ala Trp Val Pro Gly Ala Phe Glu Il - #e Pro Leu Val Ala Lys 
# 60 
- Lys Met Ala Asn Ser Gly Lys Tyr Asp Ala Va - #l Ile Cys Leu Gly Thr 
# 80 
- Val Ile Arg Gly Ser Thr Thr His Tyr Asp Ty - #r Val Cys Asn Glu Ala 
# 95 
- Ala Lys Gly Ile Gly Ala Val Ala Leu Glu Th - #r Gly Val Pro Val Ile 
# 110 
- Phe Gly Val Leu Thr Thr Glu Asn Ile Glu Gl - #n Ala Ile Glu Arg Ala 
# 125 
- Gly Thr Lys Ala Gly Asn Lys Gly Ser Glu Cy - #s Ala Leu Gly Ala Ile 
# 140 
- Glu Ile Val Asn Val Leu Lys Ala Ile 
145 1 - #50 
__________________________________________________________________________