Nobuyuki Terakado

Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid.

Virulent typical strains (Shikan, Morioka, Shizuoka) and Pasteur vaccine strains (no. 1, no. 2-H, no. 2-17JB) of Bacillus anthracis harboured two plasmid species with molecular masses of 110 MDal and 60 MDal. All of the 110 MDal plasmids isolated from the various strains showed indistinguishable patterns of digestion with restriction endonucleases. All the 60 MDal plasmids were also indistinguishable. Strain Davis, which is encapsulated but is asporogenous and avirulent, harboured only the 60 MDal plasmid while three non-encapsulated vaccine strains (34F2, Smith, Mukteswer) harboured only the 110 MDal plasmid. Four non-encapsulated variant strains obtained from the encapsulated strains Shikan, Pasteur no. 1, Pasteur no. 2-17JB and Davis had lost the 60 MDal plasmid, suggesting that encapsulation of B. anthracis may be associated with the 60 MDal plasmid.

Virulence and immunogenicity in experimental animals of Bacillus anthracis strains harbouring or lacking 110 MDa and 60 MDa plasmids

A comparative study was made of the virulence and immunogenicity in mice or guinea pigs of Bacillus anthracis strains harbouring 110 MDa and/or 60 MDa plasmids. Strains cured of the 110 MDa or the 60 MDa plasmid were more than 100-fold less virulent to mice than were the parental strains harbouring these plasmids. Guinea-pigs immunized with plasmid-free derivatives of the non-encapsulated vaccine strain 34F2 showed no resistance to challenge with strain 17JB, which harbours both 110 MDa and 60 MDa plasmids, suggesting that the derivative strains had lost their immunizing ability against anthrax.

R plasmid with carbadox resistance from Escherichia coli of porcine origin.

Escherichia coli isolates of porcine fecal origin from a farm where the antibacterial agent carbadox was used were examined for resistance to carbadox (Cdxr). Of 72 strains examined, 24 showed resistance to this drug. All 24 Cdxr strains, except one, were also resistant to tetracycline (Tcr), streptomycin (Smr), spectinomycin (Spcr), sulfadimethoxine (Sur), kanamycin (Kmr), ampicillin (Apcr), or a combination of tetracycline, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin. The Cdxr character was invariably transmissible by conjugation to E. coli K-12 jointly with other drug resistance, with the resistance patterns present in transconjugants being Cdxr Smr Spcr Apcr or Cdxr Smr Spcr Sur Apcr. About 25% of these transconjugants simultaneously lost the resistance to carbadox, streptomycin, spectinomycin, and ampicillin or carbadox, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin or carbadox, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin in the presence of acriflavine. Agarose gel electrophoretic analysis of deoxyribonucleic acid from a transconjugant showed a single plasmid deoxyribonucleic acid molecule with a molecular weight of about 28 x 10(6), which was capable of transforming E. coli C to Cdxr Smr Spcr Apcr. This resistance was transmissible by conjugation as a unit to other K-12 strains. These results confirmed the presence of an R plasmid specifying the Cdxr character in the host strain. Images

Positive regulator for the expression of Mba protein of the virulence plasmid, pKDSC50, of Salmonella choleraesuis

A positive regulator was identified within a 2.3 kb fragment of the 6.4 kb mouse bacteremia region (mba region) of the virulence pKDSC50 plasmid of Salmonella choleraesuis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that Escherichia coli K-12 carrying the recombinant plasmids of the 2.3 kb fragment produced Mba1 protein with a molecular mass of 32 kDa. The recombinant plasmids carrying a 4.1 kb fragment, the other part of 6.4 kb region, produced Mba2 (32 kDa), Mba3 (70 kDa) and Mba4 (29 kDa) proteins. All three proteins were expressed by using the lacZ promoter under isopropyl thiogalactoside induction. In contrast to this, Mba3 protein was overexpressed independently of the lacZ promoter when the 2.3 kb fragment coexisted either in cis or trans. These results suggest that Mba1 is a trans-acting positive regulator for the expression of the Mba3 protein of mba region of pKDSC50.

Identification and mapping of mba regions of the Salmonella choleraesuis virulence plasmid pKDSC50 responsible for mouse bacteremia.

A restriction cleavage map was constructed for pKDSC50, the 50 kb virulence plasmid of Salmonella choleraesuis. Using Tn1 transposon-insertion mutagenesis, we obtained 19 mutant strains of S. choleraesuis with a diminished ability to cause bacteremia in the mouse. pKDSC50::Tn1 DNA of the mutant strains was extracted and introduced into a pKDSC50-cured S. choleraesuis strain. Nine of the 19 transformants showed the diminished ability to cause mouse bacteremia. Tn1-insertion of the nine mutant strains was clustered within a 6.2 kb segment of pKDSC50, demonstrating that this region was necessary for conferring ability to cause mouse bacteremia (Mba) on the host organism. Two regions, mba-1 and mba-2, were identified within the 6.2 kb fragment. In E. coli minicells, the cloned fragments containing the mba region expressed at least three proteins with apparent molecular weights of 29,000, 32,000 and 32,000.

Antibiotic resistance of Erysipelothrix rhusiopathiae isolated from pigs with chronic swine erysipelas.

The susceptibility of 258 isolates of Erysipelothrix rhusiopathiae from slaughtered pigs affected with chronic erysipelas in Japan to antimicrobial agents was determined. A total of 111 (43.0%) strains showed resistance to erythromycin, oleandomycin, oxytetracycline, or dihydrostreptomycin. Plasmids were not detected. This is the first report of resistance of E. rhusiopathiae to these antibiotics.

Plasmid-mediated Serum Resistance and Alterations in the Composition of Lipopolysaccharides in Salmonella dublin

Survival rates of Salmonella dublin in rabbit serum after culture for 1 h at 37 degrees C were compared between a wild-type strain (5240) carrying a 50 MDa plasmid, a plasmid-cured strain (C524), and a cured strain containing the 50 MDa plasmid tagged with Tn1 (5241). Strain C524 was more susceptible to the bactericidal activity of normal serum than its parent strain 5240 (percentage survival less than 1% and 52.5 +/- 9.2%, respectively). On the other hand, the percentage survival of strain 5241 was significantly increased (90.4 +/- 4.0%), indicating that the reintroduction of the plasmid into the cured strain restored the serum resistance. Moreover, this change in the serum resistance properties correlated with changes in the neutral sugar composition of the lipopolysaccharides (LPS) of these strains, suggesting that the 50 MDa plasmid is necessary for O-side chain expression in the LPS of S. dublin.

Molecular cloning of an Actinobacillus pleuropneumoniae outer membrane lipoprotein (Oml A) from serotype 5a

The gene encoding an outer membrane lipoprotein (OmIA) was cloned from Actinobacillus pleuropneumoniae strain NG-8 (serotype 5a). The deduced amino acid sequence of OmIA from strain NG-8 showed 61% identity to the OmIA from serotype 1 strain, which confers protective immunity to pigs. Southern blot analysis showed the presence of a sequence highly homologous to the omIA gene of strain NG-8 in strains of serotype 5a, 5b and 10. A specific serum against OmIA of NG-8 also detected a homologous protein in the strains of these serotypes. These data shows the presence of antigenic variability among A. pleuropneumoniae OmIA proteins.

Properties of R Factors from Bordetella bronchiseptica

Genetic properties and host ranges of R factors derived from Bordetella bronchiseptica of pig origin were examined. All of 61 R factors tested could confer resistance to streptomycin, sulfonamide, and aminobenzyl penicillin on their host bacteria. All of them were identified as fi− (no fertility inhibition) type and were found to exhibit no restriction of phages λ, φ80, P1, P2, T1, T3, T6, T7, W31, and BF-23. They could confer macarbomysin susceptibility on their host cells when infected. An Rte16, a representative R factor, was incompatible with both RP4 and R40a, which are classified as compatibility groups P and C, respectively. An Rte16 was conjugally transmissible to B. bronchiseptica, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, and Yersinia enterocolitica, but not to Shigella flexneri, S. sonnei, Proteus mirabilis, P. vulgaris, P. rettgeri, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

Restriction map of a capsule plasmid of Bacillus anthracis

The capsule plasmid pTE702 of Bacillus anthracis has been physically mapped with the restriction endonucleases HindIII, PstI, BamHI, SalI, and XhoI. A HindIII fragment map of pTE702 (96.5 kb) was obtained by analysis of the recombinant plasmids and cosmids containing overlapping fragments partially digested with HindIII. The physical map for PstI, BamHI, SalI, and XhoI was obtained by double digestion mapping of these sites in relation to the HindIII sites. The replication region of pTE702 was determined by in vitro genetic replicon labeling in B. subtilis.