Alistair J. Lax

A Salmonella dublin virulence plasmid locus that affects bacterial growth under nutrient-limited conditions

This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal‐medium containing various sole carbon/energy sources, unlike the wild‐type and plasmid‐cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the pheno‐typic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal‐medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.

Identification of an essential virulence region on Salmonella plasmids

An 8 kilobase pair (kbp) fragment from the Salmonella dublin 2229 plasmid is sufficient to restore virulence for mice to cured strains of both S. dublin and S. typhimurium but only when cloned on a low copy vector. Two regions previously shown to be associated with virulence lie outside the fragment and complementation results suggest that one of these mutated regions affects expression from the 8 kbp fragment. It therefore appears that there are control elements both within and adjacent to the essential virulence fragment.

Cloning of the toxin gene from Pasteurella multocida and its role in atrophic rhinitis.

The gene for the osteolytic toxin of Pasteurella multocida has been cloned into a plasmid vector and expressed off its own promoter in Escherichia coli. Particular restriction endonucleases failed to cut the gene and regions flanking it, suggesting an A + T base ratio significantly greater than the remaining genome of P. multocida. Cloned toxin was indistinguishable from the native toxin with respect to molecular mass, antigenicity and toxicity in different tests. A single intraperitoneal injection of toxin purified from the recombinant E. coli reproduced in gnotobiotic pigs the pathological changes characteristic of atrophic rhinitis. The recombinant E. coli produced at least 10 times as much toxin as P. multocida.

Sequence analysis of the potent mitogenic toxin of Pasteurella multocida.

Pasteurella multocida toxin is a potent mitogen for cultured Swiss 3T3 cells where it causes an accumulation of inositol phosphates and activation of protein kinase C. The gene sequence described here coded for a 146 kDa protein. The ORF was preceded by a ribosome binding site and followed by a stem loop. There was no evidence for a signal sequence. The gene had a low G + C base ratio which differs from the rest of the Pasteurella genome. There was no significant homology with other known proteins, although a motif found in certain bacterial toxins which are ADP‐ribosyl transferases is present. A recombinant expressing only part of the PMT gene was not mitogenic.

The virulence plasmid of Salmonella dublin: detailed restriction map and analysis by transposon mutagenesis.

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.

Can scrapie titres be calculated accurately from incubation periods

Since endpoint titrations of scrapie material are costly and time-consuming, several workers have estimated titre from the correlation between the incubation period of the disease and the infectivity titre. However, we show here that the relationship between incubation period and titre cannot be assumed to be constant for all scrapie preparations. Our results indicate that sodium deoxycholate treatment of scrapie preparations does not reduce the titre, but can lengthen the incubation period by about 10 days. This is equivalent to a discrepancy of 1 log LD50 unit if the estimation of titre was based on the incubation period.

Plasmids related to RSF1010 from Bordetella bronchiseptica

Six out of 14 Bordetella bronchiseptica isolates from U.K. pigs each contained one plasmid, of 8.7-44 kb. All plasmid-containing isolates were sulfonamide resistant, and this property was shown to be plasmid-encoded. Five of the plasmids were related; two were indistinguishable from the broad-host-range plasmid, RSF1010. The other three, two of which appeared to be identical, were shown to have regions of homology with RSF1010. One of these regions encompassed the sulfonamide resistance determinant while the other contained oriV, which also determines plasmid incompatibility. None of the plasmids could be associated with virulence or phase variation, and it appears likely that they have been acquired in response to antibiotic pressure.

Identification of proteins expressed by the essential virulence region of the Salmonella dublin plasmid

An 8 kilobase pair (kb) fragment from the Salmonella dublin 2229 plasmid is sufficient to restore virulence for mice to a cured strain of S. dublin. Deletion analysis of this virulence fragment identified at least one specific region required for virulence expression. Plasmid-directed protein synthesis in minicells has indicated the presence of at least four genes within the essential virulence region of the S. dublin plasmid, encoding proteins of 70, 33, 30 and 26 kDa. Analysis of the proteins expressed by the deletion derivatives suggested that expression of the 33 kDa polypeptide was linked to that of the 30 kDa polypeptide. The proteins expressed by the essential virulence region of the S. dublin plasmid appeared to be similar to the plasmid-encoded virulence proteins recently identified in S. typhimurium.

Plasmid Genes Involved in Virulence in Salmonella

Salmonella virulence plasmids were first recognised in 1970 (Dowman and Meynell, 1970), but it was 10 years before a role in virulence was identified (Jones et al., 1982). A series of papers followed which indicated that several serotypes contained such plasmids and that curing reduced virulence in mice or chickens, which could be restored by reintroduction of the plasmid (Table 1: Chikami et al., 1985; Gulig and Curtiss, 1987; Barrow et al., 1987; Barrow and Lovell; 1988, Kawahara et al. 1988; Hovi et al., 1988). Originally referred to as “cryptic” or serotype-specific, they are now generally and more accurately referred to as virulence plasmids. Although virulence plasmids are generally serotype-specific, there are exceptions. The closely related S. rostock and S. dublin carry an identical plasmid (Platt, D.J., personal communication), and different virulence plasmids are found within both S. enteritidis and S. pullorum (Williamson et al., 1988a). The virulence plasmids range in size from about 54kb to 98kb, and therefore represent up to 2% of the genetic information of the Salmonella genome. So far only 12 serotypes have been found to contain virulence plasmids (Table 1), and within a serotype there are phage-type differences in plasmid carriage (Brown et al., 1986; Woodward et al., 1989). The relative importance of these plasmids for virulence in mice varies in different serotypes (Williamson et al. 1988a). Discussion of the role of the plasmid genes in virulence must also take into account that serotypes without virulence plasmids are not avirulent.

Polyadenylated RNA and scrapie in mice

Abstract It has been reported that infection of mice with scrapie results in changes in the polyadenylated RNA metabolism of the host. We have attempted to repeat some aspects of this work, using the same methods, but have failed to confirm the earlier fundamental conclusions.