S Makino

A Genetic Determinant Required for Continuous Reinfection of Adjacent Cells on Large Plasmid in S. flexneri 2a

We have identified a region (virG) on the 230 kb virulence plasmid of S. flexneri that is required for cell-to-cell spread of the bacterium. Tn5 insertions into this region result in avirulent mutants that can initially invade and multiply in epithelial cells, but tend to lose active movement and tend to localize within the cytoplasm, where they are gradually extinguished without infecting adjacent cells. The virG region was localized to within 4 kb and may contain a single cistron. Sequences hybridizing to this region were found in all intact virulence plasmids of Shigellae and enteroinvasive E. coli.

A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence‐associated antigens of Shigella flexneri

The expression of plasmid‐encoded, invasion‐related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence‐associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230kb plasmid‐encoded virulence genes.

Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis

Bacillus anthracis produces a gamma‐linked poly‐D‐glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low‐molecular‐weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region atone caused production of a high‐molecular‐weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51460. By in vitro transcription‐translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian γ‐glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly‐D‐glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low‐molecular‐weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.

CROSS-TALK TO THE GENES FOR BACILLUS ANTHRACIS CAPSULE SYNTHESIS BY ATXA, THE GENE ENCODING THE TRANS-ACTIVATOR OF ANTHRAX TOXIN SYNTHESIS

The two major virulence factors of Bacillus anthracis are the tripartite toxin and the polyglutamate capsule, which are encoded by genes on the large plasmids, pX01 and pX02, respectively. The genes atxA, located on pX01, and acpA, located on pX02, encode positive frans‐acting proteins that are involved in bicarbonate‐mediated regulation of toxin and capsule production, respectively. A derivative strain cured of pX01 produced less capsular substance than the parent strain harbouring both pX01 and pX02, and electroporation of the strain cured of pX01 with a plasmid containing the cloned atxA gene resulted in an increased level of capsule production. An acpA‐null mutant was complemented by not only acpA but also the atxA gene. The cap region, which is essential for encapsulation, contains three genes capB, capC, and cap A, arranged in that order. The atxA gene stimulated capsule synthesis from the cloned cap region. Transcriptional analysis of cap by RNA slot‐blot hybridization and primer‐extension analysis revealed that atxA activated expression of cap in trans at the transcriptional level. These results indicate that cross‐talk occurs, in which the pX01‐located gene, atxA, activates transcription of the cap region genes located on pX02. We identified two major apparent transcriptional start sites, designated P1 and P2, located at positions 731 bp and 625 bp, respectively, upstream of the translation‐initiation codon of capB. Transcription initiated from P1 and P2 was activated by both atxA and acpA, and activation appeared to be stimulated by bicarbonate. Deletion analysis of the upstream region of the cap

Cloning and CO2‐dependent expression of the genetic region for encapsulation from Bacillus anthracis

The capsule of Bacillus anthracis is an important virulence factor consisting of poly‐o‐glutamic acid. The genetic region required for the encapsulation was cloned in Escherichia coli from the capsule plasmid pTE702, using a selection procedure based on an immunodlffusion assay. The cloned region directed synthesis of the capsule both In E. coli and B. anthracis. Capsule synthesis from these clones, as in the wild type, was dependent upon the presence of CO2. However, encapsulation directed by the cloned fragment was less marked than from pTE702. Another region enhancing capsulation was shown to exist on pTE702. The minimum size of the encapsulation region was defined to within 2.7 kb DNA and shown to be essential for the encapsulation in B. anthracis.

Genetic relatedness of the basic replicon of the virulence plasmid in shigellae and enteroinvasive Escherichia coli.

The regions required for replication (Rep) and for stable maintenance (Stb) of the 230 kilobase virulence plasmid, pMYSH6000, in Shigella flexneri 2a YSH6000 were identified on the restriction enzyme Sa/l fragments, C and O, respectively. The stable plasmid, pMYSH6610, consisting of adjacent fragments C and O was found to belong to the incompatibility group Fl. In all strains of shigellae and enteroinvasive Escherichia coli (EIEC) tested, virulence plasmids were found to be highly homologous with the Rep region of pMYSH6000. The restriction map of this probe was very similar to that of the inc region of RepFIIA replicon. The virulence plasmid of S. flexneri 6, S. boydii, S. dysenteriae and EIEC all coexisted stably with pMYSH6610, whereas that of S. flexneri other than serotype 6 and S. sonnei did not. These findings indicate that all the virulence plasmids of shigellae and EIEC are highly related but can be divided into at least 2 groups.

Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri

In Shigella flexneri, in addition to several well‐recognized plasmid‐borne virulence loci, at least three genetic loci implicated in pathogenesis have been recognized on the chromosome. To understand more about the pathogenesis of bacillary dysentery at a molecular level, the genetically recognized but previously unidentified KcpA region (one of the chromosomal regions near purE) was cloned and sequenced. A single translatable open reading frame encoding a 12310 Dalton protein corresponding to the minicell product was found. Immunofluorescence microscopy, as well as optical and electron microscopic comparison of tissue‐cultured cells and guinea‐pigs’eyes infected with wild‐type or kcpA mutant bacteria, revealed that the kcpA product is required by invading bacteria for spread into adjacent cells.

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.

Analysis of the plasmids of Escherichia coli O148:H28 from travellers with diarrhea☆

98 Escherichia coli strains of serotype O148:H28 isolated from diarrheal patients from 10 Asian countries and Mexico at Osaka Airport Quarantine were analyzed for enterotoxigenicity and plasmid profile. They were classified into three groups. The first group contained 44 strains that were non-enterotoxigenic and carried 3.9 kb and 50 kb non-enterotoxin plasmids. The second group contained 9 strains that produced LT and ST. They carried a 45 kb enterotoxin plasmid, and 4.6 kb and 9.2 kb non-enterotoxin plasmids. The third group contained 45 strains that produced ST. They carried a 40 kb enterotoxin plasmid, and non-enterotoxin plasmids other than 3.9 kb, 4.6 kb, 9.2 kb and 50 kb. Southern blot hybridization demonstrated that all the non-enterotoxin or enterotoxin plasmids carried by the strains of the same group were identical or similar. These results suggested that the 98 E. coli strains with O148:H28 serotype were derived from three clones, and that the individual strains among each group were derived from a single clonal strain.

Loss of virulence in Shigella strains preserved in culture collections due to molecular alteration of the invasion plasmid

Fifty-two Shigella strains long preserved by three Japanese culture collections were examined for virulence. All of them were avirulent when judged by the focus-plaque assay and the ability to bind Congo red. Fifteen strains had a plasmid comparable in size to that responsible for epithelial invasiveness and were positive in hybridization tests with a probe derived from a plasmid cistron, virG. Twenty-four strains had a similar plasmid but were negative in hybridization tests. The remaining 13 strains were negative in all the five criteria for virulence. Similar studies made on one hundred Shigella strains isolated from 1967 to 1985 clearly demonstrated loss of virulence with prolonged time of storage.