Identification of multiple integration sites for Stx-phage Phi24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressor.

The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to lambda phage. However, contrary to the lambda superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Phi24(B). Here, the Phi24(B) integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Phi24(B) integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Phi24(B) gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Phi24(B) E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Phi24(B) to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle.

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.