Yersinia pestis, the causative agent of plague, diverged from Yersinia pseudotuberculosis, an enteric pathogen, an estimated 1500–20,000 years ago. Genetic characterization of these closely related organisms represents a useful model to study the rapid emergence of bacterial pathogens that threaten mankind. To this end, we undertook genome-wide DNA microarray analysis of 22 strains of Y. pestis and 10 strains of Y. pseudotuberculosis of diverse origin. Eleven Y. pestis DNA loci were deemed absent or highly divergent in all strains of Y. pseudotuberculosis. Four were regions of phage origin, whereas the other seven included genes encoding a vitamin B12 receptor and the insect toxin sepC. Sixteen differences were identified between Y. pestis strains, with biovar Antiqua and Mediaevalis strains showing most divergence from the arrayed CO92 Orientalis strain. Fifty-eight Y. pestis regions were specific to a limited number of Y. pseudotuberculosis strains, including the high pathogenicity island, three putative autotransporters, and several possible insecticidal toxins and hemolysins. The O-antigen gene cluster and one of two possible flagellar operons had high levels of divergence between Y. pseudotuberculosis strains. This study reports chromosomal differences between species, biovars, serotypes, and strains of Y. pestis and Y. pseudotuberculosis that may relate to the evolution of these species in their respective niches.

Summary RovA is a transcriptional activator of

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode~1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalianpathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 6C and 37 6C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multinucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

We generated an ORF65 deletion mutant by using a cosmid system constructed from the genome of a low-passage clinical isolate (P-Oka). Using the SCID-hu mouse model, we demonstrated that the ORF65 protein is dispensable for viral replication in skin and T cells, which are critical host cell targets during primary varicella-zoster virus infection.Varicella-zoster virus (VZV) is a human herpesvirus that causes varicella (chickenpox) as the primary infection, establishes latency in sensory nerve ganglia, and may reactivate as herpes zoster (shingles) (1, 2). Open reading frame (ORF) 65 of VZV is one of four genes located in the short unique region of the genome and is homologous to Us9 in herpes simplex virus type 1 and the other alphaherpesviruses (3, 4, 7-9, 12, 16, 18). VZV ORF65 is predicted to encode an 11-kDa protein with 20% serine and threonine residues and a hydrophobic carboxyl terminus (6). Cohen et al. demonstrated that a partial deletion of ORF65 in a recombinant vaccine Oka strain was dispensable for viral replication in melanoma cells (5). The role of VZV ORF65 protein in vivo has not been reported.In previous work, Moffat et al. found that the vaccine Oka strain was attenuated in its growth in skin xenografts compared with the low-passage clinical isolate P-Oka (15). P-Oka was isolated from a varicella lesion and used to develop the attenuated vaccine Oka strain (17). To introduce mutations into a VZV genome from a wild-type virus, we made a cosmid system for VZV by using DNA derived from the P-Oka clinical isolate. This cosmid system was used to construct a complete deletion of ORF65. We evaluated the effects of the complete FIG. 1. Construction of P-Oka cosmid vectors with full deletion of VZV ORF65. Line 1 shows a schematic diagram of the P-Oka VZV genome and the location of ORF65 in the unique short region. Line 2 depicts the overlapping segments of the VZV genome used to construct the P-Oka VZV cosmids. Line 3 shows the AatII fragment which includes ORF65. Line 4 indicates the deleted region (open box), resulting in cosmid pvSpe23⌬65.

Insecticide resistance to the microbial insecticides Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) represents a serious threat to their success. Available evidence indicates that the risk for resistance to Bti is low due to the makeup of its parasporal crystal, which contains Cyt1A, Cry4A, Cry4B, and Cry11A toxic proteins. Disrupting the toxin complex in Bti enables resistance to evolve, especially in the absence of the key factor, the cytolytic toxin, Cyt1A. Cross-resistance is widespread among mosquitocidal Bacillus thuringiensis Cry toxins and the mechanisms of Cry resistance in mosquitoes are not known. Bacillus sphaericus (Bs) is at higher risk for resistance due to its singlesite action and field cases have been reported from a number of locations worldwide. Cross-resistance is reported among the various Bs isolates, although some isolates produce additional toxic proteins that can reduce cross-resistance and slow resistance evolution. Field and lab evolved resistant populations consistently show recessive and monofactorial inheritance of resistance. Resistant populations, however, have evolved a variety of molecular mechanisms causing that resistance. Traditional resistance management strategies with promise include rotations and mixtures of Bti and Bs, as well as untreated areas that provide natural refuges for susceptible alleles. Promising new strategies include genetic engineering to increase the toxin complexity targeted toward mosquito larvae, to enhance the host range of the mosquito control product, and to avoid the evolution of insecticide resistance. Regardless of the control strategy, a resistancemonitoring program alongside an integrative pest management approach is the best strategy to delay insecticide resistance.

The varicella-zoster virus (VZV) genome has unique long (U L ) and unique short (U S ) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in U S allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant Oka⌬62 (rOka⌬62) or rOka⌬71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.Varicella-zoster virus (VZV) belongs to the alphaherpesvirus subfamily of the Herpesviridae. VZV is the causative agent of varicella, which is characterized by cell-associated viremia and a cutaneous vesicular rash (4). VZV establishes latency in cells within sensory ganglia during primary infection. VZV reactivation from latency results in herpes zoster, a localized skin rash in the distribution of nerves from the affected ganglion. VZV is the first human herpesvirus for which a vaccine has been developed to prevent primary infection (58). This live attenuated varicella vaccine was created by serial passage of a wild-type clinical isolate, the parent Oka (pOka) strain, in guinea pig embryo cells and human fibroblasts to generate the varicella vaccine virus (vOka).The VZV genome consists of approximately 125 kb and has at least 70 unique open reading frames (ORFs) (52). As is characteristic of herpesviruses, the double-stranded DNA genome has unique long (U L ) and unique short (U S ) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. Three duplicated genes, ORF62/71, ORF63/ 70, and ORF64/69, are located in repeats at each end of the U S segment. The two VZV origins of replication, designated OriS, are also located in these repeat regions. The immediate-early 62 (IE62) protein,...

Varicella-zoster virus (VZV) is the only human herpes virus for which a vaccine has been licensed. A clinical VZV isolate, designated the parent Oka (pOka) strain was passed in human and non-human fibroblasts to produce vaccine Oka (vOka). The pOka and vOka viruses exhibit similar infectivity in cultured cells but healthy susceptible individuals given vaccines derived from vOka rarely develop the cutaneous vesicular lesions characteristic of varicella. Inoculation of skin xenografts in the SCIDhu mouse model of VZV pathogenesis demonstrated that vOka had a reduced capacity to replicate in differentiated human epidermal cells in vivo (Moffat, J.F., Zerboni, L., Kinchington, P.R., Grose, C., Kaneshima, H., Arvin A.M., 1998a. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol. 72:965-74). In order to investigate the attenuation of vOka in skin, we made chimeric pOka and vOka recombinant viruses from VZV cosmids. Six chimeric pOka/vOka viruses were generated using cosmid sets that incorporate linear overlapping fragments of VZV DNA from cells infected with pOka or vOka. The cosmid sets consist of pOka and vOka DNA segments that have identical restriction sites. As expected, the growth kinetics and plaque morphologies of the six chimeric pOka/vOka viruses were indistinguishable in vitro. However, the chimeric viruses exhibited varying capacities to replicate when evaluated in skin xenografts in vivo. The presence of ORFs 30-55 from the pOka genome was sufficient to maintain wild-type infectivity in skin. Chimeric viruses containing different vOka components retained the attenuation phenotype, suggesting that vOka attenuation is multi-factorial and can be produced by genes from different regions of the vOka genome.

DNA microarrays represent a powerful technology that enables whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. Such studies have dramatically increased our ability to differentiate between bacteria, highlighting previously undetected genetic differences and population structures and providing new insight into virulence and evolution of bacterial pathogens. Recent results from such studies have generated insights into the evolution of bacterial pathogens, the levels of diversity and plasticity in the genome of a species, as well as the differences in virulence amongst pathogenic bacteria.