The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague

Identification of Retinal Transformation Hot Spots in Developing Drosophila Epithelia

Background: The retinal determination (RD) network is an evolutionarily conserved regulatory circuit that governs early events in the development of eyes throughout the animal kingdom. Ectopic expression of many members of this network leads to the transformation of non-retinal epithelia into eye tissue. An often-overlooked observation is that only particular cell-populations within a handful of tissues are capable of having their primary developmental instructions superseded and overruled. Methodology/Preliminary Findings: Here we confirm that indeed, only a discrete number of cell populations within the imaginal discs that give rise to the head, antenna, legs, wings and halteres have the cellular plasticity to have their developmental fates altered. In contrast to previous reports, we find that all transformable cell populations do not lie within the TGFb or Hedgehog signaling domains. Additionally neither signaling cascade alone is sufficient for non-retinal cell types to be converted into retinal tissue. The transformation ‘‘hot spots’ ’ that we have identified appear to coincide with several previously defined transdetermination ‘‘weak spots’’, suggesting that ectopic eye formation is less the result of one network overriding the orders of another, as previously thought, but rather is the physical manifestation of redirecting cell populations of enormous cellular plasticity. We also demonstrate that the initiation of eye formation in non-retinal tissues occurs asynchronously compared to that of the normal eye suggesting that retinal development is not under the control o

Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence

Diversity of 23S rRNA Genes within Individual Prokaryotic Genomes

The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes.These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy

On the typology and the worship status of sacred trees with a special reference to the Middle East

This article contains the reasons for the establishment of sacred trees in Israel based on a field study. It includes 97 interviews with Muslim and Druze informants. While Muslims (Arabs and Bedouins) consider sacred trees especially as an abode of righteous figures' (Wellis') souls or as having a connection to their graves, the Druze relate sacred trees especially to the events or deeds in the lives of prophets and religious leaders. A literary review shows the existence of 24 known reasons for the establishment of sacred trees worldwide, 11 of which are known in Israel one of these is reported here for the first time. We found different trends in monotheistic and polytheistic religions concerning their current worship of sacred trees

Septic arthritis of the knee:the use and effect of antibiotics prior to diagnostic aspiration

Septic arthritis of the native knee joint and total knee arthroplasty both cause diagnostic and treatment issues. There is no gold standard test to diagnose a joint infection and the use of joint aspiration is commonly relied on. It is widely accepted by orthopaedic surgeons that antibiotics should be withheld until aspiration has been performed to increase the odds of identifying an organism. Patients often present to other specialties that may not be as familiar with these principles. Our study found that 25 (51%) of the 49 patients treated for septic arthritis of the native or prosthetic knee in our unit over a 3-year period had received antibiotics prior to discussion or review by the on-call orthopaedic service. Patients were significantly less likely to demonstrate an organism on initial microscopy (entire cohort: p=0.001, native knees: p=0.006, prosthetic knees: p=0.033) or on subsequent culture (entire cohort: p=0.001, native knees: p=0.017, prosthetic knees: p=0.012) of their aspirate if they had received antibiotics. The sensitivity of microscopy in all patients dropped from 58% to 12% when patients had received antibiotics (native knees: 46% to 0%, prosthetic knees: 72% to 27%). The sensitivity of the culture dropped from 79% to 28% in all patients when the patient had received antibiotics (native knees: 69% to 21%, prosthetic knees: 91% to 36%). This study demonstrated how the management of patients with suspected cases of septic arthritis of the knee may be compromised by empirical administration of antibiotics. These patients were significantly less likely to demonstrate an organism on microscopy and culture of their initial aspirate. There is a significant high false negative rate associated with knee aspiration with prior administration of antibiotic therapy

Sequencing and functional annotation of avian pathogenic Escherichia coli serogroup O78 strains reveal the evolution of E. coli lineages pathogenic for poultry via distinct mechanisms.

Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes