Sandy mice have a deletion mutation in the gene encoding dysbindin-1, Dtnbp1, with consequent reduction of the protein in heterozygotes and its loss in homozygotes. The sandy mouse thus serves as an animal model of dysbindin-1 function. Since this protein is concentrated in synaptic tissue and affects transmitter release, it may affect neuronal processes that mediate behavior. To investigate the neurobehavioral effects of the Dtnbp1 mutation, we studied littermate sandy and wild-type controls on a C57BL/6J genetic background. The three animal groups were indistinguishable in their external physical characteristics, sensorimotor skills, and indices of anxiety-like behaviors. In the open field, however, homozygous animals were hyperactive and appeared to show less habituation to the initially novel environment. In the Morris water maze, homozygous animals displayed clear deficits in spatial learning and memory with marginal deficits in visual association learning. Apart from the last mention deficits, these abnormalities are consistent with hippocampal dysfunction and in some cases with elevated dopaminergic transmission via D2 dopamine receptors. Since similar deficits in spatial learning and memory have been found in schizophrenia, where decreased dysbindin-1 has been found in the hippocampus, the sandy mouse may also model certain aspects of cognition and behavior relevant to schizophrenia.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP UV ) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP UV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP UV fluorescence.Rickettsia prowazekii is the etiologic agent of epidemic typhus, a historically significant human disease that continues to threaten populations subjected to nonhygienic, vector-laden conditions. Tragically, due to the continuing human legacy of war and extreme poverty, such conditions are not rare and recent outbreaks of this disease have been reported (13). In addition, R. prowazekii is a category B select agent and thus is classified as a potential agent of bioterrorism.R. prowazekii is an obligate intracellular bacterium that grows directly within the cytoplasm of eukaryotic host cells, unbounded by a vacuolar membrane. The pathogenicity of R. prowazekii is dependent on its ability to grow to high numbers within the rich cytoplasmic environment, leading to lysis of the host cell and the release of rickettsiae that can then invade and destroy additional host cells. It accomplishes this feat, in organisms as diverse as humans and arthropods, with a small genome of approximately 10 6 base pairs predicted to encode 835 proteins (1,18).Defining the importance of specific rickettsial genes in intracellular invasion and growth has been hampered by the lack of genetic tools. However, progress in developing such tools has been made. Rickettsial genome manipulation has been performed using both homologous recombination mechanisms and transposon-based systems (3, 10-12, 14, 17). Recently, Felsheim et al. (7) described the use of a Himar1-based system to generate transposon insertions in the genome of another obligate intracellular pathogen, Anaplasma phagocytophilum. In this study, we report the use of the mari...

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL P ), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL P -Rparr-2 was combined with transposase, and the resulting DNAprotein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows directly within the cytoplasm of its eukaryotic host cell, unbounded by a vacuolar membrane. R. prowazekii is exquisitely adapted to this cytoplasmic environment, as evidenced by its expression of specialized systems, such as those for the transport of high-energy compounds, that exploit this metabolically rich environment (24, 25). However, rickettsial obligate dependence on the intracytoplasmic milieu does not prevent this versatile pathogen from infecting organisms as diverse as humans, flying squirrels, and the arthropod vector of epidemic typhus, the human body louse. Rickettsial pathogenicity, in both the arthropod vector and the human host, is due to intracellular growth of the rickettsiae followed by the lysis of the host cell and the infection of additional cells. Thus, an understanding of rickettsial growth and metabolism within the eukaryotic cytoplasm is essential to understanding the basis of rickettsial pathogenicity.Previous studies on the physiology of rickettsial intracellular growth are now complemented by the genome sequences of several rickettsial species, including that of R. prowazekii (2, 19). The R. prowazekii genome contains 834 open reading frames (ORFs), a number of pseudogenes, and a high proportion of noncoding regions (2). While some of the 834 gene products can be annotated confidently by their extensive identity to proteins of known fu...

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K T of 2.3 M. Transport was inhibited by S-adenosylethionine and Sadenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.