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 etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.
Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.
The authors studied the distribution of alanine aminotransferase (ALT) levels in 10,034 volunteer blood donors. The mean +/- SD ALT value was 21.4 +/- 19.3 IU/liter; 549 (5.5%) of the donors had a ALT value greater that 45 IU; 2.5 per cent had ALT values greater than 60 IU. In general, ALT levels were higher in males than in females, and were age related; peak values occurred in the third decade of life for males and between 50-60 years of age in females. ALT values greater than 45 IU were found significantly more often in males, in donors of both sexes 30-40 years of age, in married donors, in non-Caucasians, and in those whose education level was no higher than high school. Follow-up samples in donors with an initial ALT greater than 45 IU, revealed that 67% continued to have ALT values above 45 IU 2-8 weeks following initial sampling, and 40% had an ALT greater than 45 IU when tested again six months after entry into the study. ALT values greater than 60 IU were associated with a significantly increased prevalence of antibody of hepatitis B surface antigen (anti-HBs) and antibody to hepatitis B core antigen (anti-HBc) occurring together. No statistically significant association was found between transaminase activity and the prevalence of anti-HBs or anti-HBc alone, or with hepatitis A antibody. These findings demonstrate that there are defined sociodemographic and serologic features of donors with elevated ALT values.
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