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Scaphoid dislocation represents a rare injury with only a few case reports and limited case series reported in the literature. The majority of scaphoid dislocations result from a high-energy trauma causing hyperextension and ulnar deviation of the wrist. The severity of a scaphoid dislocation depends on the degree of periscaphoid ligamentous injury as well as the presence of concomitant injuries, such as axial carpal dissociation. The most common complication after a scaphoid dislocation is scapholunate dissociation, which emphasizes the importance of scapholunate ligament repair/reconstruction in these cases. We report a case of an open scaphoid dislocation with the associated injuries of a hamate fracture and fourth metacarpal fracture treated with an open reduction of the scaphoid, open ligamentous repair and augmentation of the involved carpal ligaments, and open reduction internal fixation of both the hamate and the fourth metacarpal fractures.

Objectives: We sought to compare the prevalence of serious bacterial infections (SBI; urinary tract infection, bacteremia, and/or meningitis) and invasive bacterial infections (IBI; bacteremia and/or meningitis) among infants with hypothermia with positive vs negative respiratory pathogen testing. Study design: We conducted a multicenter retrospective cohort study of infants ≤90 days presenting to an emergency department or directly admitted to a hospital from September 1, 2016, to May 5, 2021, with reported or documented hypothermia (≤36°C). Positive respiratory pathogen testing included positive single or multiplex nucleic acid amplification testing. The primary outcome was prevalence of SBI, defined as positive blood, urine, and/or cerebrospinal fluid culture and antibiotic treatment course; the prevalence of SBI and IBI was compared between infants with positive vs negative respiratory pathogen tests using the χ2 test. Results: Respiratory pathogen testing was obtained in 40.6% (446/1098) of infants with hypothermia; of those, 24.9% (111/446) had a positive respiratory pathogen result. Infants with a positive respiratory pathogen result were more often older, ill-appearing at presentation, and evaluated during fall/winter months. The prevalence of SBI in our cohort was 7.4%, and the prevalence of IBI was 4.5%. There were no associations between respiratory pathogen test result and SBI or IBI. Conclusions: Most young infants with hypothermia evaluated did not have respiratory pathogen testing performed. The diagnosis of SBI or IBI was not associated with a positive respiratory pathogen test. Further research is needed to understand the utility of respiratory pathogen testing in risk stratification for infants with hypothermia.

Abstract Background Medicare hospital Value-based purchasing (VBP) program that links Medicare payments to quality of care will become effective from 2013. It is unclear whether specific hospital characteristics are associated with a hospital’s VBP score, and consequently incentive payments. The objective of the study was to assess the association of hospital characteristics with (i) the mean VBP score, and (ii) specific percentiles of the VBP score distribution. The secondary objective was to quantify the associations of hospital characteristics with the VBP score components: clinical process of care (CPC) score and patient satisfaction score. Methods Observational analysis that used data from three sources: Medicare Hospital Compare Database, American Hospital Association 2010 Annual Survey and Medicare Impact File. The final study sample included 2,491 U.S. acute care hospitals eligible for the VBP program. The association of hospital characteristics with the mean VBP score and specific VBP score percentiles were assessed by ordinary least square (OLS) regression and quantile regression (QR), respectively. Results VBP score had substantial variations, with mean score of 30 and 60 in the first and fourth quartiles of the VBP score distribution. For-profit status (vs. non-profit), smaller bed size (vs. 100–199 beds), East South Central region (vs. New England region) and the report of specific CPC measures (discharge instructions, timely provision of antibiotics and beta blockers, and serum glucose controls in cardiac surgery patients) were positively associated with mean VBP scores (p Conclusions Although hospitals serving the poor and the elderly are more likely to score lower under the VBP program, the correlation appears small. Profit status, geographic regions, number and type of CPC measures reported explain the most variation among scores.

Abstract Background Current practice is to perform a completion axillary lymph node dissection (ALND) for breast cancer patients with tumor-involved sentinel lymph nodes (SLNs), although fewer than half will have non-sentinel node (NSLN) metastasis. Our goal was to develop new models to quantify the risk of NSLN metastasis in SLN-positive patients and to compare predictive capabilities to another widely used model. Methods We constructed three models to predict NSLN status: recursive partitioning with receiver operating characteristic curves (RP-ROC), boosted Classification and Regression Trees (CART), and multivariate logistic regression (MLR) informed by CART. Data were compiled from a multicenter Northern California and Oregon database of 784 patients who prospectively underwent SLN biopsy and completion ALND. We compared the predictive abilities of our best model and the Memorial Sloan-Kettering Breast Cancer Nomogram (Nomogram) in our dataset and an independent dataset from Northwestern University. Results 285 patients had positive SLNs, of which 213 had known angiolymphatic invasion status and 171 had complete pathologic data including hormone receptor status. 264 (93%) patients had limited SLN disease (micrometastasis, 70%, or isolated tumor cells, 23%). 101 (35%) of all SLN-positive patients had tumor-involved NSLNs. Three variables (tumor size, angiolymphatic invasion, and SLN metastasis size) predicted risk in all our models. RP-ROC and boosted CART stratified patients into four risk levels. MLR informed by CART was most accurate. Using two composite predictors calculated from three variables, MLR informed by CART was more accurate than the Nomogram computed using eight predictors. In our dataset, area under ROC curve (AUC) was 0.83/0.85 for MLR (n = 213/n = 171) and 0.77 for Nomogram (n = 171). When applied to an independent dataset (n = 77), AUC was 0.74 for our model and 0.62 for Nomogram. The composite predictors in our model were the product of angiolymphatic invasion and size of SLN metastasis, and the product of tumor size and square of SLN metastasis size. Conclusion We present a new model developed from a community-based SLN database that uses only three rather than eight variables to achieve higher accuracy than the Nomogram for predicting NSLN status in two different datasets.

Rickettsia is a genus of Gram-negative bacteria that has for centuries caused large-scale morbidity and mortality. In recent years, the resurgence of rickettsial diseases as a major cause of pyrexias of unknown origin, bioterrorism concerns, vector movement, and concerns over drug resistance is driving a need to identify novel treatments for these obligate intracellular bacteria. Utilizing an uvGFP plasmid reporter, we developed a screen for identifying anti-rickettsial small molecule inhibitors using Rickettsia canadensis as a model organism. The screening data were utilized to train a Bayesian model to predict growth inhibition in this assay. This two-pronged methodology identified anti-rickettsial compounds, including duartin and JSF-3204 as highly specific, efficacious, and noncytotoxic compounds. Both molecules exhibited in vitro growth inhibition of R. prowazekii , the causative agent of epidemic typhus. These small molecules and the workflow, featuring a high-throughput phenotypic screen for growth inhibitors of intracellular Rickettsia spp. and machine learning models for the prediction of growth inhibition of an obligate intracellular Gram-negative bacterium, should prove useful in the search for new therapeutic strategies to treat infections from Rickettsia spp. and other obligate intracellular bacteria.

It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host-pathogen interactions.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.

Rapid advancements in the genetic manipulation of obligate intracellular bacterial pathogens have been made over the past two years. In this paper we attempt to summarize the work published since 2011 that documents these exciting accomplishments. Although each genus comprising this diverse group of pathogens poses unique problems, requiring modifications of established techniques and the introduction of new tools, all appear amenable to genetic analysis. Significantly, the field is moving forward from a focus on the identification and development of genetic techniques to their application in addressing crucial questions related to mechanisms of bacterial pathogenicity and the requirements of obligate intracellular growth., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Legionella pneumophila effector protein RalF functions as a guanine nucleotide exchange factor (GEF) that activates the host small GTPase protein ADP-ribosylation factor (Arf), and recruits this host protein to the vacuoles in which this pathogen resides. GEF activity is conferred by the Sec7 domain located in the N-terminal region of RalF. Structural studies indicate that the C-terminal region of RalF makes contacts with residues in the Sec7 domain important for Arf interactions. Theoretically, the C-terminal region of RalF could prevent nucleotide exchange activity by blocking the ability of Arf to interact with the Sec7 domain. For this reason, the C-terminal region of RalF has been termed a capping domain. Here, the role of the RalF capping domain was investigated by comparing biochemical and effector activities mediated by this domain in both the Legionella RalF protein (LpRalF) and in a RalF ortholog isolated from the unrelated intracellular pathogen Rickettsia prowazekii (RpRalF). These data indicate that both RalF proteins contain a functional Sec7 domain and that the capping domain regulates RalF GEF activity. The capping domain has intrinsic determinants that mediate localization of the RalF protein inside of host cells and confer distinct effector activities. Localization mediated by the capping domain of LpRalF enables the GEF to modulate membrane transport in the secretory pathway, whereas, the capping domain of RpRalF enables this bacterial GEF to modulate actin dynamics occurring near the plasma membrane. Thus, these data reveal that divergence in the function of the C-terminal capping domain alters the in vivo functions of the RalF proteins.

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.

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.

Termination of transcription is an important component of bacterial gene expression. However, little is known concerning this process in the obligate intracellular pathogen and model for reductive evolution, Rickettsia prowazekii. To assess transcriptional termination in this bacterium, transcripts of convergent gene pairs, some containing predicted intrinsic terminators, were analyzed. These analyses revealed that, rather than terminating at a specific site within the intervening region between the convergent genes, most of the transcripts demonstrated either a lack of termination within this region, which generated antisense RNA, or a putative non-site-specific termination that occurred throughout the intervening sequence. Transcripts terminating at predicted intrinsic terminators, as well as at a putative Rho-dependant terminator, were also examined and found to vary based on the rickettsial host environment. These results suggest that transcriptional termination, or lack thereof, plays a role in rickettsial gene regulation.

A radiographic system is optimized for the contrast inherent to small animals and is developed for a multi-modal imaging system devised for in-vivo studies. The range of X-ray energies utilized (generally considered "soft X-rays") enables enhanced spatial resolution and superior contrast for detailed study of the mouse anatomy and smaller specimens. Despite the difficulties presented by the complicated energy spectrum of soft X-rays, relevant system calibrations for bone measures are described in detail and applied to the mouse. Further, long-bone symmetry modeling using a cylindrical projection is applied to the planar density image, providing convenient bone density estimates that are consistent with other methodologies., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

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 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.

M-DNA is a complex formed between duplex DNA and divalent metal ions (Zn2+, Cu2+ or Ni2+) at pHs above 8. Previous results showed that the fluorescence of an electron donor fluorophore was quenched when an acceptor flourophore was placed in the opposite end of an M-DNA duplex suggesting electron transfer through the duplex and indicating M-DNA may operate as a better conductor than B-DNA. To further investigate the properties of M-DNA, oligodeoxynucleotides were prepared with fluorescein (Fl) as an electron donor placed at different positions along the helix. An internal position of the chromophore was made possible by attaching it to the extra hydroxyl arm in the branched monomer 4'-C-hydroxymethylthymidine. Upon excitation of the donor fluorophore, it was demonstrated that electrons could be injected into the side of an M-DNA helix thereby extending the range of nanoelectronic structures that can be prepared from DNA.

The obligate nature of Rickettsia prowazekii intracellular growth places severe restrictions on the analysis of rickettsial gene function and gene expression. Fortunately, this situation is improving as methods for the genetic manipulation and proteomic analysis of this fascinating human pathogen become available. In this paper, we review the current status of rickettsial genetics and the isolation of rickettsial mutants using a genetic approach. In addition, the examination of rickettsial gene expression through characterization of the rickettsial proteome will be described. This will include a description of a high-throughput, accurate mass approach that has identified 596 rickettsial proteins in a complex rickettsial protein sample.

M-DNA, a complex formed in solution between divalent metal ions (M) and duplex DNA, has been studied extensively using fluorescence quenching. This review examines the methods used to examine the formation of M-DNA, and its ability to serve as a pathway for electron transfer between donor and acceptor chromaphores. A mass action model for M-DNA formation is presented based upon the results of fluorescence quenching studies using fluorescein/QSY-7 labeled duplexes. From the mass action analysis, it was determined that approximately 1.4 protons are released per base pair, with k(eq) on the order of 10(-8), indicative of a strong interaction. As resonance energy transfer is shown to be unlikely over the distances involved in this work, the observed quenching in M-DNA is discussed in terms of an electron hopping mechanism for electron transfer, with k(hop)=2.5 x 10(11)s(-1).

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.

Ni(II) and Zn(II) M-DNA formation and denaturation of double-stranded DNA (dsDNA) by Cd(2+) were monitored by surface plasmon resonance (SPR). When exposed to immobilized 30 bp 50% GC dsDNA, Zn(2+) and Ni(2+) were found to give signals indicative of a conformational change at pH 8.5 but not 7.5, while Mg(2+) and Ca(2+) caused small changes at both pHs. The concentrations that gave 50% of the maximum responses were 0.06 and 0.50 mM for Zn(2+) and Ni(2+), respectively. At pH 8.5, Cd(2+) denatured over 40% of the dsDNA, while other metals denatured less than 5% of the DNA. Smaller pH-dependent signals were induced by Zn(2+), Ni(2+) or Cd(2+) with 50% GC single-stranded DNA (ssDNA), and with a homopolymer of d(T)30. Homopolymers d(A)30 and d(C)30 showed small signals that were largely independent of pH in the presence of Zn(2+) or Ni(2+).

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 DNA-protein 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 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 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine 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.

The thermodynamics of formation of a novel divalent metal ion-DNA complex known as M-DNA have been investigated using an ethidium bromide (EB) fluorescence assay, and with isothermal titration calorimetry. The process of M-DNA formation was observed from the EB assay to be strongly temperature-dependent. The binding of Zn(2+) to calf thymus (42% GC content) and Escherichia coli (50% GC content) DNA at pH 8.5 exhibited an endothermic cooperative binding process at Zn(2+) concentrations of approximately 0.1 mM, indicating an entropy driven process. This binding process is consistent with a site-specific binding interaction, similar in nature to Z-DNA formation; however, the interaction occurs at much lower metal ion concentrations. The enthalpy of M-DNA formation for calf thymus DNA was determined to be 10.5+/-0.7 and 9+/-2 kJ/mbp at DNA concentrations of 100 and 50 microg ml(-1), respectively. An enthalpy of 13+/-3 kJ/mbp was obtained for M-DNA formation for 50 microg ml(-1) E. coli DNA. No evidence of M-DNA formation was observed in either DNA at pH 7.5 with Zn(2+) or at either pH 7.5 or 8.5 with Mg(2+).

M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of approximately 8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)*poly(dC) and poly(dA)*poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)*(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage lambda DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from T(m) measurements. It is evident that the lower the pK(a) of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion.

Objective: To examine the relationship between child maltreatment and cognitive development in extremely low birth weight infants, adjusting for perinatal and parental risk factors., Methods: A total of 352 infants with birth weight of <1000 g were followed prospectively for 4 years. The data were analyzed with regard to perinatal and parental risk factors and referrals for suspected child maltreatment to government agencies. Perinatal risk factors included birth weight, gestation, gender, periventricular hemorrhage, ventricular dilation, home oxygen requirement, and necrotizing enterocolitis. Parental risk factors included maternal age, race, marital status, education, and hospital insurance status. Cognitive z scores were calculated at 1, 2, and 4 years, and head circumference z scores were calculated at birth, 2 years, and 4 years., Results: Fifteen percent of infants were referred to child protective services for suspected child maltreatment. The adjusted general cognitive index at 4 years was significantly reduced in infants who were referred for neglect (-17.6; 95% confidence interval: -3.3, -31.9). Infants whose neglect was substantiated had a progressive decline in their cognitive function over time (cognitive z scores: -0.97, -1.37, and -2.05 standard deviations at 1, 2, and 4 years, respectively), compared with non-neglected infants (z scores: -0.04 to -0.36). They had a significantly smaller head circumference at 2 and 4 years but not at birth (adjusted z score at 4 years: -0.812; 95% confidence interval: -0.167, -1.458). Perinatal risk factors and physical disability were not related to maltreatment referral; only parental factors were independent predictors., Conclusions: Childhood neglect is associated significantly with delayed cognitive development and head growth. Addressing risk factors antenatally and in early childhood may improve outcomes.

Objectives: To look for features of non-accidental fractures in infants aged under I year and assess the risk of subsequent morbidity and mortality., Methodology: A retrospective analysis of 99 children aged under 1 year who presented to the Mater Children's Hospital, Brisbane, between January 1990 and December 1993, and were found to have a fracture. The 99 infants were divided into non-accidental and accidental groups. Comparison was made between the two groups for age, sex and type of fracture. Deaths, subsequent injuries and child protection notifications until March 1997 were compared between groups., Results: Of the 99 infants with fracture (64 males, 35 female), the skull and femur were the most prevalent sites of fracture. Twenty-six infants had fractures assessed as non-accidental. This group was younger but did not differ significantly in gender or site of fracture. Infants aged under 4 months had a significantly greater risk of their fracture being non-accidental (P = 0.0007). Subsequent substantiated child protection notifications occurred in nine of the non-accidental group and in one of the accidental group (P = 0.000001). There was no significant difference in the rate of subsequent notifications between those infants with abuse who were removed from their carers and those not removed. Subsequent injuries presenting to hospital occurred in 17 of the accidental group and three of the non-accidental group (P = 0.20). There were no deaths., Conclusion: Infants aged under 1 year with fractures have a high prevalence of abuse. The risk of abuse as cause for the fracture is greater in those aged under 4 months. Infants with non-accidental fractures have a high risk of further abuse even with intervention.

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.

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.

The dnaA gene encoding the initiator protein of DNA replication was isolated from the obligate intracellular bacterium, Rickettsia prowazekii. Comparison of the deduced amino acid sequence of R. prowazekii DnaA with other bacterial DnaA proteins revealed extensive similarity. However, the rickettsial sequence is unique in the number of basic lysine residues found within a highly conserved portion of the putative DNA binding region, suggesting that the rickettsial protein may recognize a DNA sequence that differs from the consensus DnaA box sequence identified in other bacteria. Consensus DnaA box sequences, found upstream of many bacterial dnaA genes, were not identified upstream of rickettsial dnaA gene. In addition, gene organization within this region differed from that of other bacteria. The putative start of transcription of the rickettsial dnaA gene was localized to a site 522 nucleotides (nt) upstream of the DnaA start codon.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism's unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R. prowazekii RNA polymerase beta subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifr region of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.

Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.

The nuclear gene encoding the mitochondrial citrate transport protein (i.e., CTP1) has been deleted from a haploid yeast strain. The stable yeast deletion strain was constructed by homologous recombination of the HIS3 gene at the CTP1 gene locus. Deletion of the CTP was confirmed by PCR. Immunoblot analysis provided the first quantitative estimate of the level of the CTP in wild-type yeast mitochondria and indicated the absence of expressed CTP in mitochondria isolated from the deletion strain. Deletion of CTP1 did not lead to a phenotype on any carbon source tested, indicating that CTP1 is not an essential gene. This suggests that either known alternative pathways are able to produce sufficient acetyl-CoA to support biosynthetic reactions, or there exists a second CTP gene. The ability of the deletion strain to serve as a host for the correct targeting and overexpression of a mutated CTP was then demonstrated. These studies provide a system which permits the use of site-directed mutagenesis to examine both CTP targeting to mitochondria, as well as the molecular basis underlying CTP function. Moreover, this system will not only facilitate the study of the yeast CTP, but also CTPs expressed from the cDNAs of higher eukaryotes.

The transcripts of the citrate synthase-encoding gene (gltA) in Rickettsia prowazekii (Rp), an obligate intracellular parasitic bacterium, were analyzed by RNase protection (RP), primer extension (PE) and in vitro transcription assays. Analysis of the 5' end of the gltA mRNA by RP and PE assays revealed that there were two gltA mRNAs with the 5' ends located at 16 bp and 307 bp upstream from the gltA coding region. Since these two mRNAs might represent two species of mRNA transcribed from two different promoters or a single transcript that was processed to give two mRNAs, an in vitro transcription analysis with purified Rp RNA polymerase (RNAP) was performed to distinguish these two possibilities. Purified Rp RNAP catalyzed the formation of two transcripts initiated from the same nucleotides indicated by RP and PE. Sequence analysis identified Escherichia coli (Ec) promoter-like sequences immediately upstream from both transcription start points (tsp). The first promoter (promoter P1) had the core sequence TTCTAA-N17-TATACT, was 6 bp upstream from the tsp (base A) and was centered at 37 bp upstream from the coding region. The second promoter (promoter P2) had the core sequence ATGAAA-N17-TAAAGT, was 7 bp upstream from the tsp (base T) and was centered at 329 bp upstream from the coding region. This is the first demonstration of multiple promoters in this obligate intracellular parasite which has implications concerning transcriptional regulation.

The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e. Km = 0.36 mM and Vmax = 2.5 mumol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.

The rat liver mitochondrial tricarboxylate transport protein has been overexpressed in E. coli. The expressed transporter, which contains a 21 amino acid N-terminal fusion sequence, accumulates in inclusion bodies. Subsequent extraction of the tricarboxylate transporter from isolated inclusion bodies yields approximately 90 mg of transport protein per liter of E. coli culture at a purity of greater than 90%. Upon incorporation into phospholipid vesicles the purified, overexpressed transporter catalyzes a 1,2,3-benezenetricarboxylate-sensitive citrate/citrate exchange (i.e., the defining reaction of the mitochondrial tricarboxylate transporter). Kinetic characterization of the reconstituted transporter indicates a Km of 0.37 mM and a Vmax of 101 nmol/min/mg protein. The substrate specificity of the reconstituted, expressed transporter is virtually identical to that of the native transporter. These studies represent the first overexpression of the rat liver mitochondrial tricarboxylate transporter. By providing a large amount of highly-purified, functionally competent transporter this system will now enable a variety of structural studies, including site-directed mutagenesis, which heretofore could not be performed.

The Rickettsia prowazekii (Rp) gyrA gene, which codes for a subunit of DNA gyrase in this obligate intracellular bacterium, has been isolated and characterized. Nucleotide sequence analysis revealed an open reading frame (ORF), initiating with a GTG start codon, of 2718 bp that could encode a protein of 905 amino acids (aa) with a calculated M(r) of 101,048. The Rp gyrase subunit A (GyrA), when compared to GyrA analogs of other bacterial species, exhibited 43 to 50% identity. Alignment of the Rp GyrA aa sequence with the other analogs revealed the presence of a span of additional aa within the putative DNA-binding domain. The lack of an ORF within 865 bp upstream from the Rp gyrA demonstrates a Rp gene organization different from that of characterized gyrA from other species. Despite the similarity to Escherichia coli GyrA, Rp GyrA did not complement an E. coli gyrA temperature-sensitive mutant. However, Rp gyrA was dominant to an E. coli gyrA96 nalidixic-acid-resistant (NalR) mutant, conferring Nal sensitivity when introduced into the NalR E. coli strain.

The firA and lpxA genes, as well as an ORF coding for a putative 16-kDa protein of unknown function, have been identified and characterized in the obligate intracellular bacterium. Rickettsia rickettsii. This is the first description of these genes, which code for enzymes involved in the biosynthesis of lipid A, in a species outside of the Enterobacteriaceae. The deduced amino acid (aa) sequences of FirA, ORF16 and LpxA of R. rickettsii, when compared to their Escherichia coli analogs, exhibited 35, 44 and 41% aa identity, respectively. In addition, the order of genes in R. rickettsii, firA-ORF16-lpxA, was identical to that found in E. coli; however, the spacing between the rickettsial genes was greater. Interestingly, the R. rickettsii FirA and LpxA deduced proteins retain an unusual hexapeptide repeat pattern found in E. coli and Salmonella typhimurium FirA/Ssc and E. coli LpxA, as well as other acyltransferases, providing additional support for the importance of this structure.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.

The gene (sdhA) coding for the flavoprotein subunit (SdhA) of succinate dehydrogenase of the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated using an oligodeoxyribonucleotide probe to the conserved flavin adenine dinucleotide (FAD)-binding region of characterized flavoproteins. Nucleotide (nt) sequence analysis revealed an open reading frame (ORF) of 1791 bp capable of encoding a protein of 596 amino acids (aa) with a deduced M(r) of 65,444. The deduced aa sequence, when compared to the flavoprotein subunits of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Bos taurus, revealed 52.8, 34.0, 65.8 and 52.0% aa identity, respectively. R. prowazekii SdhA produced in E. coli minicells and analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) migrated as a protein of approximately 63 kDa, comparable to the size of the deduced protein. In addition, two proteins of approximately 12 and 41 kDa were also produced in the E. coli minicells. The production of these proteins resulted from additional translational starts within the SdhA coding sequence, suggesting differences between the translational start signals of E. coli and R. prowazekii. Despite the similarity of R. prowazekii SdhA to that of E. coli, the R. prowazekii SdhA did not complement an E. coli sdhA mutant. In addition, analysis of the nt sequence immediately upstream from R. prowazekii sdhA revealed that the rickettsial sdh gene organization differs from that of E. coli and B. subtilis.

The amino acid sequence of the rat liver mitochondrial tricarboxylate transport protein has been deduced from its corresponding cDNA. Using the polymerase chain reaction, with primers derived from amino acid sequence information that we obtained by direct sequencing of the purified transporter and its internal peptides, a cDNA fragment was amplified that encodes approximately two-thirds of the tricarboxylate transport protein. This cDNA fragment was used to screen a rat liver lambda gt11 cDNA library which permitted the isolation and sequencing of a cDNA clone that encodes the entire tricarboxylate transporter. The clone is 1927 base pairs in length with 5'- and 3'-untranslated regions of 419 and 572 base pairs, respectively. The open reading frame encodes a mature transport protein of 298 amino acids preceded by a presequence of 13 residues. Analysis of the tricarboxylate transporter sequence indicates that it contains three related sequence domains, each of approximately 100 amino acid residues in length. Dot plot comparisons and sequence alignment indicate that these domains are related to each other, as well as to domains of similar length that are present in other mitochondrial transporters. Hydrophobicity analysis predicts that the tricarboxylate carrier contains six membrane-spanning alpha-helices (two per 100-amino acid sequence domain) and has permitted the construction of an initial model for the topography of this transporter within the mitochondrial inner membrane. Finally, Southern blot analysis of both rat and human genomic DNA demonstrates the presence of multiple sequences related to the tricarboxylate transporter in both genomes. These studies provide the first information on the primary structure of the mitochondrial tricarboxylate transport protein. We are now able, on the basis of both structural and functional considerations, to assign this metabolically important transporter to the mitochondrial carrier family, the members of which are likely to have evolved from a common genetic origin.

The gene (dnaG) coding for DNA primase in the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated and characterized. An open reading frame (ORF) of 1848 bp capable of encoding 616 amino acids (aa) is located 18 bp upstream from the gene coding for the major sigma factor of R. prowazekii, sigma 73. Based on aa sequence comparisons of DNA primase from R. prowazekii, Escherichia coli, Salmonella typhimurium and Bacillus subtilis, we propose that R. prowazekii dnaG begins 69 bp into the ORF and encodes 593 aa with a calculated M(r) of 68,683. An upstream ORF overlaps 66 of the first 69 bp of the larger R. prowazekii dnaG ORF, suggesting either an overlapping gene structure or the generation of the smaller protein product of 593 aa. Predicted aa sequence of R. prowazekii primase compared to E. coli, S. typhimurium and B. subtilis primases reveals 30.5%, 30.5% and 29.7% aa identity, respectively. The R. prowazekii dnaG gene failed to complement an E. coli dnaG temperature sensitive mutation perhaps due to poor expression of the gene or inability to function properly in E. coli. The gene organization of an ORF followed by DNA primase (dnaG) and then the major sigma factor (rpoD) is consistent with the major macromolecular synthesis operons of E. coli, S. typhimurium and B. subtilis.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.

The established criteria for the determination of the optimum time for the sacrifice of guinea pigs inoculated with samples of cooling tower water were found to be inadequate for the detection of low levels of Legionella pneumophila. By ignoring the requirement for fever and by sequentially sacrificing the infected guinea pigs on days 3 through 5 postinoculation, we simplified the procedure, and the sensitivity of detection was improved a great deal.

Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator. Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with [alpha-32P]ATP in 100 mM sodium phosphate buffer. This clone carried a plasmid, pMW286, containing a 9-kilobase-pair insert of rickettsial DNA, as established by DNA X DNA hybridizations. Transformation studies with purified pMW286 established that the ability of E. coli cells to accumulate radioactivity was mediated by the recombinant plasmid. Results from experiments in which [3H]ATP was substituted for [alpha-32P]ATP strongly suggested that the radiolabeled ATP was transported intact. Furthermore, [3H]ATP was incorporated into 10% (wt/vol) trichloroacetic acid-precipitable material in a time-dependent manner. Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP. Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides. The successful cloning and expression of the rickettsial ADP/ATP translocator in E. coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism.