Protein synthesis by intact Coxiella burnetii cells.

Coxiella burnetii was isolated from persistently infected fibroblast host cells by a rapid mechanical lysis technique. Macromolecular synthesis was initiated in these otherwise dormant cells by incubation at pH 4.5. The synthesis of protein proceeded for as long as 24 h. Initiation of protein synthesis in C. burnetii was dependent upon RNA synthesis. Approximately 24 species of polypeptides were synthesized, and some of these appeared to be major synthetic products. Increases in protein biomass of 15 to 30% were calculated to occur during incubation. Inhibition of DNA synthesis affected protein synthesis after 12 h of incubation. The results suggest that although these parasitic bacteria did not grow in the axenic media devised, significant biosynthetic processes occurred.     

Physiology of Rickettsiae VI. Host-independent Synthesis of Polyribonucleotides by Coxiella burnetii

Preparations of purified and disrupted suspensions of Coxiella burnetii are able to incorporate ribonucleotides into polymers in the presence of adenosine, guanosine, cytidine, and uridine triphosphates. Nucleotide incorporation requires the presence of all four ribonucleoside triphosphates. The reaction is enhanced by the addition of phosphoenolpyruvate and pyruvic kinase, and exogenous deoxyribonucleic acid, and is inhibited by deoxyribonuclease and actinomycin D. Incorporation is maximal between pH 7.0 and 8.0, and at 37 C. The synthesized polymer is relatively insensitive to deoxyribonuclease and is sensitive to ribonuclease and dilute alkaline hydrolysis. The data indicate the presence of an autonomous deoxyribonucleic acid-dependent ribonucleic acid polymerase in the rickettsial agent.     

Physiology of rickettsiae. VII. Amino acid incorporation by Coxiella burnetii and by infected hosts

Protein synthesis, in terms of (14)C-labeled amino acid incorporation into a hot trichloroacetic acid fraction, was studied in cell-free preparations of Coxiella burnetii, and in uninfected and Q fever-infected guinea pig and chick embryo hosts. Purified and disrupted suspensions of C. burnetii incorporated (14)C-labeled l-leucine, l-phenylalanine and algal hydrolysate. Livers of infected guinea pigs and chick embryos had a greater incorporation rate at the height of infection than comparable preparations from uninfected animals. As chick embryonic development continued during infection, the rate of incorporation progressively decreased below that of uninfected embryos.     

Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase.

The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.     

Biochemistry of Coxiella burnetii: 6-phosphogluconic acid dehydrogenase

Purified preparations of the rickettsial agent, Coxiella burnetii, have been examined for their ability to decarboxylate 6-phosphogluconate. The enzyme 6-phosphogluconic acid dehydrogenase [6-phospho-d-gluconate: NADP (nicotinamide adenine dinucleotide phosphate) oxidoreductase (decarboxylating), EC 1.1.1.44] was detected in extracts, but not in whole-cell preparations of C. burnetii. Both extracts and whole cells were shown to be free from contaminating host enzyme activity. Partial characterization of the enzyme has shown that it is substrate-dependent, specific for NADP, and requires magnesium for activity. The pH optimum of the rickettsial enzyme is 8.0.     

Active transport of proline by Coxiella burnetii

The obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3.0 to 4.5, a pH range approximately that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent Km for proline transport from 45 microM to 15 microM, with the Vmax increasing from 3.6 pmol s-1 (mg dry wt)-1 to 4.8 pmol s-1 (mg dry wt)-1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. D-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.     

Inactivation of Coxiella burnetii by gamma irradiation

The gamma radiation inactivation kinetics for Coxiella burnetii at -79 degrees C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10(11) C. burnetii ml-1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.     

Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M_r was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (small-cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M_r of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.     

Survival of Coxiella burnetii in soil

For the first time the survival of Coxiella burnetii of five types in soils has been studied. The survival of C. burnetii has been found to depend on the content of organic substances in black earth, as well as soil temperature. The method for the prevention of an epidemic outbreak of Q fever directly under the natural conditions has been proposed.     

Efficacy of translation in the rickettsia Coxiella burnetii.

The addition of coliphage Q beta ribonucleic acid to a cell-free translation system obtained from Coxiella burnetii cells caused the formation of monosomes and disomes from ribosomal subunits and resulted in synthesis of viral coat protein. Rickettsial ribosomes and associated translation factors functioned together, with fidelity, efficiency, and a specificity similar to those of free-living gram-negative bacteria.     

Genome size of the rickettsia Coxiella burnetii.

The genome size of Coxiella burnetii Nine Mile strain was determined by the method of initial rate of deoxyribonucleic acid renaturation. The mean value obtained was 1.04 X 10(9) daltons.     

Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.     

PCR法对鸡卵黄中Coxiella burnetii菌的测定

[目的]通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii (C.b)CoMl基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义.[方法]提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物.[结果]用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Coml基因,用此方法可测出鸡卵中Coxiella burnetii Coml基因达104-106个,阳性率为5％-22％;对阳性鸡卵Coml基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物.[结论]由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源.