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Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis. I. Processing of B. pumilus and B. licheniformis DNA in B. subtilis.

Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donor-recipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.

Bacillus↗

Evaluation of two commercial diagnostic kits for Mycobacterium tuberculosis completely based on bacterial DNA and rRNA amplification for direct detection of tubercle bacilli in sputum specimens.

The Roche Amplicor Mycobacterium Tuberculosis Kit (Amplicor PCR) and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) were compared for efficiency in the direct detection of tubercle bacilli in sputum specimens. In total, 72 sputum specimens obtained from patients with active tuberculosis were examined with the Amplicor PCR and MTD kits. Of the 72 test specimens, 45 and 41 samples were positive by the Amplicor PCR and MTD methods, respectively. These values were larger than those obtained by the culture method (Septi-Chek) and acid-fast bacilli-smear test, yielding 37 and 20 positive results, respectively, and the sensitivities were as follows: smear, 39.2%; culture, 72.5%; Amplicor PCR, 88.2%; MTD, 80.4%. Amplicor PCR was similarly or somewhat more sensitive in direct detection of M. tuberculosis in sputum samples from patients with clinically active tuberculosis than was MTD.

DNA, Bacterial↗

Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages.

LPS is well recognized for its potent capacity to activate mouse macrophages to produce NO, an important inflammatory mediator in innate host defense. We demonstrate here that, although inducing little NO alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce NO in cultures of RAW 264.7 macrophages. The effects of the DNA are mimicked by synthetic CpG-containing oligodeoxynucleotides but not by non-CpG-containing oligodeoxynucleotides. This synergistic activity is not inhibited by neutralizing Abs against IFN. Preincubation of macrophages with DNA for 8-24 h suppresses subsequent synergistic macrophage responses to DNA/LPS, whereas prolonged pretreatment with LPS enhances synergy. RT-PCR analysis indicates that the mRNA levels of the inducible NO synthase gene are also coordinately suppressed or induced. These findings indicate that temporally controlled, synergistic interactions exist between microbial DNA and LPS in the induction of macrophage NO via enhanced inducible NO synthase gene expression.

Animals↗