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The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.
A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.
Th1 immune responses, characterized by production of gamma interferon (IFN-gamma), are associated with protective immunity to viruses and intracellular bacteria. Heat-killed Brucella abortus promotes secretion of Th1-inducing cytokines such as interleukin-12 (IL-12) and IFN-gamma and has been used as a carrier to induce Th1 responses to vaccines. To explore which bacterial constituents could mediate this response and how it is regulated, murine spleen cells were cultured with B. abortus derived DNA, lipopolysaccharide (LPS), or whole killed organisms. Each constituent induced similar, substantial amounts of IL-10. However, only B. abortus and B. abortus DNA induced high levels of IFN-gamma and IL-12. B. abortus and B. abortus DNA-stimulated IL-12 production was maximal by 6 to 18 h, while IL-10 production steadily accumulated over this time period. These kinetics suggested that IL-10 may eventually downmodulate the Th1-like cytokine response to B. abortus and B. abortus DNA, which was confirmed by using neutralizing antibody. In the absence of IL-10, B. abortus LPS induced strong IFN-gamma responses, but IL-12 p70 levels were still undetectable from BALB/c spleen cells. LPS induced IL-12 if the spleen cells were primed with IFN-gamma and IL-10 was neutralized, indicating that LPS can stimulate IL-12 production under the most favorable conditions. Responses to Escherichia coli LPS and DNA mirrored the responses to B. abortus components, suggesting that immune effects observed with these constituents may be generalizable to many microbial species. In vivo experiments demonstrated the same hierarchy of responses for IL-12 production. These findings support the likelihood that microbial components, if used as carriers or adjuvants, can differ substantially in their ability to effect a Th1 response.
Recombinant lambda phages containing the genes for dnaZ protein (the gamma subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichia coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.
The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.
Coupling sequences are the 6 bp flanking the conjugative transposon Tn916 and are thought to play a role in determining the frequency of conjugative transposition. The affinity of binding of a chimeric protein, which consisted of maltose binding protein fused to the carboxy-terminal DNA binding domain of Tn916 integrase (Int), to different double-stranded oligonucleotide substrates containing coupling sequences associated with high- and low-frequency conjugative transposition was measured using a competition binding assay. The relative affinity of the chimeric protein was unaffected by the nature of the coupling sequences tested. The same results were obtained when the coupling sequences were placed in a different surrounding sequence context. It therefore appears that the effects of different coupling sequences on the frequency of conjugative transposition are not due simply to differences in Int binding.
Escherichia coli is a model system to study the mechanism of DNA replication and its regulation during the cell cycle. One regulatory pathway ensures that initiation of DNA replication from the chromosomal origin, oriC, is synchronous and occurs at the proper time in the bacterial cell cycle. A major player in this pathway is SeqA protein and involves its ability to bind preferentially to oriC when it is hemi-methylated. The second pathway modulates DnaA activity by stimulating the hydrolysis of ATP bound to DnaA protein. The regulatory inactivation of DnaA function involves an interaction with Hda protein and the beta dimer, which functions as a sliding clamp for the replicase, DNA polymerase III holoenzyme. The datA locus represents a third mechanism, which appears to influence the availability of DnaA protein in supporting rifampicin-resistant initiations.
Accurate monitoring and identification of Legionella species, the causative agents of Legionnaires' and other diseases, in environmental water sources is an important public health issue. Traditional culture methods often lack the sensitivity and specificity that can be attained using the polymerase chain reaction (PCR) to amplify targeted regions of the bacterial genome. Matrix-assisted laser desorption/ionization combined with time-of-flight (MALDI-TOF) mass spectrometry is shown to be useful for detection of 108- and 168-base PCR products specific to Legionella. A rapid purification aimed at removal of salts and unreacted primers is demonstrated. The addition of a synthetic DNA 20-mer to the MALDI sample facilitates aiming the laser at a favorable spot on the sample probe from which the PCR products can be detected.
To confirm the sensitivity of the polymerase chain reaction (PCR) technique (versus blood cultures) and to gain a better understanding of the incidence of true- and false-positive results when using this technique, one hundred randomly chosen patients treated operationally because of valve defects were examined. In our studies we found that PCR techniques using universal primers complementary to the bacterial 16S rDNA showed promise as being more sensitive than conventional blood culture (BC) techniques. From the time that a blood culture is positive, conventional methods of culture and antibiotic susceptibility testing require at least 64 h for the detection of infection or colonization. Rapid identification of bacteria from blood using PCR technique accelerates the microbiological diagnosis.
DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.
As a result of an epidemiological survey on the periodontal status of young adults, a school was found with a relatively high number of subjects showing loss of attachment (18 out of 87). In 10 of these subjects the microflora of one pocket and of the dorsum of the tongue was investigated with special reference to black-pigmented species and Actinobacillus actinomycetemcomitans. Prevotella intermedia was the most prevalent species, being found in 9 patients. A. actinomycetemcomitans was isolated from 3 subjects and Porphyromonas gingivalis from 2 subjects. To determine whether cross-infection had occurred, the P. intermedia, P. gingivalis, and A. actinomycetemcomitans isolates were examined by restriction endonuclease analysis of the DNA after digesting with one or a combination of the endonucleases Pst I, Bam HI, and Hind III. Evaluation of the restriction endonuclease typing method showed it to be sensitive and useful for the typing of P. intermedia, P. gingivalis, and A. actinomycetemcomitans. Using this restriction endonuclease typing method, all isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia obtained from single individuals showed different digest patterns. We conclude that in the present study bacterial cross-infection did not occur among subjects in this school.
BACKGROUND: The etiology of inflammatory bowel diseases is unknown. Mycobacteria spp., Bacteroides vulgatus, and Escherichia coli have been suspected to be involved. The aim of the present study was to examine the possible relationship between inflammatory bowel diseases and these microbes. METHODS: We studied 45 patients; 16 with Crohn's disease, 11 with ulcerative colitis, and 18 with colon cancer as controls. We used a real-time quantitative polymerase chain reaction to detect and estimate numbers of bacterial genomes in formalin-fixed, paraffin-embedded tissue samples from the subjects. The bacteria studied were Mycobacterium tuberculosis, M. avium, M. paratuberculosis, B. vulgatus, and E. coli. Immunohistochemical staining was done to locate B. vulgatus and E. coli in tissue samples. RESULTS: The three Mycobacterium species were not detected. B. vulgatus and E. coli were detected more frequently and in greater numbers in samples from patients with inflammatory bowel diseases than in samples from control patients with colon cancer. The frequency and numbers were not related to the severity of the disease. Many bacteria of these species were found within the mucous layer, underneath erosions, in necrotic ulcer bed tissues, and in abscesses. E. coli cells were found in perivascular areas in the proper muscle layer and in germinal centers of lymph follicles. CONCLUSIONS: Our results suggest that Mycobacteria spp. are not involved in the etiology of Crohn's disease and that mucosa-associated B. vulgatus and E. coli are not a direct cause of inflammatory bowel diseases, although they may contribute to the diseases by preventing or delaying remission.
Previous studies with a mycobacterial heat shock protein (hsp-65) have demonstrated some efficacy using cationic liposome-mediated gene transfer in murine i.p. sarcoma models. To further analyze the efficacy of hsp-65 immunotherapy in clinically relevant models of localized cancer, immunocompetent mice bearing i.p. murine mesothelioma were treated with four i.p. doses of a cationic lipid complexed with plasmid DNA (pDNA) containing hsp65, LacZ, or a null plasmid. We observed >90% long-term survival (median survival, 150 days versus approximately 25 days, treated versus saline control, respectively) in a syngeneic, i.p. murine mesothelioma model (AC29). Long-term survivors were observed in all groups treated with lipid complexed with any pDNA. Lipid alone or DNA alone provided no demonstrable survival advantage. In a more aggressive i.p. model of mesothelioma (AB12), we observed >40% long-term survival in groups treated with lipid:pDNA complexes, again irrespective of the transgene. To ask whether these antitumor effects had led to an adaptive immune response against the tumor cell, we rechallenged long-term survivors in both murine models s.c. with the parental tumor cell line. Specific, long-lasting systemic immunity against the tumor was readily demonstrated in both models (AB12 and AC29). Consistent with these results, splenocytes from long-term survivors specifically lysed the parental tumor cell lines. Depleting the CD8+ T-cells from the splenocyte pool eliminated this lytic activity. Lipid:pDNA treatment of athymic, SCID, and SCID/Beige mice bearing a murine i.p. mesothelioma (AC29) resulted in only a slight survival advantage, but there were no long-term survivors. Treatment of immunocompetent mice depleted of specific immune effector cells demonstrated roles for CD8+ and natural killer cells. Although the exact mechanism(s) responsible for these antitumor effects is unclear, the results are consistent with roles for both innate and adaptive immune responses. An initial tumor cell killing stimulated by cationic lipid:pDNA complexes appears to be translated into long-term, systemic immunity against the tumor cell. These results are the first to demonstrate that adaptive immunity against a tumor cell can be induced by the administration of lipid:pDNA complexes. Multiple administrations of cationic lipid complexed with pDNA lacking an expressed transgene could provide a promising generalized immune-mediated modality for treating cancer.
The cDNA for the chloroplast-located homolog of bacterial RecA protein, designated recA-AT, was placed in a plasmid appropriate for in vitro transcription and translation. Translation with 35S-labeled Met permitted demonstration of uptake of the protein product into isolated pea chloroplasts, and processing to a mature size. Preliminary evidence for the first amino acid was estimated from results using both 35S-Met and 3H-Leu for in vitro transcription and translation, followed by uptake into chloroplasts and processing. The labeled protein was subject to sequential amino acid hydrolyses, and radioactivity was measured in each round. Induction of gene transcription in leaves infiltrated with the DNA-damaging agent, methyl methane-sulfonate was shown by Northern blot analysis. Further constructs were made for over-expression of the gene in E. coli; and one out of many tried permitted production of some soluble protein. Extracts from transformed bacteria were shown to have RecA activity using the "POM" assay [Bertrand et al. (1993) Nucl. Acids Res. 21:3653] for DNA strand transfer. The protein was purified to close to homogeneity using methods developed for E. coli RecA isolation.
In this era of coexistence of high-throughput sequencing technologies and serious difficulties in the management of both common and novel infectious syndromes, new techniques which improve the study of micro-organisms is timely. In bacteriology, the most important subjects are bacterial pathogenicity, discovery of the genomic complexity of bacteria, and the epidemiology of antimicrobial resistance traits. From the clinical point of view, genetic testing is flanking phenotypic testing for the assessment of new, difficult to test antibiotic resistance traits, and for correlations with the microbial behaviour in vivo. The demand for faster, comprehensive and highly parallel microbial diagnostics is also cogent even at the basic laboratory level, where the ultimate objective is saving lives. In this setting, DNA microarrays offer a pivotal contribution by allowing performance of hybridization experiments in highly parallel formats, with an increasing reliability. Not only they are useful in deciphering host and microbial pathophysiology, they can also make the difference in the management of prognostic and therapeutic aspects of many diseases. Here, we provide an overview of the current use and the potential of DNA microarrays in clinical bacteriology, and several applications and technical solutions are discussed.
Bacteria ensure the fidelity of genetic inheritance by the coordinated control of chromosome segregation and cell division. Here, we review the molecules and mechanisms that govern the correct subcellular positioning and rapid separation of newly replicated chromosomes and plasmids towards the cell poles and, significantly, the emergence of mitotic-like machineries capable of segregating plasmid DNA. We further describe surprising similarities between proteins involved in DNA partitioning (ParA/ParB) and control of cell division (MinD/MinE), suggesting a mechanism for intracellular positioning common to the two processes. Finally, we discuss the role that the bacterial cytoskeleton plays in DNA partitioning and the missing link between prokaryotes and eukaryotes that is bacterial mechano-chemical motor proteins.
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Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 from Escherichia coli on the synthesis of the coat protein of the RNA-containing phages Qbeta and MS2, on that of an "early" and a "late" enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicate that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.