Although most Escherichia coli strains occur in the mammalian intestine as commensals, some of them, including enterohemorrhagic E. coli (EHEC), are capable of causing disease in humans. The most notorious virulence factors of EHEC are Shiga toxins, encoded by genes located on genomes of lambdoid prophages. Production and release of these toxins is strongly stimulated after the induction of these prophages. Many antibiotics used to treat bacterial infections stimulate induction of Shiga toxin-converting prophages, enhancing severity of the disease symptoms. Hence, treatment with antibiotics is not recommended if infection with EHEC is confirmed or even suspected. In this light, rapid detection of EHEC is crucial, and understanding the mechanisms of prophage induction and phage development in the human intestine is important to facilitate development of procedures preventing or alleviating Shiga toxin-caused diseases.

No abstract

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

No abstract

Development of bacteriophage T4 depends on the physiological state of its host cell. Based on previous studies performed under laboratory conditions with different media determining various growth rates of Escherichia coli, a mathematical model was developed which suggested that phage T4 development cannot proceed efficiently in bacteria growing with a doubling time longer than 160 min. Contrary to this prediction, using a chemostat culture system allowing for culturing E. coli at different growth rates without changes in the medium composition, we found that T4 can yield progeny in host cells growing with a doubling time as long as 21 h. Our results indicate that the actual limiting growth rate of the host culture for the development of phage T4 is about 0.033 h(-1) , corresponding to the doubling time of about 21 h.

Metagenomics approaches and recent improvements in the next‐generation sequencing methods, have become a method of choice in establishing a microbial population structure. Many commercial soil DNA extraction kits are available and due to their efficiency they are replacing traditional extraction protocols. However, differences in the physicochemical properties of soil samples require optimization of DNA extraction techniques for each sample separately. The aim of this study was to compare the efficiency, quality, and diversity of genetic material extracted with the use of commonly used kits. The comparative analysis of microbial community composition, displayed differences in microbial community structure depending on which kit was used. Statistical analysis indicated significant differences in recovery of the genetic material for 24 out of 32 analyzed phyla, and the most pronounced differences were seen for Actinobacteria. Also, diversity indexes and reproducibility of DNA extraction with the use of a given kit, varied among the tested methods. As the extraction protocol may influence the apparent structure of a microbial population, at the beginning of each project many extraction kits should be tested in order to choose one that would yield the most representative results and present the closest view to the actual structure of microbial population.

No abstract

The exo–xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ϕ24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ϕ24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo–xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo–xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.