Elucidation of a non-thermal mechanism for DNA/RNA fragmentation and protein degradation when using Lyse-It.

Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.

Differential decay of human faecal Bacteroides in marine and freshwater.

Genetic markers from Bacteroides and other faecal bacteria are being tested for inclusion in regulations to quantify aquatic faecal contamination and estimate public health risk. For the method to be used quantitatively across environments, persistence and decay of markers must be understood. We measured concentrations of contaminant molecular markers targeting Enterococcus and Bacteroides spp. in marine and freshwater microcosms spiked with human sewage and exposed to either sunlight or dark treatments. We used Bayesian statistics with a delayed Chick-Watson model to estimate kinetic parameters for target decay. DNA- and RNA-based targets decayed at approximately the same rate. Molecular markers persisted (could be detected) longer in marine water. Sunlight increased the decay rates of cultured indicators more than those of molecular markers; sunlight also limited persistence of molecular markers. Within each treatment, Bacteroides markers had similar decay profiles, but some Bacteroides markers significantly differed in decay rates. The role of extracellular DNA in persistence appeared unimportant in the microcosms. Because conditions were controlled, microcosms allowed the effects of specific environmental variables on marker persistence and decay to be measured. While marker decay profiles in more complex environments would be expected to vary from those observed here, the differences we measured suggest that water matrix is an important factor affecting quantitative source tracking and microbial risk assessment applications.

Responses of the bacterial community to chronic gamma radiation in a rice paddy ecosystem.

PURPOSE: Public concern about radiation protection of the environment and non-human species is increasing. The potential impact of chronic gamma radiation on the bacterial community in a rice paddy ecosystem was investigated. MATERIALS AND METHODS: Paddy field microcosm samples were continuously exposed at a dose rate of 1 Gy day(-1) for five days after which the bacterial community structures in them were compared with those of unirradiated control samples by denaturing gradient gel electrophoresis (DGGE) of the 16S ribosomal ribonucleic acid (rRNA) gene. Dissolved ions in ponding waters of the microcosm samples were determined by ion chromatography. RESULTS: The DGGE band profiles were clearly different between the control and exposure samples. Sequencing of selected DGGE bands revealed that a specific species of the genus Clostridium and of the genus Massilia were grown under radiation exposure. Additionally, the concentrations of sulphate ion in the ponding water of the microcosm samples increased under radiation exposure. Concentrations of Na(+), Ca(2+), and F(-) inversely decreased. CONCLUSIONS: The data presented suggest that chronic gamma radiation affects the bacterial community structure and material cycles in the rice paddy ecosystem.

The effects of high-power microwaves on the ultrastructure of Bacillus subtilis.

AIMS: To investigate the microbicidal mechanisms of high-power microwave (2.0 kW) irradiation on Bacillus subtilis and to determine the effect of this procedure on the ultrastructure of the cell wall. METHODS AND RESULTS: We performed viability test, examined cells using transmission electron microscopy (TEM), and measured the release of intracellular proteins and nucleic acids. The inactivation rate of B. subtilis by 2.0-kW microwave irradiation was higher than that of a domestic microwave (0.5 kW). Few proteins were released from either microwaved or boiled cells. However, the leakage of nucleic acids from 2.0-kW-microwaved cells was significantly higher than that of 0.5-kW-microwaved or boiled cells. Therefore, we examined ultrastructural alterations of microwaved or boiled cells to analyse the pattern of release of cytoplasmic contents. Although boiled cells did not show any ultrastructural changes on TEM, 2.0-kW-microwaved cells showed disruption of the cell wall. CONCLUSION: The microbicidal mechanisms of 2.0-kW microwave irradiation include damage to the microbial cell wall, breakage of the genomic DNA, and thermal coagulation of cytoplasmic proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: TEM images showed that the cytoplasmic protein aggregation and cell envelope damage by microwave irradiation were different from the ultrastructural changes observed after boiling.

Importance of considering injured microorganisms in sterilization validation.

Disinfection or sterilization treatment by heating, irradiation, or chemicals can cause injury to microorganisms at sublethal levels. Microbial injury is the inability to grow under conditions suitable for the uninjured microorganisms. This inability of injured microorganisms to grow is explained in terms of more complex or different nutritional requirements or in terms of increased sensitivity to environmental conditions such as incubation conditions (time or temperature) or to chemical agents such as halogen compounds. Injured microorganisms can be distinguished from those that are dead or mutated by their ability to regain normal physiological activity when placed in appropriate conditions for cultivation. The return to normal physiological function has been termed repair. The extent and severity of sublethal injury, the mechanisms of injury, and the mechanisms and degree of recovery vary with the sterilization procedures, the species, the strains, the condition of the microorganism, and the methods of repair. Injury to spore formers has been detected at different stages of the spore cycle. The sites of injury include damage to enzymes, membrane disruption, and/or damage to DNA or RNA. Information on the sublethal injury and recovery of microorganisms is very important in evaluating sterilization/disinfection procedures. This paper supplies academic as well as practical information dealing with the repair, and detection of injured microorganisms for performing reproducible sterilization validation.

Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics.

UV light-induced RNA photocrosslinks are formed at a limited number of specific sites in the Escherichia coli and in other eubacterial 16 S rRNAs. To determine if unusually favorable internucleotide geometries could explain the restricted crosslinking patterns, parameters describing the internucleotide geometries were calculated from the Thermus thermophilus 30 S subunit X-ray structure and compared to crosslinking frequencies. Significant structural adjustments between the nucleotide pairs usually are needed for crosslinking. Correlations between the crosslinking frequencies and the geometrical parameters indicate that nucleotide pairs closer to the orientation needed for photoreaction have higher crosslinking frequencies. These data are consistent with transient conformational changes during crosslink formation in which the arrangements needed for photochemical reaction are attained during the electronic excitation times. The average structural rearrangement for UVA-4-thiouridine (s4U)-induced crosslinking is larger than that for UVB or UVC-induced crosslinking; this is associated with the longer excitation time for s4U and is also consistent with transient conformational changes. The geometrical parameters do not completely predict the crosslinking frequencies, implicating other aspects of the tertiary structure or conformational flexibility in determining the frequencies and the locations of the crosslinking sites. The majority of the UVB/C and UVA-s4U-induced crosslinks are located in four regions in the 30 S subunit, within or at the ends of RNA helix 34, in the tRNA P-site, in the distal end of helix 28 and in the helix 19/helix 27 region. These regions are implicated in different aspects of tRNA accommodation, translocation and in the termination reaction. These results show that photocrosslinking is an indicator for sites where there is internucleotide conformational flexibility and these sites are largely restricted to parts of the 30 S subunit associated with ribosome function.

Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA.

The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome.

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding.

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.

Action spectra for UV-light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome.

UV irradiation induces intramolecular crosslinks in ribosomal RNA in the ribosome. These crosslinks occur between nucleotides distant in primary sequence and they are specific, limited in number and have crosslinking efficiencies sufficient to allow their use in monitoring conformational changes. In this work, the frequency of crosslinking for eight 16S rRNA crosslinks was determined as a function of wavelength of irradiation. For six of the crosslinks, the action spectra correspond to the absorption spectra of at least one of the participating nucleotides. For a crosslink between nucleotides C967 and C1400 the maximum frequency of crosslinking occurs at wavelengths blue-shifted from the absorbance maximum of cytidine and for a crosslink between C1402 and C1501 the maximum frequency of crosslinking is red-shifted. Photoreversal of the crosslinks was also studied by deproteinizing crosslinked RNA under mild conditions and then re-irradiating it with specific wavelengths under conditions in which the crosslinks were reversed but not formed. The different crosslinks exhibit significantly different extents of photoreversal versus wavelength profiles. The differences in the crosslinking action spectra can be accounted for in the absorbance spectra of the nucleotides that are involved in the crosslink as well as by the photoreversal action spectra.

Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli.

tRNA sulfurtransferase activity was assayed in Escherichia coli cell extracts obtained from bacterial suspensions exposed to a sub-lethal dose of ultraviolet-A radiation (fluence 148 kJ m(-2)) imparted at a low fluence rate (41 W m(-2)). We found that the irradiation reduced the enzymatic activity to one fourth of the control value, indicating that ultraviolet-A exposure inhibits the synthesis of 4-thiouridine, the most abundant thionucleoside in E. coli tRNA. Changes in the tRNA content of 4-thiouridine and its derived photoproduct 5-(4'-pyrimidin 2'-one) cytosine were studied in bacteria growing under ultraviolet-A irradiation. In these conditions the accumulation of photoproduct was limited, and the kinetics of this process was non-coincident with disappearance of 4-thiouridine. The results, which are compatible with the fact that ultraviolet-A induces an inhibition of the 4-thiouridine synthesis, suggest that the effect of radiation on tRNA modification is relevant to tRNA photo-inactivation in growing bacteria.

Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae.

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.

Three-dimensional folding of the tRNA-like domain of Escherichia coli tmRNA.

UV irradiation of Escherichia coli tmRNA both on and off the ribosome induced covalent cross-links between its 3'- and its 5'-terminal segments. Cross-linking was unaffected in a molecule that lacked the tag-peptide codon region and pseudoknots 2, 3, and 4. Intact and truncated cross-linked tmRNAs were aminoacylated as efficiently as the respective nonirradiated molecules, suggesting that the added UV-induced bonds did not disturb tmRNA conformation. Using RNase H digestion followed by primer extension with reverse transcriptase, two cross-linked sites were identified within the tRNA-like region of tmRNA. The first was formed between nucleotides U9/U10 near the 5' end and nucleotides C346/U347 in the T loop. The second cross-link involved residues at positions 25-28 and 326-329 within helix 2a. Together with comparative sequence analysis, these findings yielded a three-dimensional model of the tRNA-like domain of E. coli tmRNA. Despite significant reduction of the D domain and the proximity of U9/U10 and C346/U347, the model closely resembles the L-shaped structure of canonical tRNA.

UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry.

Sixteen long-range crosslinks are induced in Escherichia coli 16S rRNA by far-UV irradiation. Crosslinking patterns in two other organisms, Bacillus subtilis and Thermus aquaticus, were investigated to determine if the number and location of crosslinks in E.coli occur because of unusually photoreactive nucleotides at particular locations in the rRNA sequence. Thirteen long-range crosslinks in B.subtilis and 15 long-range crosslinks in T.aquaticus were detected by gel electrophoresis and 10 crosslinks in each organism were identified completely by reverse transcription analysis. Of the 10 identified crosslinks in B.subtilis, eight correspond exactly to E.coli crosslinks and two crosslinks are formed close to sites of crosslinks in E.coli. Of the 10 identified crosslinks in T.aquaticus, five correspond exactly to E.coli crosslinks, three are formed close to E.coli crosslinking sites, one crosslink corresponds to a UV laser irradiation-induced crosslink in E.coli and the last is not seen in E.coli. The overall similarity of crosslink positions in the three organisms suggests that the crosslinks arise from tertiary interactions that are highly conserved but with differences in detail in some regions.

Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure.

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.

Characterization of U6 snRNA-protein interactions.

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.

Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30 S ribosomal subunit.

Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967xC1400 completely, increases the frequency of cross-link C1402x1501 twofold, and decreases the cross-link G894xU244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934xU1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect.

Three-dimensional placement of the conserved 530 loop of 16 S rRNA and of its neighboring components in the 30 S subunit.

Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved. Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding. Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits. The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base. The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure.

rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37 degrees C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1, 400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121 degrees C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80 degrees C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

Magnetic field exposure enhances mRNA expression of sigma 32 in E. coli.

The mechanism of interaction between weak electromagnetic fields and cells is not understood. As a result, the health effect(s) induced by exposure to these fields remains unclear. In addition to questions relating to the site of initial magnetic field (MF) interactions, the nature of the cell's response to these perturbations is also unclear. We examined the hypothesis that the cells respond to MFs in a manner similar to other environmental stressors such as heat. Using the bacterium Escherichia coli, we examined the mRNA levels of sigma 32, a protein that interacts with RNA polymerase to help it recognize a variety of stress promoters in the cell. Our data show that the intracellular level of sigma 32 mRNA is enhanced following a 15-min exposure to a 60 Hz, 1.1 mT magnetic field.