rDNA Ratio Research Articles

Eukaryotic plankton size structure and species composition in the wetland lakes

Eukaryotic plankton play an important role in the wetland lake ecosystems. However, the size structure and species composition of eukaryotic plankton in shallow wetland lake ecosystems remains largely unexplored. To address this gap, our study employed high-throughput sequencing and quantitative PCR techniques to investigate the size structure (pico-0.7–5 μm and nano-5–20 μm) and species composition of eukaryotic plankton in two shallow wetland lakes in China during August 2016. Our findings reveal that the diversity index values of the pico-size fraction were significantly higher than those of the nano-size fraction. Additionally, there was a significant difference in community structure between these two size fractions. Furthermore, different dominant taxa were associated with each particle size, with certain lineages being enriched exclusively in one of the size fractions. In particular, biomarkers differed between the pico- and nano- fractions. The abundance of the 18S rDNA gene did not differ significantly between the two fractions. However, there was a significant difference in the abundance of gene transcripts. Moreover, the pico- fraction exhibited a significantly higher rRNA: rDNA ratio compared to the nano- fraction. It is important to note that certain taxa had lower absolute abundance in specific samples, despite their higher relative abundance.

Underlying function regulators of anaerobic granular sludge: Starvation and dormancy

Anaerobic granular sludge (AnGS) is the core of anaerobic granular sludge bed system. In this study, the effect and its mechanism of stopping substrate supply on function of AnGS were investigated. The cutoff of exogenous substrate supply triggered AnGS to enter the dormant state. Some methanization microorganisms sporulated. The number and activity of methanization microorganisms based on 16S rDNA and 16S rRNA/16S rDNA ratio declined and stayed at 45.5% and 0.06% (bacteria), 48.7% and 0.39% (archaea) of the initial vegetative value, respectively. The resuming of exogenous substrate supply promoted AnGS to restore the vegetative state. The spores disappeared. The specific methanization activity of AnGS returned to the original level of 35.82 mL-CH4/g-VSS·d, but the delay time for gas production (DTGS) was prolonged from 9.54 to 18.04 h (0–132 d). The dormancy of methanization microorganisms was the main cause for the fluctuation of apparent function and the stability of intrinsic function of AnGS under starvation stress. The dormancy stabilized the structure and sustained the methanization community of AnGS via the reduction of EPS (structure binder/energy reserve) consumption.

Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean

Microbial eukaryotes are key components of the ocean plankton. Yet, our understanding of their community composition and activity in different water layers of the ocean is limited, particularly for picoeukaryotes (0.2–3 µm cell size). Here, we examined the picoeukaryotic communities inhabiting different vertical zones of the tropical and subtropical global ocean: surface, deep chlorophyll maximum, mesopelagic (including the deep scattering layer and oxygen minimum zones), and bathypelagic. Communities were analysed by high-tthroughput sequencing of the 18S rRNA gene (V4 region) as represented by DNA (community structure) and RNA (metabolism), followed by delineation of Operational Taxonomic Units (OTUs) at 99% similarity. We found a stratification of the picoeukaryotic communities along the water column, with assemblages corresponding to the sunlit and dark ocean. Specific taxonomic groups either increased (e.g., Chrysophyceae or Bicosoecida) or decreased (e.g., Dinoflagellata or MAST-3) in abundance with depth. We used the rRNA:rDNA ratio of each OTU as a proxy of metabolic activity. The highest relative activity was found in the mesopelagic layer for most taxonomic groups, and the lowest in the bathypelagic. Altogether, we characterize the change in community structure and metabolic activity of picoeukaryotes with depth in the global ocean, suggesting a hotspot of activity in the mesopelagic.

Metabarcoding reveals that rhizospheric microbiota of Quercus pyrenaica is composed by a relatively small number of bacterial taxa highly abundant

Melojo oak (Quercus pyrenaica Willd.) is a key tree species of Mediterranean forests; however, these forests show an advanced stage of deterioration in the Iberian Peninsula. Plant-associated microorganisms play an essential role improving their host’s fitness, hence, a better understanding of oak rhizospheric microbiome, especially of those active members, could be the first step towards microbiome-based approaches for oak-forest improvement. Here we reported, for the first time, the diversity of total (DNA-based) and potentially active (RNA-based) bacterial communities of different melojo-oak forest formations through pyrosequencing of 16S rRNA gene amplicons. We found that potentially active bacterial communities were as rich and diverse as total bacterial communities, but different in terms of relative abundance patterns in some of the studied areas. Both core microbiomes were dominated by a relatively small percentage of OTUs, most of which showed positive correlation between both libraries. However, the uncoupling between abundance (rDNA) and potential activity (rRNA) for some taxa suggests that the most abundant taxa are not always the most active, and that low-abundance OTUs may have a strong influence on oak’s rhizospheric ecology. Thus, measurement of rRNA:rDNA ratio could be helpful in identifying major players for the development of bacterial bioinoculants.

A rAAV2-producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield.

Recombinant adeno-associated viral vectors (rAAV) are promising therapies for genetic diseases. Although current platforms for recombinant vector production can generate drug material for pre-clinical and clinical studies, rAAV biomanufacturing will eventually face commercial supply challenges if per cell vector productivity and process scalability are not improved. Because considerable efforts have traditionally focused on optimizing rAAV plasmid design, herein we investigate the impact of host cell proteins on vector production to identify proteins that may enhance rAAV yield. Using a rAAV2-GFP-producing Saccharomyces cerevisiae model in combination with the yeast Tet Hughes Collection screening library, we identified 22 gene candidates that improved rAAV DNA replication (rAAV-GFP/18s rDNA ratio) and vector yield (benzonase-resistant rAAV DNA vector genome titer) as high as 6-fold and 15-fold relative to control, respectively. The candidate proteins participate in biological processes such as DNA replication, ribosome biogenesis, and RNA and protein processing. The best five candidates (PRE4, HEM4, TOP2, GPN3, and SDO1) were further screened by generating overexpression mutants in the YPH500 yeast strain. Subsequent clone evaluation was performed to confirm the rAAV-promoting activity of selected candidates under plate-based and bioreactor-controlled fermentation conditions. Digital droplet PCR analysis of cell lysate and AVB resin-purified material confirmed HEM4 and TOP2 overexpression mutants displayed the highest per cell total rAAV DNA productivity (1.6 and 1.7-fold increase over control, respectively) and per cell vector productivity (3 and 4-fold over control, respectively). This evaluation confirmed that overexpression of HEM4 and TOP2 proteins enhanced total and benzonase-resistant rAAV DNA yield. Further studies are needed to understand their mechanism of action and to assess their potential application in molecular strategies for rAAV production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2725, 2019.

Diversity and potential activity patterns of planktonic eukaryotic microbes in a mesoeutrophic coastal area (eastern English Channel).

The diversity of planktonic eukaryotic microbes was studied at a coastal station of the eastern English Channel (EEC) from March 2011 to July 2015 (77 samples) using high throughput sequencing (454-pyrosequencing and Illumina) of the V2-V3 hypervariable region of the 18S SSU rDNA gene. Similar estimations of OTU relative abundance and taxonomic distribution for the dominant higher taxonomic groups (contributing >1% of the total number of OTUs) were observed with the two methods (Kolmogorov-Smirnov p-value = 0.22). Eight super-groups were identified throughout all samples: Alveolata, Stramenopiles, Opisthokonta, Hacrobia, Archeaplastida, Apusozoa, Rhizaria, and Amoebozoa (ordered by decreasing OTU richness). To gain further insight into microbial activity in the EEC, ribosomal RNA was extracted for samples from 2013–2015 (30 samples). Analysis of 18S rDNA and rRNA sequences led to the detection of 696 and 700 OTUs, respectively. Cluster analysis based on OTUs’ abundance indicated three major seasonal groups that were associated to spring, winter/autumn, and summer conditions. The clusters inferred from rRNA data showed a clearer seasonal representation of the community succession than the one based on rDNA. The rRNA/rDNA ratio was used as a proxy for relative cell activity. When all OTUs were considered, the average rRNA:rDNA ratio showed a linear trend around the 1:1 line, suggesting a linear relation between OTU abundance (rDNA) and activity (rRNA). However, this ratio was highly variable over time when considering individual OTUs. Interestingly, the OTU affiliated with P. globosa displayed rRNA:rDNA ratio that allowed to delimit high vs low abundance and high vs low activity periods. It unveiled quite well the Phaeocystis bloom dynamic regarding cell proliferation and activity, and could even be used as early indicator of an upcoming bloom.

P872 BaiCD gene abundance is negatively correlated with Clostridium difficile infection

Clostridium difficile infection (CDI) poses an increased risk to patients with inflammatory bowel disease. Preconditions are colonisation with C. difficile, but also a breakdown of the colonisation resistance, of which secondary bile acids are believed to constitute a decisive component. 7α-dehydroxylation is one of the key steps in primary to secondary bile acid transformation, and required genes have been located in a single bile acid inducible (baiCD) operon in C. scindens as well as in C. hiranonis. The prevalence of these species in human fecal samples—particularly with regard to samples tested positive for C. difficile—and in the setting of fecal microbiota transfer (FMT) in CDI patients has not been studied. A species-specific qPCR for detection of baiCD in C. scindens and C. hiranonis was established. Fecal samples of patients with CDI, toxigenic C. difficile colonisation (TCD), non-toxigenic C. difficile colonisation (NTCD), of C. difficile negative (NC) patients, and of two patients before and after fecal microbiota transfer (FMT) for recurrent CDI (rCDI) were tested for the presence of the baiCD gene cluster. In NC samples, the prevalence of the baiCD gene cluster was significantly higher than in CDI samples, with 72.5% (100/138) vs. 35.9% (23/64; p < 0.0001), respectively. No differences were seen between NC compared with NTCD samples or TCD samples. Both rCDI patients were baiCD negative at baseline, but one patient turned positive after successful FMT from a baiCD positive donor. BaiCD/16S rDNA ratio calculation showed no significant difference between the relative abundances in both groups (NC vs. CDI; p = 0.3244). The median of the NC group was 0.006% (IQR 0.0005%-0.376%) and for the CDI group 0.009% (IQR 0.0002%-7.8%). In addition, stool consistency did not measurably influence the relative abundance of bacterial species in this context of complex microbial communities. BaiCD gene positive species are reduced in fecal samples of patients with C. difficile infection as compared with asymptomatic carriers or C. difficile negative fecal samples. Furthermore, we present a case of baiCD gene positivity observed after successful fecal microbiota transfer for recurrent CDI.

Total and Active Core Microbiomes of Melojo Oak Are Composed by a Minority of Bacterial Taxa Highly Abundant

Melojo oak (Quercus pyrenaica Willd.) is a key tree species of Mediterranean forests with high landscape value in the Iberian Peninsula. However, these forests exhibit an advanced stage of deterioration and their distribution area has been seriously reduced over the last decades. Plant-associated microorganisms play an essential role improving their host’s fitness, hence, a better understanding of oak rhizospheric microbiome, especially of those active members, could be the first step towards microbiome-based approaches for the improvement of the survival and growth of oak trees. Here we reported, for the first time, the diversity of total (DNA-based) and potentially active (RNA-based) bacterial communities of three types of Quercus pyrenaica Willd. forest formations through 454 pyrosequencing of 16S rDNA amplicons, and defined the rhizospheric core microbiome of Q. pyrenaica at Sierra Nevada National and Natural Park during spring season. In addition rRNA:rDNA ratio was calculated as a proxy of the contribution of each core taxon to the rhizosphere environment. We found that potentially active bacterial communities were as rich and diverse as DNA-based populations, but different in terms of relative abundance patterns in some of the studied areas. Both microbiomes were dominated by a relatively small percentage of OTUs, most of which showed positive correlation between both libraries. However, the uncoupling between abundance (rDNA) and potential activity (rRNA) for some taxa suggests that the most abundant taxa are not always the most active, and that low-abundance OTUs may have a strong influence on oak’s rhizospheric ecology. Thus, measurement of rRNA:rDNA ratio could be helpful in characterizing rhizospheric bacterial communities, their potential involvement in the functioning of the soil ecosystem and, therefore, in identifying major bacterial players for the development of microbiome-based strategies for future afforestation tasks.

Single Cell Analysis Linking Ribosomal (r)DNA and rRNA Copy Numbers to Cell Size and Growth Rate Provides Insights into Molecular Protistan Ecology.

Ribosomal (r)RNA and rDNA have been golden molecular markers in microbial ecology. However, it remains poorly understood how ribotype copy number (CN)‐based characteristics are linked with diversity, abundance, and activity of protist populations and communities observed at organismal levels. Here, we applied a single‐cell approach to quantify ribotype CNs in two ciliate species reared at different temperatures. We found that in actively growing cells, the per‐cell rDNA and rRNA CNs scaled with cell volume (CV) to 0.44 and 0.58 powers, respectively. The modeled rDNA and rRNA concentrations thus appear to be much higher in smaller than in larger cells. The observed rRNA:rDNA ratio scaled with CV 0.14. The maximum growth rate could be well predicted by a combination of per‐cell ribotype CN and temperature. Our empirical data and modeling on single‐cell ribotype scaling are in agreement with both the metabolic theory of ecology and the growth rate hypothesis, providing a quantitative framework for linking cellular rDNA and rRNA CNs with body size, growth (activity), and biomass stoichiometry. This study also demonstrates that the expression rate of rRNA genes is constrained by cell size, and favors biomass rather than abundance‐based interpretation of quantitative ribotype data in population and community ecology of protists.

Quantifyingin situgrowth rate of a filamentous bacterial species in activated sludge using rRNA:rDNA ratio

If the in situ growth rate of filamentous bacteria in activated sludge can be quantified, researchers can more accurately assess the effect of operating conditions on the growth of filaments and improve the mathematical modeling of filamentous bulking. We developed a method to quantify the in situ specific growth rate of Sphaerotilus natans (a model filament) in activated sludge using the species-specific 16S rRNA:rDNA ratio. Primers targeting the 16S rRNA of S. natans were designed, and real-time PCR and RT-PCR were used to quantify DNA and RNA levels of S. natans, respectively. A positive linear relationship was found between the rRNA:rDNA ratio (from 440 to 4500) and the specific growth rate of S. natans (from 0.036 to 0.172 h-1) using chemostat experiments. The in situ growth rates of S. natans in activated sludge samples from three water reclamation facilities were quantified, illustrating how the approach can be applied in a complex environment such as activated sludge.

The influence of environmental factors on protistan microorganisms in grassland soils along a land-use gradient

In this study, we investigated the effect of land use intensity, soil parameters and vegetation on protistan communities in grassland soils. We performed qualitative (T-RFLP) and quantitative (qPCR) analyses using primers specifically targeting the 18S rRNA gene for all Eukarya and for two common flagellate groups, i.e. the Chrysophyceae and the Kinetoplastea. Both approaches were applied to extracted soil DNA and RNA, in order to distinguish between the potentially active protists (i.e. RNA pool) and the total protistan communities, including potentially inactive and encysted cells (i.e. DNA pool). Several environmental determinants such as site, soil parameters and vegetation had an impact on the T-RFLP community profiles and the abundance of the quantified 18S rRNA genes. Correlating factors often differed between quantitative (qPCR) and qualitative (T-RFLP) approaches. For instance the Chrysophyceae/Eukarya 18S rDNA ratio as determined by qPCR correlated with the C/N ratio, whereas the community composition based on T-RLFP analysis was not affected indicating that both methods taken together provide a more complete picture of the parameters driving protist diversity. Moreover, distinct T-RFs were obtained, which could serve as potential indicators for either active organisms or environmental conditions like water content. While site was the main determinant across all investigated exploratories, land use seemed to be of minor importance for structuring protist communities. The impact of other parameters differed between the target groups, e.g. Kinetoplastea reacted on changes to water content on all sites, whereas Chrysophyceae were only affected in the Schorfheide. Finally, in most cases different responses were observed on RNA- and DNA-level, respectively. Vegetation for instance influenced the two flagellate groups only at the DNA-level across all sites. Future studies should thus include different protistan groups and also distinguish between active and inactive cells, in order to reveal causal shifts in community composition and abundance in agriculturally used systems.

Evidence for an active rare biosphere within freshwater protists community.

Studies on the active rare biosphere at the RNA level are mainly focused on Bacteria and Archaea and fail to include the protists, which are involved in the main biogeochemical cycles of the earth. In this study, the richness, composition and activity of the rare protistan biosphere were determined from a temporal survey of two lakes by pyrosequencing. In these ecosystems, the always rare OTUs represented 77.2% of the total OTUs and 76.6% of the phylogenetic diversity. From the various phylogenetic indices computed, the phylogenetic units (PUs) constituted exclusively by always rare OTUs were discriminated from the other PUs. Therefore, the rare biosphere included mainly taxa that are distant from the reference databases compared to the dominant ones. In addition, the rarest OTUs represented 59.8% of the active biosphere depicted by rRNA and the activity (rRNA:rDNA ratio) increased with the rarity. The high rRNA:rDNA ratio determined in the rare fraction highlights that some protists were active at low abundances and contribute to ecosystem functioning. Interestingly, the always rare and active OTUs were characterized by seasonal changes in relation with the main environmental parameters measured. In conclusion, the rare eukaryotes represent an active, dynamic and overlooked fraction in the lacustrine ecosystems.

Identification of nitrifiers and nitrification performance in drinking water biological activated carbon (BAC) filtration

In this study laboratory scale biological activated carbon (BAC) columns were operated with water taken from a surface water reservoir in Istanbul. The aim was to evaluate the efficiency of nitrification in columns packed with two different granular activated carbon grades (open superstructure/chemically activated and closed superstructure/steam activated carbon) and to examine the probable beneficial effect of pre-ozonation. The occurrence and diversity of ammonia-oxidizing bacteria were investigated using 16S rDNA and amoA gene based molecular techniques. Nearly complete removal of NH 4 +-N was achieved by nitrification in both carbon types. The nitrification efficiency did not change in columns fed with ozonated water. However, the type of feed (either raw or ozonated) played a more important role than the type of GAC with respect to the dominance of nitrifier species in BAC columns. In biofilters ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were most closely related to Nitrosomonas spp. and Nitrospira spp. as determined by cloning and slot-blot analysis, respectively. The fraction of the AOB population in the biomass was high as detected by real-time PCR. The amoA/16S rDNA ratio varied from 28.7% to 2.1% along the depth of filters. In spite of similar removal efficiencies, BAC columns fed with ozonated water harbored different types of AOB than columns that were receiving raw water.

Temporal changes in bacterial rRNA and rRNA genes in Delaware (USA) coastal waters

The diversity and abundance of bacterial 16S rRNA genes (rDNA) and associated 16S rRNA were assessed to examine community structure and potential activity in a 2 yr seasonal study of marine surface waters along the Delaware (USA) coast. The majority of the ribotypes appearing as rRNA or rDNA were present at least 50% of the time, and their variation over time was less than that of most biotic and abiotic factors. Even though there were no predictable seasonal patterns in com- munity structure, environmental parameters explained between 40 and 50% of the observed varia- tion in community structure. In general, summer communities correlated positively with temperature, light, bacterial production, and phosphate levels, while winter communities correlated with dissolved organic carbon concentrations. The abundance of 3 select ribotypes (SAR11, a Cytophaga subgroup, SAR116-like) varied some over time as measured by quantitative PCR (qPCR). The SAR11 clade was 10 times more abundant than the other 2 groups. However, the 16S rRNA to rDNA ratios, as mea- sured by qPCR and reverse transcription-qPCR, varied greatly over time and were highest in the SAR11 population and the Cytophaga subgroup. The rRNA to rDNA ratio of the Cytophaga subgroup and SAR116-like group significantly correlated with some environmental parameters. Taken together, these results suggest that the community composition of the major members does not change much over time, but individual members do vary in abundance and activity and can be influ- enced by environmental factors.

Bioaugmentation with the resin acid-degrading bacterium Zoogloea resiniphila DhA-35 to counteract pH stress in an aerated lagoon treating pulp and paper mill effluent

Efficient and reliable removal of resin acids such as dehydroabietic acid (DhA), which are the major toxicants in pulp and paper mill effluents and form pitch interfering with papermaking, is critically important to prevent toxicity discharge and failure of paper machines. Low- and high-pH stresses sometimes occur in effluent treatment systems due to the use of large amounts of acids and alkalines in the pulping processes. We found that both low- and high-pH stresses (pH 3 and 10, respectively) decreased the removal of total organic carbon and completely inhibited the removal of DhA by the biomass of an aerated lagoon treating pulp mill effluent. The pH stresses caused changes in the bacterial community structure as assessed by ribosomal intergenic spacer length polymorphism. The pH stresses greatly reduced the indigenous DhA-degrading populations in the lagoon community. Bioaugmentation with Zoogloea resiniphila DhA-35, a DhA-degrading bacterium originally isolated from a pulp mill treatment system, restored the DhA removal by both the low- and high-pH-stressed lagoon biomass. This bacterium was persistent after introduction into the lagoon microbial community, and its cellular rRNA:rDNA ratio increased during the period of DhA removal. The introduction of strain DhA-35 changed the microbial community structure, but did not adversely affect the TOC removal by the community. This study suggests that it is feasible and potentially useful to use bioaugmentation with resin-acid-degrading bacteria such as DhA-35 to restore and enhance resin acid removal by aerated lagoon microbial communities.

Population dynamics and metabolic activity of Pseudomonas abietaniphila BKME-9 within pulp mill wastewater microbial communities assayed by competitive PCR and RT-PCR

Resin acids contribute to the acute toxicity of pulp and paper mill effluent. We monitored the abundance and metabolic activity of the resin acid degrader, Pseudomonas abietaniphila BKME-9, within complex communities of laboratory-scale effluent treatment systems using competitive PCR and competitive RT-PCR, targeting the 16S rDNA and rRNA. In continuous culture, a positive linear relationship was found between the rRNA:rDNA ratio and specific growth rate of BKME-9. In batch culture, the rRNA:rDNA ratio peaked briefly during early exponential growth of BKME-9. During continuous growth of an activated sludge community inoculated with BKME-9, the rRNA:rDNA ratio of BKME-9 decreased when the community was stressed with an alkaline pH shock. After the pH shock, the metabolic activity and population of BKME-9 recovered concomitantly with recovery of resin acid removal activity in the system. Thus, BKME-9 has characteristics that could be useful for bioaugmentation of biotreatment systems, and it is possible to monitor the population and activity of BKME-9 in such complex systems.

Population dynamics and metabolic activity of Pseudomonas abietaniphila BKME-9 within pulp mill wastewater microbial communities assayed by competitive PCR and RT-PCR

Resin acids contribute to the acute toxicity of pulp and paper mill effluent. We monitored the abundance and metabolic activity of the resin acid degrader, Pseudomonas abietaniphila BKME-9, within complex communities of laboratory-scale effluent treatment systems using competitive PCR and competitive RT-PCR, targeting the 16S rDNA and rRNA. In continuous culture, a positive linear relationship was found between the rRNA:rDNA ratio and specific growth rate of BKME-9. In batch culture, the rRNA:rDNA ratio peaked briefly during early exponential growth of BKME-9. During continuous growth of an activated sludge community inoculated with BKME-9, the rRNA:rDNA ratio of BKME-9 decreased when the community was stressed with an alkaline pH shock. After the pH shock, the metabolic activity and population of BKME-9 recovered concomitantly with recovery of resin acid removal activity in the system. Thus, BKME-9 has characteristics that could be useful for bioaugmentation of biotreatment systems, and it is possible to monitor the population and activity of BKME-9 in such complex systems.

Monitoring the Size and Metabolic Activity of the Bacterial Community during Biostimulation of Fuel-Contaminated Soil using Competitive PCR and RT-PCR.

Efforts to understand and improve soil bioremediation are limited by our ability to determine how treatment variables affect microbial communities. A method was developed to monitor the density and metabolic activity of the total bacterial community in soil. This method was used to monitor the bacterial community in microcosms of Arctic soil after addition of N plus P to stimulate biodegradation of hydrocarbon contaminants. During 29 days of incubation, the total petroleum hydrocarbon level in the soil was reduced from 850 to 360 mg/g of soil. DNA and RNA were extracted from soil using a bead beating method, purified by ammonium acetate precipitation, and assayed by competitive PCR and RT-PCR assays with universal bacterial primers. The copy number of 16S rDNA in the soil microbial community was relatively stable and ranged from 1.7 x 109 to 4.5 x 109/g of soil throughout the incubation. The copy number of 16S rRNA changed substantially and ranged from 5.6 x 1010 to 1.0 x 1012/g of soil. The rRNA:rDNA ratio was highest during the phase of fastest hydrocarbon biodegradation. These results suggest that the treatment to stimulate hydrocarbon biodegradation did not substantially change the density of the bacterial community but did transiently increase its overall metabolic activity.

Cu(II)/H2O2-induced DNA damage is enhanced by packaging of DNA as a nucleosome.

Copper is a physiologically important, redox-active metal that may be involved in endogenous DNA damage and mutagenesis. To understand the factors that affect the location and quantity of copper-induced oxidative DNA damage in cells, we used the 5S rDNA nucleosome as a model to assess the effect of chromatin structure on DNA damage produced by Cu(II)/H2O2. Packaging of DNA into a nucleosome increased the extent of Cu(II)/H2O2-induced strand breaks by a factor of 2, while the extent of base lesions sensitive to Fpg and endo III glycosylases increased 8-fold. We also observed that Cu(II)/H2O2 caused slightly more strand breaks than base lesions in isolated 5S rDNA (ratio of base lesions to strand breaks of approximately 0.6), while base lesions outnumbered strand breaks by a factor of 3-4 when the DNA was incorporated into a nucleosome. Apart from several sites of enhanced or diminished DNA damage, there were no major changes in the sequence selectivity of Cu(II)/H2O2, and there was no apparent footprinting effect associated with nucleosome structure, such as that observed with the Fe(II)-EDTA complex. Possible mechanisms for explaining these observations include (1) an increase in Cu(II) concentration in the vicinity of nucleosomal DNA caused by binding of Cu to histone proteins or (2) increased reactivity or accessibility of nucleobases caused by DNA conformational changes associated with nucleosome structure. The enhancement of Cu(II)/H2O2-induced DNA damage in nucleosomes stands in contrast to the protective effect afforded DNA by proteins in chromatin against radiation-induced DNA damage.

Quantitation of the Population Size and Metabolic Activity of a Resin Acid Degrading Bacterium in Activated Sludge Using Slot-Blot Hybridization to Measure the rRNA:rDNA Ratio.

The 16S rRNA:rDNA ratio is a useful parameter for measuring metabolic activity of a selected member of a complex microbial community, as in pulp effluent activated sludge systems. The RNA:DNA ratio of Sphingomonas sp. DhA-33, previously isolated from a sequencing batch reactor treating pulp mill effluent, is positively correlated with its growth rate (µ) under steady-state conditions. DhA-33 was grown in a chemostat with growth rates ranging from 0.04 to 0.15 cell divisions per hour. DhA-33 was also able to degrade dehydroabietic acid in bleached kraft mill effluent (BKME) plus mineral medium in batch culture. Slot-blot hybridization with radioactively labeled species-specific oligonucleotide probes for 16S rRNA and 16S rDNA was used to measure rRNA, rDNA, and the RNA:DNA ratio of this strain when in a mixed sludge community. An increase in DhA-33 rDNA indicated growth of DhA-33 within the community. The RNA:DNA ratio of DhA-33 increased sharply during exponential growth and declined as cells entered stationary phase. The RNA:DNA ratio decreased earlier and faster in DhA- 33/sludge co-cultures than in DhA-33 pure cultures, presumably due to an earlier depletion of nutrients. The species-specific quantification of the RNA:DNA ratio makes it possible to estimate the metabolic activity of selected members of a microbial community in situ.