The ecological roles of bacterial populations in the surface sediments of coastal lagoon environments in Japan as revealed by quantification and qualification of 16S rDNA.

Based on quantification and qualification of bacterial 16S rDNA, we verified the bacterial ecological characteristics of surface sediments of Lakes Shinji and Nakaumi, which are representative of coastal lagoons in Japan. Quantification and qualification of the 16S rDNA sequences was carried out using real time polymerase chain reaction and polymerase chain reaction denaturing gradient gel electrophoresis and non-metric multidimensional scaling, respectively. The results revealed that the copy number per gram of sediment ranged from 8.33 × 10(8) (Lake Nakaumi) to 1.69 × 10(11) (Honjo area), suggesting that bacterial carbon contributed only 0.05-9.64 % of the total carbon content in the samples. Compared with other aquatic environments, these results indicate that sedimentary bacteria are not likely to be important transporters of nutrients to higher trophic levels, or to act as carbon sinks in the lagoons. The bacterial compositions of Lake Shinji and Lake Nakaumi and the Honjo area were primarily influenced by sediment grain sizes and salinity, respectively. Statistical comparisons of the environmental properties suggested that the areas that were oxygen-abundant (Lake Shinji) and at a higher temperature (Honjo area) presented efficient organic matter degradation. The 16S rDNA copy number per gram of carbon and nitrogen showed the same tendency. Consequently, the primary roles of bacteria were degradation and preservation of organic materials, and this was affected by oxygen and temperature. These roles were supported by the bacterial diversity rather than the differences in the community compositions of the sedimentary bacteria in these coastal lagoons.

Analysis of bacterial populations in the environment using two-dimensional gel electrophoresis of genomic DNA and complementary DNA.

Two-dimensional gel electrophoresis (2-DGE) mapping of genomic DNA and complementary DNA (cDNA) amplicons was attempted to analyze total and active bacterial populations within soil and activated sludge samples. Distinct differences in the number and species of bacterial populations and those that were metabolically active at the time of sampling were visually observed especially for the soil community. Statistical analyses and sequencing based on the 2-DGE data further revealed the relationships between total and active bacterial populations within each community. This high-resolution technique would be useful for obtaining a better understanding of bacterial population structures in the environment.

Bacterial community structure analysis of sediment in the Sagami River, Japan using a rapid approach based on two-dimensional DNA gel electrophoresis mapping with selective primer pairs.

A rapid approach based on two-dimensional DNA gel electrophroesis (2-DGE) mapping with selective primer pairs was employed to analyze bacterial community structure in sediments from upstream, midstream and downstream of Sagami River in Japan. The 2-DGE maps indicated that Alpha- and Delta-proteobacteria were major bacterial populations in the upstream and midstream sediments. Further bacterial community structure analysis showed that richness proportion of Alpha- and Delta-proteobacterial groups reflected a trend toward decreasing from the upstream to downstream sediments. The biomass proportion of bacterial populations in the midstream sediment showed a significantly difference from that in the other sediments, suggesting that there may be an environmental pressure on the midstream bacterial community. Lorenz curves, together with Gini coefficients were successfully applied to the 2-DGE mapping data for resolving evenness of bacterial populations, and showed that the plotted curve from high-resolution 2-DGE mapping became less linear and more an exponential function than that of the 1-DGE methods such as chain length analysis and denaturing gradient gel electrophoresis, suggesting that the 2-DGE mapping may achieve a more detailed evaluation of bacterial community. In conclusion, the 2-DGE mapping combined with the selective primer pairs enables bacterial community structure analysis in river sediment and thus it can also monitor sediment pollution based on the change of bacterial community structure.

Identification and validation of suitable reference genes for quantitative expression of xylA and xylE genes in Pseudomonas putida mt-2.

Reference genes are used to normalize target genes for relative quantification in gene expression studies. However, different experimental conditions may affect the expression of reference genes, which could lead to erroneous quantitative results. In this study, we performed real-time polymerase chain to investigate the expression of eight reference genes (rpoN, rpoD, dbhA, phaF, 16S rRNA, gst, lexA, and atkA) in Pseudomonas putida mt-2 during degradation of p-xylene. According to their expression stability, geNorm software analysis revealed that rpoN, rpoD, 16S rRNA, and atkA were suitable reference genes with highly stable expression, whereas phaF and dbhA were not suitable due to unstable expression. When normalized either to phaF or dbhA, xylA and xylE expression were significantly different compared to the expression levels normalized with the normalization factor (NF(4)) obtained from the four most stable reference genes (rpoN, -rpoD, -16S rRNA, and -atkA). The use of unstably expressing reference genes resulted in an over- or underestimation of target gene expression, a delay in maximal gene expression, and an increase in gene expression in the absence of inducer. While experimental results indicated that the relative maximum expression of xylA and xylE occurred at different times, unstable reference genes indicated that the maximum expression occurred at the same time. Our study indicates that a valid set of reference genes covering a broad expression range is recommended to accurately normalize and quantify the relative expression levels of the target gene(s) transcripts in many microbial processes.

Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples.

Real-time quantitative PCR (RTQ-PCR) was used to quantify the bacterial target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in presence of liquid nitrogen and hot detergent SDS based enzymatic lysis). For the purpose of our study, pure culture of Bacillus cereus (model organism), sterilized soil seeded with a known amount of B. cereus (model soil system) and samples from woodland and grassland (environmental samples) were chosen to extract DNA by three different protocols. The extracted DNA was then quantified by RTQ-PCR using 16S rDNA specific universal bacterial primers. The standard curve used for the quantification by RTQ-PCR was linear and revealed a strong linear relationship (r(2)=0.9968) with a higher amplification efficiency, e5=1.02. High resolution gel electrophoresis was also carried out to observe the effect of these extraction methods on diversity analysis. For the model soil system, the liquid nitrogen method showed the highest target DNA copy number (1.3 x 10(9) copies/microl). However, for both the environmental samples, the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (5.38 x 10(9) and 4.01 x 10(8) copies/ml for woodland and grassland respectively), high yield (6.4 microg/g and 1.76 microg/g of soil for woodland and grassland respectively) and different band patterns on high resolution gel electrophoresis suggesting an overall high extraction efficiency. This difference in the extraction efficiency between the model soil system and environmental samples may be attributed to different affinity of seeded and native DNA to soil particles.

Interference of contaminating DNA in the quantification of a toluene-induced tod gene in Pseudomonas putida.

To determine the extent of interference of co-extracted DNA contamination in the quantification of the tod gene transcript, two different concentrations of RNA (high, 500 ng/microl; low, 250 ng/microl) from a toluene-induced culture of Pseudomonas putida were treated with different amounts of DNase (2, 4, 6 and 8 U) and incubated for 30 and 60 min. The highly sensitive and reproducible TaqMan system was used to quantify the transcript of the tod gene, the tod gene in contaminating DNA and the 16S rRNA gene in DNase-treated RNA samples. For the high RNA concentration, the shorter incubation time (30 min) lowered the level of contaminating DNA as evidenced by the presence of 2.5 x 10(6) copies of the tod gene before treatment to 1.4 x 10(5) copies/microl (8 U), whereas, irrespective of the DNase units used, the longer incubation time (60 min) considerably lowered the level of DNA contamination (2.5 x 10(6) to 6.5 x 10(2) copies of the tod gene/microl). However, for the low RNA concentration, DNase treatment was found to be equally effective in lowering the level of contaminating DNA (10(6) to 10(2) copies of the tod gene/mu), irrespective of the incubation time and the amount of DNase used. Although the results of gel electrophoresis of conventional PCR amplification of the low RNA concentration revealed the absence of the target gene in contaminating DNA, the results of the TaqMan PCR indicated that a very low amount of contaminating DNA (less than 10(3) copies of the tod gene/mul) was still present in RNA samples, even after the DNase treatment. The number of copies of the tod gene transcript in RNA samples did not show any marked variation because of the DNase treatment. However, the proportion of contaminating DNA in RNA samples considerably decreased due to the treatment (0.01 to 0.000001). Furthermore, these results suggested that the extent of the removal of contaminating DNA from RNA samples depends on the concentration of RNA, the amount of DNase used and the incubation time. It is also suggested that the copies of the catabolic genes in contaminating DNA have to be quantified along with the target genes in RNA samples to have a more accurate quantification of the target genes for better understanding of their roles in many microbial processes.

Quantitative measurement of fungal DNA extracted by three different methods using real-time polymerase chain reaction.

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount ofA. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships (R2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 x 10(9) copies/microl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 x 10(8) and 2.7 x 10(8) copies/microl for woodland and grassland, respectively), high DNA yield (6.4 microg/g and 1.8 microg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.