Potential of pmoA Amplicon Pyrosequencing for Methanotroph Diversity Studies

We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.     

Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.     

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10³ to 10⁴ oocysts, and the nested PCR method was able to detect 10⁰ to 10² oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.     

Diversity of amoA and pmoA Genes in Extremely Saline Alkaline Soils of the Former Lake Texcoco

Abstract

Ammonia-oxidizing bacteria (AOB) and aerobic methane oxidizing-bacteria (MOB) were studied in three extreme soils of the former Lake Texcoco, Mexico, with pH ranging from 8.5 to 10.5 and electrolytic conductivity (EC) from 0.67 to 84.76 dS m^?1, and in two arable soils. Soil DNA was extracted with three different methods and total DNA was used as a template to amplify the pmoA and amoA functional genes and subsequently sequenced by pyrosequencing. The amoA gene sequences clustered as uncultured AOB dominated in the Texcoco soils, while Nitrosospira was dominant in the arable soils. Sequences of MOB associated with Nitrosococcus-rel (Type I) dominated (>85%) in the Texcoco soils, but they were more diverse in the arable soils, for example, JR2, JR3, Methylocaldum USC-g (Type I), USC-a (Type II) and gp23 (pxmA). Aerobic methane oxidizing-bacteria and AOB microbial diversity were significantly related to EC and pH (p?<?0.05). As such, the lower MOB and AOB microbial diversity in the Texcoco soil compared to the arable soil was determined by its higher EC and pH.     

Novel Diabetic Mouse Models as Tools for Investigating Diabetic Retinopathy

Objective

Mouse models possessing green fluorescent protein (GFP) and/or human aldose reductase (hAR) in vascular tissues have been established and crossed with naturally diabetic Akita mice to produce new diabetic mouse models.

Research Design and Methods

Colonies of transgenic C57BL mice expressing GFP (SMAA-GFP), hAR (SMAA-hAR) or both (SMAA-GFP-hAR) in vascular tissues expressing smooth muscle actin were established and crossbred with C57BL/6-Ins2^Akita/J (AK) mice to produce naturally diabetic offspring AK-SMAA-GFP and AK-SMAA-GFP-hAR. Aldose reductase inhibitor AL1576 (ARI) was administered in chow. Retinal and lenticular sorbitol levels were determined by HPLC. Retinal functions were evaluated by electroretinography (ERGs). Growth factor and signaling changes were determined by Western Blots using commercially available antibodies. Retinal vasculatures were isolated from the neural retina by enzymatic digestion. Flat mounts were stained with PAS-hematoxylin and analyzed.

Results

Akita transgenics developed DM by 8 weeks of age with blood glucose levels higher in males than females. Sorbitol levels were higher in neural retinas of AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice also had higher VEGF levels and reduced ERG scotopic b-wave function, both of which were normalized by AL1576. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors bFGF, IGF-1, and TGFβ, as well as signaling changes in P-Akt, P-SAPK/JNK and P-44/42 MAPK that were also reduced by ARI treatment. Quantitative analysis of flat mounts in 18 week AK-SMAA-GFP-hAR mice revealed increased loss of nuclei/capillary length and a significant increase in the percentage of acellular capillaries present which was not seen in AK-SMAA-GFP-hAR treated with ARI.

Conclusions/Significance

These new mouse models of early onset diabetes may be valuable tools for assessing both the role of hyperglycemia and AR in the development of retinal lesions associated with diabetic retinopathy.     

Bacterial Diversity in Three Different Antarctic Cold Desert Mineral Soils

A bacterial phylogenetic survey of three environmentally distinct Antarctic Dry Valley soil biotopes showed a high proportion of so-called “uncultured” phylotypes, with a relatively low diversity of identifiable phylotypes. Cyanobacterial phylotypic signals were restricted to the high-altitude sample, whereas many of the identifiable phylotypes, such as the members of the Actinobacteria, were found at all sample sites. Although the presence of Cyanobacteria and Actinobacteria is consistent with previous culture-dependent studies of microbial diversity in Antarctic Dry Valley mineral soils, many phylotypes identified by 16S rDNA analysis were of groups that have not hitherto been cultured from Antarctic soils. The general belief that such “extreme” environments harbor a relatively low species diversity was supported by the calculation of diversity indices. The detection of a substantial number of uncultured bacterial phylotypes showing low BLAST identities (<95%) suggests that Antarctic Dry Valley mineral soils harbor a pool of novel psychrotrophic taxa.     

Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities

ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng μl⁻¹, while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.     

Comparison of Different Denaturing Gradient Gel Electrophoresis Primer Sets for the Study of Marine Bacterioplankton Communities

An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment.     

通用引物PCR检测临床常见致病菌的实验研究

通用引物可一次性扩增18种临床常见致病菌和耐药菌株的DNA,扩增片段长度在220bp左右,18种特异性探针分别与18种标准菌株的PCR扩增产物杂交结果显示探针都具有高度特异性;5种37例经法国梅里埃API细菌鉴定系统确定的临床分离菌株进行杂交鉴定,鉴定结果与分离株一致,表明设计的探针具有高度特异性及准确性。80例临床标本分别用法国梅里埃API细菌鉴定系统及PCR杂交法进行检测,阳性率分别为(52.5%)和(67.5%),表明PCR结合寡核苷酸杂交法比传统的生物学培养法更为灵敏,值得推广。     

Comparison of Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Aquatic Bacterial Communities: an Ecological Perspective

Two primer sets for automated ribosomal intergenic spacer analysis (ARISA) were used to assess the bacterial community composition (BCC) in Lake Mendota, Wisconsin, over 3 years. Correspondence analysis revealed differences in community profiles generated by different primer sets, but overall ecological patterns were conserved in each case. ARISA is a powerful tool for evaluating BCC change through space and time, regardless of the specific primer set used.     

Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.     

Primer design for PCR and sequencing in high-throughput analysis of SNPs

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.     

Effects of Water Level Management on Autumn Staging Waterbird and Macrophyte Diversity in Three Danish Coastal Lagoons

Many Danish wetlands frequently experience algae blooms and oxygen deficiencies because of eutrophication caused by enhanced nitrogen and phosphorous levels. As a consequence, wetland managers have focused on improving water quality, but often without considering the suitability of wetlands for waterbirds. In this study, managers improved water quality in two Danish lagoons by opening a floodgate to the sea. We studied the responses of autumn staging waterbirds and submerged vegetation, to resultant higher water replacement rates and water levels. A third lagoon with no change in management was studied for comparisons. Lagoons with rise in water levels experienced declines in bird species diversity, a decline in benthivore species abundances and an increase in herbivore species abundances. Macrophyte biomass increased but seagrass diversity was low due to high salinity. The lagoon with no changes in management control had high and stable waterbird diversity and bird-days spent was increasing during the study period. Explanations for this were diverse typography and low water levels. Furthermore, the macrophyte community was more diverse due to low salinity. In order to improve both water quality and to increase waterbird diversity in the managed lagoons, we suggest water levels should be managed actively during peak migration in autumn. This could secure more shallow-water areas to waders and create better congruence between appropriate water levels and timing of peak bird migration in autumn. The rest of the year the floodgate should be left open in order to secure a high water quality.     

Geogenic Factors as Drivers of Microbial Community Diversity in Soils Overlying Polymetallic Deposits

This study shows that the geogenic factors landform, lithology, and underlying mineral deposits (expressed by elevated metal concentrations in overlying soils) are key drivers of microbial community diversity in naturally metal-rich Australian soils with different land uses, i.e., agriculture versus natural bushland. One hundred sixty-eight soil samples were obtained from two metal-rich provinces in Australia, i.e., the Fifield Au-Pt field (New South Wales) and the Hillside Cu-Au-U rare-earth-element (REE) deposit (South Australia). Soils were analyzed using three-domain multiplex terminal-restriction-fragment-length-polymorphism (M-TRFLP) and PhyloChip microarrays. Geogenic factors were determined using field-mapping techniques and analyses of >50 geochemical parameters. At Fifield, microbial communities differed significantly with geogenic factors and equally with land use (P < 0.05). At Hillside, communities in surface soils (0.03- to 0.2-m depth) differed significantly with landform and land use (P < 0.05). Communities in deeper soils (>0.2 m) differed significantly with lithology and mineral deposit (P < 0.05). Across both sites, elevated metal contents in soils overlying mineral deposits were selective for a range of bacterial taxa, most importantly Acidobacteria, Bacilli, Betaproteobacteria, and Epsilonproteobacteria. In conclusion, long-term geogenic factors can be just as important as land use in determining soil microbial community diversity.     

Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.     

Feasibility Study of Using Centrifuge for Investigating LNAPL Migration in Unsaturated Soils

Centrifuge modeling appears potentially useful for studying geo-environmental problems such as pollutant migration in subsurface systems. In this study, the “modeling of models” technique was used to validate the feasibility of using a geotechnical centrifuge to model the transport behavior of light non-aqueous phase liquids (LNAPLs) in unsaturated soils. All the experiments were conducted to simulate a gasoline spill from a leaking underground storage tank (UST) and the subsequent subsurface migration of the gasoline. When the gravity in the centrifuge reached the desired g-level, the gasoline was released from the UST and then it migrated in the unsaturated soil corresponding to a prototype time equivalent of one year. After the centrifuge tests, soil samples were collected using sampling tubes and the concentrations of individual constituent in the LNAPL were directly measured by means of gas chromatograph analysis. Results obtained from the centrifuge tests at different g-levels show that similar migration patterns are found for LNAPL transport in unsaturated porous media. The location of the peak concentration and the behavior of lateral spreading can be adequately described. In addition, centrifuge test data show that the migration pattern of LNAPLs is related to the soil type and the physical properties of individual constituents in the LNAPLs.     

Diversity of Basidiomycetes in Michigan Agricultural Soils

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with ≥99% identity in the 5′ 650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.     

Detection of Fiber-Digesting Bacteria in the Ceca of Ostrich Using Specific Primer Sets

The purpose of this study was to detect three fibrolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus, in the cecal digesta of the ostrich (Struthio camelus) by PCR using a species-specific primer set for each 16S ribosomal RNA gene (16S rDNA). Although amplified DNA fragments obtained from each primer set had the expected size, the clone library derived from the amplimer contained non-specific sequences. The F. succinogenes-specific primer set recovered a partial 16S rDNA sequence of an uncultivated Fibrobacter with low similarity (<95%) and distantly related phylogenetic positioning to Fibrobacter sequences deposited in the databases, indicating a novel species of Fibrobacter. The sequence was considered to be identical to a clone detected in our previous experiment. Thus, we confirm that the gastrointestinal tract of the ostrich is one of the habitats of Fibrobacter species. The clone library derived from the R. flavefaciens-specific primer set contained a 16S rDNA sequence with 97% similarity to R. flavefaciens, indicating it could be one of a major fibrolytic bacterium in the ostrich ceca. No R. albus 16S rDNA sequence was found in the clone library of the R. albus-specific primer set.