CiteULike: vrich's lh-pcr

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Jeanette Norton — 2008-05-11T02:48:43-00:00

Archives of Microbiology, Vol. 177, No. 2. (1 February 2002), pp. 139-149.

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the &#35- and the &#37-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the &#35-subdivision representatives but in single copies in the &#37-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the &#35-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

Marcelino Suzuki — 2008-05-11T01:05:46-00:00

Appl. Environ. Microbiol., Vol. 64, No. 11. (1 November 1998), pp. 4522-4529.

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5' domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Stewart — 2007-11-14T09:59:10-00:00

Journal of Molecular Evolution, Vol. 65, No. 1. (July 2007), pp. 44-67.

Assessing Microbial Community Diversity Using Amplicon Length Heterogeneity Polymerase Chain Reaction

Deetta Mills — 2008-05-07T21:59:32-00:00

Soil Sci Soc Am J, Vol. 71, No. 2. (12 March 2007), pp. 572-578.

It is thought that a microbial community is an assemblage of organisms, genes, and gene functions. Transient, acute signals such as excessive nutrient loads or disturbance and chronic signals such as seasonal temperature or rainfall impact the total environmental system. The goal of many microbial ecologists is to determine if a finely resolved study of microbial dynamics can be used as a large-scale biosensor to follow diversity patterns in the environment. With the development of new genomic tools, community-level studies have been designed that can interrogate the finer details of the biological components of a given habitat. Amplicon length heterogeneity polymerase chain reaction (LH-PCR) interrogates the hypervariable domains of the ribosomal small-subunit genes and separates these domains based on the naturally occurring sequence lengths of DNA. The amplicons are phylogenetically relevant in that the various amplicons generated can be directly associated with specific taxonomic sequences archived in the databases. The application of the LH-PCR technique as a monitoring tool for microbial ecology has been shown to enhance and extend the current understanding of the dynamics of microbial communities in their specific environments. 10.2136/sssaj2006.0147

Use of Length Heterogeneity PCR and Fatty Acid Methyl Ester Profiles To Characterize Microbial Communities in Soil

Nancy Ritchie — 2008-05-07T21:29:09-00:00

Appl. Environ. Microbiol., Vol. 66, No. 4. (1 April 2000), pp. 1668-1675.

In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5' domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the [beta] subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition. 10.1128/AEM.66.4.1668-1675.2000

Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling

Lorenzo Brusetti — 2008-05-05T17:06:52-00:00

FEMS Microbiology Ecology, Vol. 56, No. 1. (2006), pp. 154-164.

Abstract The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.

Phylogenetic Screening of Ribosomal RNA Gene-Containing Clones in Bacterial Artificial Chromosome (BAC) Libraries from Different Depths in Monterey Bay

MT Suzuki — 2007-09-19T01:32:56-00:00

Microbial Ecology, Vol. 48, No. 4. (1 December 2004), pp. 473-488.

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.

Microbial community dynamics based on 16S rRNA gene profiles in a Pacific Northwest estuary and its tributaries

Anne Bernhard — 2008-05-05T17:03:11-00:00

FEMS Microbiology Ecology, Vol. 52, No. 1. (1 March 2005), pp. 115-128.

We analyzed bacterioplankton community structure in Tillamook Bay, Oregon and its tributaries to evaluate phylogenetic variability and its relation to changes in environmental conditions along an estuarine gradient. Using eubacterial primers, we amplified 16S rRNA genes from environmental DNA and analyzed the PCR products by length heterogeneity polymerase chain reaction (LH-PCR), which discriminates products based on naturally occurring length differences. Analysis of LH-PCR profiles by multivariate ordination methods revealed differences in community composition along the estuarine gradient that were correlated with changes in environmental variables. Microbial community differences were also detected among different rivers. Using partial 16S rRNA sequences, we identified members of dominant or unique gene fragment size classes distributed along the estuarine gradient. Gammaproteobacteria and Betaproteobacteria and members of the Bacteroidetes dominated in freshwater samples, while Alphaproteobacteria, Cyanobacteria and chloroplast genes dominated in marine samples. Changes in the microbial communities correlated most strongly with salinity and dissolved silicon, but were also strongly correlated with precipitation. We also identified specific gene fragments that were correlated with inorganic nutrients. Our data suggest that there is a significant and predictable change in microbial species composition along an estuarine gradient, shifting from a more complex community structure in freshwater habitats to a community more typical of open ocean samples in the marine-influenced sites. We also demonstrate the resolution and power of LH-PCR and multivariate analyses to provide a rapid assessment of major community shifts, and show how these shifts correlate with environmental variables.

High Temporal but Low Spatial Heterogeneity of Bacterioplankton in the Chesapeake Bay

Jinjun Kan — 2008-05-05T17:02:59-00:00

Appl. Environ. Microbiol., Vol. 73, No. 21. (1 November 2007), pp. 6776-6789.

Compared to freshwater and the open ocean, less is known about bacterioplankton community structure and spatiotemporal dynamics in estuaries, particularly those with long residence times. The Chesapeake Bay is the largest estuary in the United States, but despite its ecological and economic significance, little is known about its microbial community composition. A rapid screening approach, ITS (internal transcribed spacer)-LH (length heterogeneity)-PCR, was used to screen six rRNA operon (16S rRNA-ITS-23S rRNA) clone libraries constructed from bacterioplankton collected in three distinct regions of the Chesapeake Bay over two seasons. The natural length variation of the 16S-23S rRNA gene ITS region, as well as the presence and location of tRNA-alanine coding regions within the ITS, was determined for 576 clones. Clones representing unique ITS-LH-PCR sizes were sequenced and identified. Dramatic shifts in bacterial composition (changes within subgroups or clades) were observed for the Alphaproteobacteria (Roseobacter clade, SAR11), Cyanobacteria (Synechococcus), and Actinobacteria, suggesting strong seasonal variation within these taxonomic groups. Despite large gradients in salinity and phytoplankton parameters, a remarkably homogeneous bacterioplankton community was observed in the bay in each season. Stronger seasonal, rather than spatial, variation of the bacterioplankton population was also supported by denaturing gradient gel electrophoresis and LH-PCR analyses, indicating that environmental parameters with stronger seasonal, rather than regional, dynamics, such as temperature, might determine bacterioplankton community composition in the Chesapeake Bay. 10.1128/AEM.00541-07

Rapid lab-on-a-chip profiling of human gut bacteria.

J Bjerketorp — 2008-05-05T17:02:23-00:00

Journal of microbiological methods, Vol. 72, No. 1. (January 2008), pp. 82-90.

The human gut microbiota has a substantial impact on human health. Different factors such as disease, diet and drug use can have significant impacts on the gut microbiota. Therefore, it is of interest to have simple, rapid methods for analysis of the composition of the gut microbiota for clinical diagnostic purposes. Since only a minor fraction of the gastrointestinal bacterial community is presently possible to cultivate, molecular approaches are currently the best suited to investigate its composition. However, most of these molecular approaches require technical expertise and expensive equipment to run and they are not routinely available. Ideally, the analyses should be point-of-care options that can be run on a chip. In this study, an existing lab-on-chip (LOC) system for sizing/quantifying DNA was combined with length heterogeneity PCR (LH-PCR), a PCR-based profiling method targeting bacterial 16S rRNA gene sequences, to develop a fast, straightforward, reproducible, and economical method for profiling bacterial communities. The LOC LH-PCR method was first evaluated using a standardized gut cocktail containing genomic DNA from eight different bacterial species representing different genera of relevance for human health. The method was also tested on DNA that was directly extracted from human faecal samples and it was consistently capable of detecting alterations in the bacterial samples before and after antibiotic treatment. Although the resolution of the method needs improvement, this study represents the first step towards development of a diagnostic LOC for profiling gut bacterial communities.