CiteULike: vrich Polz

Adaptation and spectral tuning in divergent marine proteorhodopsins from the eastern Mediterranean and the Sargasso Seas

Gazalah Sabehi — 2008-05-18T20:11:48-00:00

ISME J, Vol. 1, No. 1. (0000), pp. 48-55.

PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample.

SG Acinas — 2006-03-17T19:55:57-00:00

Appl Environ Microbiol, Vol. 71, No. 12. (December 2005), pp. 8966-8969.

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.

Genotypic Diversity Within a Natural Coastal Bacterioplankton Population

Janelle Thompson — 2008-05-09T20:41:29-00:00

Science, Vol. 307, No. 5713. (25 February 2005), pp. 1311-1313.

The genomic diversity and relative importance of distinct genotypes within natural bacterial populations have remained largely unknown. Here, we analyze the diversity and annual dynamics of a group of coastal bacterioplankton (greater than 99% 16S ribosomal RNA identity to Vibrio splendidus). We show that this group consists of at least a thousand distinct genotypes, each occurring at extremely low environmental concentrations (on average less than one cell per milliliter). Overall, the genomes show extensive allelic diversity and size variation. Individual genotypes rarely recurred in samples, and allelic distribution did not show spatial or temporal substructure. Ecological considerations suggest that much genotypic and possibly phenotypic variation within natural populations should be considered neutral. 10.1126/science.1106028

Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

Eelin Lim — 2008-05-05T02:10:04-00:00

Appl. Environ. Microbiol., Vol. 67, No. 9. (1 September 2001), pp. 3897-3903.

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml[-]1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml[-]1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples. 10.1128/AEM.67.9.3897-3903.2001

Diversity and Dynamics of a North Atlantic Coastal Vibrio Community

Janelle Thompson — 2008-05-05T02:00:06-00:00

Appl. Environ. Microbiol., Vol. 70, No. 7. (1 July 2004), pp. 4103-4110.

Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains. Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation. A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period. Quantitative PCR (QPCR) with primers specific for the genus Vibrio was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE). Vibrio populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations. Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction. It further showed that CDCE populations corresponded in most cases to [~]98% rRNA similarity groups and suggested that the abundance of these follows temperature trends. Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples. Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend. 10.1128/AEM.70.7.4103-4110.2004

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'

Janelle Thompson — 2008-05-04T21:02:22-00:00

Nucl. Acids Res., Vol. 30, No. 9. (1 May 2002), pp. 2083-2088.

Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample. 10.1093/nar/30.9.2083

Bias in Template-to-Product Ratios in Multitemplate PCR

Martin Polz — 2008-05-04T21:02:13-00:00

Appl. Environ. Microbiol., Vol. 64, No. 10. (1 October 1998), pp. 3724-3730.

Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

Diversity and Heterogeneity of Epibiotic Bacterial Communities on the Marine Nematode Eubostrichus dianae

Martin Polz — 2008-05-04T19:59:30-00:00

Appl. Environ. Microbiol., Vol. 65, No. 9. (1 September 1999), pp. 4271-4275.

The diversity of a microbial community covering the surface of a marine nematode was analyzed by performing a 16S ribosomal DNA (rDNA) restriction cutting and sequencing analysis. In two clone libraries constructed by using individual nematodes, 54 and 85 restriction patterns were identified, and only 13 of these patterns were common to both libraries. Sequence analysis indicated that the common patterns belonged to four groups related to sequences of cytophagas, sulfate-reducing bacteria, members of the gamma subclass of the class Proteobacteria, and caulobacters. At least two groups appeared to be permanent members of the community as they were also detected in a 16S rDNA library constructed 3 years previously by using 100 pooled nematode specimens. A surprising outcome was that very dominant filamentous bacteria were apparently not represented in the clone libraries, as quantitative probing showed that none of the common operational taxonomic unit groups displayed the expected overwhelming dominance. Nevertheless, our analysis revealed both an unexpectedly high level of bacterial diversity and heterogeneity in samples representing presumably very similar microenvironments.