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E W Alm

Publications and source records attributed to E W Alm.

7 recordsLinked to original sources
PubMed2001

Distribution and abundance of Gram-positive bacteria in the environment: development of a group-specific probe.

We developed a 16S rRNA-targeted oligonucleotide probe (S-P-GPos-1200-a-A-13) for the Gram-positive bacteria, confirmed its specificity by database searches and hybridization studies, and investigated the effects of humic acids on membrane hybridizations with this probe. S-P-GPos-1200-a-A-13 was used to estimate the abundance of Gram-positive populations in the bovine rumen and Lake Michigan sediments. This probe should be useful for studies of the environmental distribution of Gram-positive bacteria and the detection of uncultured, phylogenetically Gram-positive bacteria with variable or negative Gram staining reactions, and could serve for Gram staining in some diagnostic settings.

Animals↗
PubMed2001

Seasonal and spatial variability in Lake Michigan sediment small-subunit rRNA concentrations.

We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4 degrees C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations.

Animals↗
PubMed2000

Critical factors influencing the recovery and integrity of rRNA extracted from environmental samples: use of an optimized protocol to measure depth-related biomass distribution in freshwater sediments.

A protocol was developed for the efficient recovery of intact, high molecular weight rRNA from different environmental matrices. Critical variables were identified in sample processing that influenced yield and integrity of recovered nucleic acid. Most notably, the order of addition and the buffer to sample volume ratio profoundly influenced the efficiency of nucleic acid recovery from sediment material when utilizing a guanidine thiocyanate-beta-mercaptoethaol extraction buffer. Addition of one sample volume to five buffer volumes contributed to an order of magnitude increase in recovery relative to reverse order of addition (buffer addition to sample). An optimized extraction protocol was used to evaluate rRNA yield by seeding samples with whole cells and radiolabeled nucleic acid. Recovery of intact rRNA was confirmed by polyacrylamide gel electrophoresis, which was also used to provide another estimate of quantity. This optimized protocol was used to measure depth-related changes in biomass distribution in Lake Michigan deep-water sediments. This revealed a biomodal biomass distribution; a maximum near the water/sediment interface and a secondary peak associated with the oxic/suboxic boundary. A significant portion of the community at the oxic/suboxic boundary was composed of non-methanogenic Archaea.

Biomass↗
PubMed2000

The presence of humic substances and DNA in RNA extracts affects hybridization results.

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.

DNA, Bacterial↗
PubMed1997

Crenarchaeota in Lake Michigan sediment.

RNA from Lake Michigan sediment was hybridized with a DNA probe for archaeal 16S rRNA. There was a peak of archaeal rRNA abundance in the oxic zone and another immediately below it. Six contributing species were identified by PCR amplification of extracted DNA with primers specific for archaeal rDNA: two related to Methanosarcina acetivorans and four related to marine crenarchaeotal sequences. rRNA quantification using a DNA probe specific for this crenarchaeotal assemblage showed it is most abundant in the oxic zone, where it accounts for about 10% of total archaeal rRNA.

Archaea↗
PubMed1996

Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.

Archaea↗