Conjugative Plasmid Transfer between Bacteria under Simulated Marine Oligotrophic Conditions

Marine Vibrio S14 strains and an Escherichia coli strain were starved in artificial seawater (NSS) with no added carbon, nitrogen, or phosphorus. The broad-host-range plasmid RP1 was transferred between the starving S14 strains and also from the E. coli donor to the S14 recipient under oligotrophic conditions, in which mixtures of donor and recipient cells were held on Nuclepore filters either floated on NSS or held such that NSS flowed through the filter. Transconjugants were obtained from S14 donors and recipients starved for at least 15 days before being mixed together for conjugation, whereas transconjugants were recovered from the E. coli donor and S14 recipient for up to 3 days of prestarvation, but not after 5 days. Transconjugants were obtained when there were as few as about 10⁵ and 10⁴ cells of starving S14 donors and recipients, respectively, per ml held on the filters. Starved donor and recipient mixtures incubated at 4 or 26°C, as well as those allowed to mate for 2, 5, or 24 h, all yielded numbers of transconjugants which were not significantly (P > 0.05) different.     

Abundance of Tn3, Tn21, and Tn501 transposase (tnpA) sequences in bacterial community DNA from marine environments.

The occurrence of the tnpA genes of the transposons Tn3, Tn21, and Tn501 was assessed in total bacterial community DNA isolated from different marine environments. The PCR technique was employed, together with most probable number statistics, to determine the abundance of the target tnpA genes. All three genes could be detected, and the Tn21 tnpA sequences predominated in all samples. The smallest amount of total community DNA in which the Tn21 tnpA sequence could be detected was 0.037 ng, and on the basis of our results, we estimated that this sequence was present in 1 of 1,000 to 10,000 bacteria. Hybridization of the PCR products with the respective tnpA probes verified the Tn21 and Tn501 tnpA sequences but only some of the Tn3 tnpA amplification products. The distribution and dissemination of transposons in natural bacterial communities are discussed.     

Determination of (R)- and (S)-Disophyramide in human plasma using a chiral α1-acid glycoprotein column

The direct resolution and quantitation of (R)- and (S)-disopyramide, isolated from human plasma, was accomplished using a chiral α₁-acid glycoprotein column. A LiChrosorb RP-2 column (50 × 3.0 mm I.D.) was used as a precolumn. Phosphate buffer, pH 6.20, containing 2-propanol and N,N-dimethyloctylamine was used as mobile phase, expressed as the relative standard deviation, was 1.8% and 3.3% for (R)- and (S)-disopyramide, respectively, at a drug level of 0.5 μg/ml. In two subjects who received a single capsule of racemic disopyramide (150 mg), the plasma levels of the (R) isomer were about half those of the (S) isomer. The half-lives of (R)- and (S)-disopyramide were similar.     

Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.     

Inter-helical hydrogen bond formation during membrane protein integration into the ER membrane

Recent work has shown that efficient di- or trimerization of hydrophobic transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a non-aqueous subcompartment within the translocon.     

The Choice of PCR Primers Has Great Impact on Assessments of Bacterial Community Diversity and Dynamics in a Wastewater Treatment Plant

Assessments of bacterial community diversity and dynamics are fundamental for the understanding of microbial ecology as well as biotechnological applications. We show that the choice of PCR primers has great impact on the results of analyses of diversity and dynamics using gene libraries and DNA fingerprinting. Two universal primer pairs targeting the 16S rRNA gene, 27F&1492R and 63F&M1387R, were compared and evaluated by analyzing the bacterial community in the activated sludge of a large-scale wastewater treatment plant. The two primer pairs targeted distinct parts of the bacterial community, none encompassing the other, both with similar richness. Had only one primer pair been used, very different conclusions had been drawn regarding dominant phylogenetic and putative functional groups. With 27F&1492R, Betaproteobacteria would have been determined to be the dominating taxa while 63F&M1387R would have described Alphaproteobacteria as the most common taxa. Microscopy and fluorescence in situ hybridization analysis showed that both Alphaproteobacteria and Betaproteobacteria were abundant in the activated sludge, confirming that the two primer pairs target two different fractions of the bacterial community. Furthermore, terminal restriction fragment polymorphism analyses of a series of four activated sludge samples showed that the two primer pairs would have resulted in different conclusions about community stability and the factors contributing to changes in community composition. In conclusion, different PCR primer pairs, although considered universal, target different ranges of bacteria and will thus show the diversity and dynamics of different fractions of the bacterial community in the analyzed sample. We also show that while a database search can serve as an indicator of how universal a primer pair is, an experimental assessment is necessary to evaluate the suitability for a specific environmental sample.