Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea

A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Δhpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of ΔproC Δhpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of β-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina. A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed ~300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.     

Evaluating Dispersal Limitation in Passive Bottomland Forest Restoration

Dispersal limitation can retard natural establishment of desirable species on restoration sites, especially where landscapes are fragmented, but dispersal limitation is assumed to become less critical with time as early colonists become reproductively mature. Distribution patterns of recruiting trees in a 20‐year‐old passively restored bottomland in northeast Louisiana suggested persistent dispersal limitation in some bottomland hardwood species and influence of dense shrub patches on colonization. To test these hypotheses, we measured seed rain as a function of distance to seed source and association with shrub cover. Seed rain of the wind‐dispersed Fraxinus pennsylvanica was highest near the forest edge, except where mature recruits occurred. Although shrub presence did not influence dispersal of F. pennsylvanica, its negative influence on probability of occurrence in the sapling layer suggests that shrub cover may limit its regeneration. The bird‐dispersed Crataegus viridis and Ilex decidua were found in the seed rain and as reproductive individuals within the field; neither had a positive relationship with shrub presence. Dispersal of heavy‐seeded Quercus spp. and Carya aquatica was limited to within 20 m of the forest edge. These results imply that dispersal limitation is diminishing in wind‐ and bird‐dispersed species with maturation of in‐field recruits and that shrub patches may influence these patterns. Heavy‐seeded species, however, remain restricted to field edges that directly abut a seed source. If canopy closure by wind‐ and bird‐dispersed species precedes dispersal of heavy‐seeded species into the field, establishment of Quercus and Carya spp. may remain low for the foreseeable future.     

TAP score: torsion angle propensity normalization applied to local protein structure evaluation

Background  

Experimentally determined protein structures may contain errors and require validation. Conformational criteria based on the Ramachandran plot are mainly used to distinguish bet ween distorted and adequately refined models. While the readily available criteria are sufficient to detect totally wrong structures, establishing the more subtle differences between plausible structures remains more challenging.     

Geochemistry of biogenic pyrite and ferromanganese coatings from a small watershed: A bacterial connection?

Present‐day groundwater in an alluvial aquifer in Holocene floodplain deposits in east‐central Alabama contains 0.1–4 mg/L Fe, 0.1–0.7 mg/L Mn, ～1–10 μg/L each of Co, Ni, As, Zn, La, and Ce, and 40–175 μ/L Ba. There is a distinct correspondence between trace elements present in groundwater and those concentrated on ferromanganese coatings on present‐day stream alluvium in the study area. This indicates that the reduction and dissolution of such coatings in the alluvial aquifer, probably mediated by Fe‐ and Mn‐reducing bacteria, has been a major control on groundwater chemistry. Authigenic euhedral pyrite crystals up to 1.5 cm in diameter replace lig‐nitic macro wood fragments near the base of the alluvial aquifer, and sulfur isotope data (δ³⁴S values from +3 to ‐40‰_CDT) indicate that pyrite precipitated as a consequence of bacterial sulfate reduction in and adjacent to the irregularly distributed wood fragments. The authigenic pyrite contains several hundred parts per million of As, Co, and Ni, indicating that these trace elements were coprecipitated in pyrite during bacterial sulfate reduction. Results suggest a strong geomicrobiological control on trace element cycling in the study area.     

The Fractured Community: Landscapes of Power and Gender in Rural Zambia

The Fractured Community: Landscapes of Power and Gender in Rural Zambia. Kate Crehan. Berkeley: University of California Press, 1997.258 pp.