Lasse D. Rasmussen

Application of a mer-lux biosensor for estimating bioavailable mercury in soil

Abstract A previously described bioassay using a mer-lux gene fusion for detection of bioavailable mercury was applied for the estimation of the bioavailable fraction of mercury in soil. The bioavailable fraction is defined here as being part of the water leachable fraction. Due to masking of light emission of soil particles leachates had to be cleaned prior to assays. Filtration of leachates through nitro-cellulose filters using pressure resulted in an underestimation of bioavailable mercury. Gravity filtration and centrifugation showed elevated (as compared with untreated leachate) and very similar responses. The utility of the mer-lux biosensor assay was tested by relating measurements of bioavailable and total mercury to the response of the soil microbial community to mercury exposure. Two different soil types (an agricultural and a beech forest soil) were spiked with 2.5 μg Hg(II) g−1 in microcosms and the frequency of mercury resistant heterotrophs and changes in community diversity, defined as the number of different 16S rDNA bands observed in DGGE gels, were monitored. In the agricultural soil the initial concentration of bioavailable mercury was estimated to be 40 ng g−1. This concentration did not change during the first 3 d and coincided with increased degrees of resistance and a decrease in diversity. The concentration of bioavailable mercury decreased subsequently rapidly and remained just above the detection level (0.2 ng g−1) for the remainder of the experiment. As a possible consequence of the decreased selection pressure of mercury, the resistance and diversity gradually returned to pre-exposure amounts. In the beech forest soil the concentration of bioavailable mercury was found to be about 20 ng g−1 throughout the experiment. This concentration did not at any time result in changes in resistance or diversity. This study showed that the bioassay using the mer-lux biosensor is a useful and sensitive tool for estimation of bioavailable mercury in soil.

The effect of longterm exposure to mercury on the bacterial community in marine sediment.

Abstract. The aim of this study was to investigate the effect of mercury contamination on bacterial community structure and function. Bacterial communities from two sites, a mercury-contaminated site inside the harbor of Copenhagen, Denmark (CH) and a unpolluted control site, Køge Buge (KB), were compared with respect to diversity indices, of antibiotic- and heavy metal-resistance patterns, abundance and self transmissibility of plasmids in resistant isolates (endogenous isolation). Furthermore, the potential for gene transfer between indigenous bacteria was assessed by the exogenous plasmid isolation approach.  It was found that resistance to all the tested compounds was higher in the mercury-polluted sediment than the control sediment. The abundance of plasmids was higher at the polluted site, where 62% of the isolates contained plasmids, whereas only 29% of the isolates from the control sediment contained plasmids. Furthermore, the frequencies of large plasmids and plasmids per isolates were found to be higher in the contaminated sediment. Exogenous plasmid isolations revealed high occurrence of Hg and tetracycline resistance, self-transmissible plasmids in CH sediment (1.8 × 10−5 transconjugants per recipients) relative to KB sediment (3.0 × 10−8 T/R). Shannon-Weaver diversity indices showed no difference in the diversity of the isolates from the two sites, and Hg-resistant isolates from CH were found to be as diverse as the CH isolates in total. This may be owing to high level of self-transmissible Hg resistance plasmids found in CH.

Cultivation of Hard-To-Culture Subsurface Mercury-Resistant Bacteria and Discovery of New merA Gene Sequences

ABSTRACT Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis

We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa). The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE). PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested. It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants. PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity. Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity. Of 43 clones, 27 different sequences were found. Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae. Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups. The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species. Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation. Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.

Acclimation of subsurface microbial communities to mercury

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.

Prey-predator dynamics in communities of culturable soil bacteria and protozoa: differential effects of mercury

We investigated whether the prey-predator dynamics of bacteria and protozoa were affected by inorganic mercury at concentrations of 0, 3.5 and 15 mg Hg(II) kg soil 21 . The amount of bioavailable Hg was estimated using a biosensor-assay based on the mer – lux gene fusion. The numbers of bacterial CFUs on the general medium 1/100 tryptic soy agar (TSA) were significantly decreased when the soil had been amended with Hg. In contrast, no effect was seen on the number of CFUs on the Pseudomonas-specific medium Gould’s S1 agar. Protozoan numbers estimated by the most probable number (MPN) method with 1/100 TSB as growth medium were also negatively affected by Hg. The different fractions of protozoa were affected to different degrees suggesting that amoebae were less sensitive than slow-growing flagellates, which again were less sensitive than the fast-growing flagellates. In contrast, Hg did not induce any detectable changes in the diversity of flagellate morphotypes. In the treatment with 15 mg Hg kg 21 a transiently increased number of bacteria was seen at day 6 probably concomitant with a decrease in the numbers of protozoa. This might indicate that Hg affected the prey-predator dynamics in communities of culturable bacteria and protozoa in soil. Furthermore, we showed that the number of Pseudomonas spp. was not affected by Hg whereas the number of bacteria growing on a general medium was. q 2003 Elsevier Ltd. All rights reserved.

An anti vimentin antibody promotes tube formation

In recent years, there has been an increasing appreciation of the importance of secreted and extracellular proteins that traditionally have been considered as intracellular components. Vimentin is a highly abundant intermediate filament protein, and its intracellular functions have been investigated in a large number of studies. Recently, however, vimentin has been shown to take part in significant processes outside the cell. Our understanding of the functions of extracellular vimentin is, however, limited. In this study we demonstrate that a vimentin specific antibody, obtained by phage antibody technology, promotes tube formation of endothelial cells in a 2D matrigel assay. By binding vimentin, the antibody increases the tube formation by 21% after 5 hours of incubation. Addition of the antibody directly to cultured endothelial cells does not influence endothelial cell migration or proliferation. The enhanced tube formation can be seen for up to 10 hours where after the effect decreases. It is shown that the antibody-binding site is located on the coil 2 domain of vimentin. To our knowledge this is the first study that demonstrates an enhanced tube formation by binding vimentin in a 2D matrigel assay under normoxic conditions.

Mercury Decreases Culturability of Pseudomonas frederiksbergensis JAJ 28 in Soil Microcosms

Mercury is a biologically potent heavy metal, which has been found to change the diversity of culturable bacteria. Therefore, we investigated whether Hg kills bacteria in soil or reduces culturability. Soil microcosms were inoculated with Pseudomonas frederiksbergensis JAJ 28 and were sampled regularly during 28 days. The total number of acridine orange-stained cells was relatively constant, and Hg reduced the number on only one sampling day. However, the fraction of culturable cells on 1/10 tryptic soy agar was lowered on days 6, 13, and 21. The number of microcolony forming units, which represents viable cells, was also affected by Hg, but this effect was delayed compared with the effects on CFUs. The amount of headspace CO2 per cell was overall increased by Hg, another indication of the toxic effects of Hg on the bacterial cells. Our results thus emphasize the need to take culturability into account when studying the effects of heavy metals on bacterial diversity. RID=”” ID=”” Correspondence to: K. Johnsen: email: kjo@vetinst.dk