Øivind Enger

Influence of long-term heavy-metal contamination on microbial communities in soil

Abstract Dot blot hybridization with group specific probes and restriction fragment length polymorphism (RFLP) were used for assessing the effect of long-term sewage sludge application on the culturable bacterial flora and total bacterial community in soil. Results obtained using dot blot hybridization with phylogenetic probes on bacterial isolates showed no dramatic difference in the community structure in two soils with different amendments of heavy metal and sludge. A decrease was seen in the percentage of isolates hybridizing with the probes ALF1b, CF319a and LGCb, with increasing sludge/metal amendment, while the percentages of isolates detected with probes BET42a, GAM42a, SRB385 and HGC69a showed an increase in soil with high-sludge/high-metal amendments. Our results support the general idea that the culturable population represents a part of the total population in soil only. A much higher percentage of clones in the 16S rDNA library of bacteria from high-metal/high-sludge than from low-metal/low-sludge amended soil hybridized with the ALF1b probe. The percentage of cloned inserts hybridizing with the probes BET42a, GAM42a, SRB385, CF319a, LGCb and HGC69a were lower in high-sludge/high-metal amended soil compared to the soil with low-sludge/low-metal amendment. Cluster analysis of the RFLP patterns of the bacterial isolates resulted in a higher diversity index in the high-sludge/high-metal soil compared to the low-sludge/low-metal soil, while cluster analysis of the cloned library from the two soils gave the opposite result.

Characterization of Alteromonas denitrificans sp. nov.

Eleven strains of gram-negative, denitrifying, prodigionine-producing marine bacteria with sheathed flagella were recovered from the fjord system off the Norwegian west coast. These isolates form a physiologically and morphologically very homogeneous group. Apart from their ability to denitrify and having sheathed flagella, the strains correspond closely to the current description of the genus Alteromonas. Their strictly respiratory metabolism precludes their inclusion in the genus Vibrio, and the low guanine-plus-cytosine contents of their deoxyribonucleic acids preclude their inclusion in the genus Pseudomonas or the genus Marinomonas. Despite their ability to denitrify, we suggest that these organisms belong to the genus Alteromonas and constitute a new species, Alteromonas denitrificans. A detailed description of the type strain of Alteromonas denitrificans, Nygaard 1977, is given. The type strain has been deposited with the American Type Culture Collection, Rockville, Md., as strain ATCC 43337.

Amplified ribosomal DNA restriction analysis in the differentiation of related species of mycobacteria.

This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra- and interspecies identification of the genus Mycobacteria. A set of primers was used to amplify part of the 16S and 23S rDNA as well as the 16S-23S rDNA spacer from 121 isolates belonging to 13 different mycobacterial species. Restriction analysis was carried out with five different restriction enzymes, namely CfoI, HaeIII, RsaI, MspI and TaqI. Restriction digestion of the PCR product using CfoI enabled differentiation between 9 of the 13 mycobacterial species, whereas the remaining four enzymes differentiated between 7 of these 13 species. None of the five enzymes distinguished between different isolates of Mycobacterium tuberculosis or between species within the M. tuberculosis complex i.e., M. tuberculosis, M. bovis, M. bovis BCG and M. africanum. Although ARDRA analysis of the 16S-23S rDNA does not seem to have a potential for intraspecies differentiation, it has proven to be a rapid and technically relatively simple method to recognise strains belonging to the M. tuberculosis complex as well as to identify mycobacterial species outside this complex.

Sequence analysis in the 23S rDNA region of Mycobacterium tuberculosis and related species

We sequenced a 516 base pair segment in the 23S rRNA gene of 54 Mycobacterium tuberculosis isolates, 52 of which were clinical isolates from Ethiopia. Sequence polymorphism was observed with 19 of the 54 strains; the polymorphic sites occurred in less than 2% (9/516) of the total sequence positions. The sequence variations represented base pair substitutions (14/23), insertions (9/23) or both (1/23). Insertions occurred at one site only, whereas substitutions were observed in various regions of the gene. There was no relation between mutational sites and drug susceptibility. However, using information from the GenBank database, comparison between the 23S rDNA sequences of M. tuberculosis and the corresponding sequences of other mycobacteria and of related non-mycobacterial species revealed considerable variation, suggesting that this region may provide a target for rapid detection and identification of mycobacteria both at the genus or species level.

Immunomagnetic beads as a tool in conjugation experiments

We describe a novel technique for counter-selection of donor cells in conjugation experiments. The technique is based on application of antibody coated magnetic beads for removal of donor cells prior to plating. This makes the technique useful in conjugation experiments where no selective growth regimes can be applied. The technique was employed in conjugation experiments where a resistance plasmid, naturally occurring in the fish pathogenic bacterium Aeromonas salmonicida, was transferred to Escherichia coli HB101 and a marine vibrio. Use of the immunomagnetic beads to remove donor cells prior to plating resulted in transconjugants constituting 8.9% to 90.9% (Aeromonas salmonicida) of total plate count.